Category Archives: Nano medicine

AN Israeli cancer drug has returned to Earth after a series of tests on the International Space Station (ISS), conducted in the hope it may prove more effective in micro-gravity.

Prof. Yehezkel Bernholtz of the Hebrew University is analysing results from the experiment to see whether there is any truth to his hypothesis that treating cancer patients with his drug in space-like conditions, where gravity is lower than normal, could have benefits.

Bernholtz, an expert in biochemistry, nanotechnology and drug development, is the inventor of Doxil, which became the first nano drug to receive approval from the US Food and Drug Administration, back in 1995.

Almost a million people around the world are believed to have been treated with the drug, including patients suffering from ovarian cancer and multiple myeloma. Bernholtz, the founder of Ayana Pharma, designed an experiment in which the drug meets tumour cells on the ISS, and packaged it in a container the size of two cigarette boxes.

It was blasted into space in early January on a SpaceX Falcon 9, as part of a smart laboratory developed by Israeli company SpacePharma, and recently arrived back on Earth.

What we want to check is whether the active molecules that kill the tumour cells are released, when in microgravity, at a rate and in a manner that makes them more effective, Bernholtz told The Times of Israel.

Prof. Yehezkel Bernholtz of the Hebrew University and founder of ay Pharma, holding a vial of Doxil (courtesy of Ayana Pharma)

He said that the microgravity of space is the best place to test the possibility, but if microgravity is found to boost the medicine, then the conditions of space can be replicated, to a degree, on Earth. If this idea proves correct, we already have equipment to imitate microgravity on Earth, and we can use it with cells, then to test with small animals, and if relevant we can try to apply microgravity to humans undergoing cancer treatment.

The samples will now undergo a series of tests, and its results will be examined and analysed so that we can better understand the effects that zero gravity has on Doxil and whether there is a change in the drug itself or in its action against cancer cells under zero-gravity conditions.

NASA and others are deeply interested in the potential of exomedicine, the study of medicine in zero gravity. NASA has explained the rationale in apost: Without gravity, cells, molecules, protein crystals, and microbes behave in very different ways.

Microgravity thus presents opportunities to explore new and potentially game-changing discoveries in areas such as human tissue regeneration, drug development, treatments for diseases such as cancer and other life-threatening and chronic conditions, as well as energy and novel materials.

The potential of exomedicine for impacting regular drugs like aspirin is thought to be limited, but its believed that some drugs with complex structures that are easily impacted by changing environments could show significant differences in zero gravity.

Bernholtz said that Doxil is well suited for space studies as it has a complex structure with qualities that could conceivably change in zero gravity. Complex drugs are much more likely to go through changes that other drugs dont go through, he said.

Bernholtz added: This is a highly exciting progress in the world of cancer treatment research as well as in our understanding of the impact of microgravity conditions on the structure and perforce of complex drugs. We are all hoping for encouraging results.

Times of Israel

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Space the next frontier for cancer treatment? - Australian Jewish News

- Ms. Levin is a seasoned CFO executive specializing in biotechnology corporate finance with broad expertise in accounting, investor relations, operations and strategy

- Dr. Porter has over 20 years of drug development expertise leading to successful NDA and sNDA product approvals for metabolic diseases

SAN DIEGO, Jan. 19, 2022 /PRNewswire/ -- ViaCyte, Inc., a clinical-stage regenerative medicine company developing novel cell replacement therapies to address diseases with significant unmet needs, today announced the appointment of Alyssa Levin as Chief Financial Officer and addition of Lisa E. Porter, M.D., to its Board of Directors.

ViaCyte logo. (PRNewsFoto/ViaCyte, Inc.)

"We are excited to welcome Alyssa and Lisa to ViaCyte to ensure we are well-positioned for the future as we continue advancing ground-breaking therapeutics for type 1 diabetes," said Michael Yang, President and Chief Executive Officer, ViaCyte. "Alyssa's business expertise will be a major asset as we execute our mission to fully realize the potential of ViaCyte's regenerative medicine platform, and our clinical programs will benefit greatly from Lisa's first-hand experience as both a physician and senior executive leading drug development strategy, clinical trials, and commercialization."

"With important milestones on the horizon, I am looking forward to working with Michael and our leadership team as we chart the course for ViaCyte as an industry leader providing potentially life-changing cell therapies," said Ms. Levin. "I am excited to lead the finance team as we pursue significant clinical and growth opportunities to create increased value across our pipeline."

"ViaCyte is leading the way into the clinic with new approaches to cell therapies that will help lessen the disease burden for people living with diabetes," said Dr. Porter. "As a physician, I am excited to see ViaCyte expand the potential of cell replacement therapies for diabetes and other disease indications to bring functional cures to patients in need."

Story continues

Appointee Bios

Most recently, Ms. Levin served as the Chief Financial Officer and Senior Vice President of Operations for Tentarix Biotherapeutics where she led the company's finance functions and played a key role in overseeing the expansion of the team as well as planning and operations setup. Previously, Ms. Levin was Chief Financial Officer at Bird Rock Bio, a clinical-stage bio-pharmaceutical company focused on the treatment of fibrotic, metabolic and inflammatory diseases. In this role, she managed all finance, accounting, investor relations, treasury and human resources. Additionally, she held positions with PwC LLP, Evomed LLC & Cosmederm Bioscience, Inc., and The Siegfried Group, LLP. Ms. Levin earned her B.A. from the University of British Columbia and an M.A. in Professional Accountancy from the University of Saskatchewan.

Dr. Porter currently serves as the Chief Medical Officer for Nano Precision Medical, a biopharmaceutical company focused on drug delivery to treat chronic metabolic diseases, where she is responsible for the strategy, direction and execution of clinical development plans for novel drug:device combinations. Prior to Nano Precision Medical, Dr. Porter was Chief Medical Officer for Eiger Biopharmaceuticals, Inc. and Dance Biopharma, Inc. (now Aerami Therapeutics). She previously held executive positions with Amylin Pharmaceuticals leading efforts that resulted in the approval of Bydureon, a GLP-1 agonist and the first once-weekly treatment for Type 2 diabetes. Additionally, Dr. Porter served in multiple clinical development and leadership roles at GlaxoSmithKline Pharmaceuticals and Zeneca Pharmaceuticals. Dr. Porter, a graduate of the College of William and Mary, earned her M.D. from Duke University School of Medicine and conducted her residency at Duke University Medical Center, then completed a fellowship in Endocrinology and Hypertension at Brigham & Women's Hospital.

About ViaCyte

ViaCyte is a privately held clinical-stage regenerative medicine company developing novel cell replacement therapies based on two major technological advances: cell replacement therapies derived from pluripotent stem cells and medical device systems for cell encapsulation and implantation. ViaCyte has the opportunity to use these technologies to address critical human diseases and disorders that can potentially be treated by replacing lost or malfunctioning cells or proteins. The Company's first product candidates are being developed as potential long-term treatments for patients with type 1 diabetes to achieve glucose control targets and reduce the risk of hypoglycemia and diabetes-related complications. To accelerate and expand ViaCyte's efforts, it has established collaborative partnerships with leading companies, including CRISPR Therapeutics and W.L. Gore & Associates. ViaCyte is headquartered in San Diego, California. For more information, please visit http://www.viacyte.com and connect with ViaCyte on Twitter, Facebook, and LinkedIn.

Cision

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Plastic pollution has become an increasing concern with microplastic and nanoplastic contamination in food being a risk for human health. A novel study has investigated the effect of these particles, with an analysis of which factor is responsible, the size, surface or dispersant, and this research has been published in the journal, Toxicology in Vitro.

Study:Microplastics and nanoplastics: Size, surface and dispersant What causes the effect? Image Credit:chayanuphol/Shutterstock.com

Plastic pollution is a global health concern. Plastic particles are commonly found in the environment, are immersed in mineral and tap water, and ultimately are ingested.

Microplastics have been detected with quantifiable methods, and knowing that these tiny particles are susceptible to further eroding and decomposition, nanoplastic contamination may also present a health risk.

Nanoplastics are defined as plastic pieces with a diameter of less than 100 nm, which is within the nanoscale, and may cause more toxicity than microplastics due to their smaller size, larger surface-to-volume and higher bioreactivity.

These nano-sized particles are considered to be a health risk, with particles of less than 1.5 m diameter being able to overcome the intestinal barrier and penetrate organs. The potential of nanoplastics to move through the gastrointestinal tract and possibly reach the small intestine holds significant health implications.

Previous research has found nanoparticles that are recognized by microfold cells can be transported to the liver through the lymphatic system;additionally, the journey of nanoplastics such as polystyrene particles can be internalized within liver cells, resulting in potential adverse effects.

Innovative research has investigated polystyrene particles for toxicity upon uptake into the intestinal barrier, through the use of differentiated Caco-2 monoculture and mucus- and microfold (M)-cell co-culture as well as liver cells via differentiated HepaRG cells.

The aim of this critical research was to differentiate between different nanoplastic properties such as size, surface, and dispersant.

This innovative polystyrene study illustrated the microplastic particles consisted of toxic dispersants, although the particles themselves were non-toxic.

However, the nanosized polystyrene particles that were tested, including a size of 20 nm, were shown to have dose-dependent effects, with high concentrations being able to result in a significant decrease in cell viability.

This conclusion was determined to be due to size and not the dispersants a reflection of the nanotoxicology paradigm of decreasing size causing more reactivity through having an increased surface to volume ratio.

Surface modification has also shown to have an effect on cellular uptake of plastic particles, which was illustrated through polystyrene particles with different surface modifications.

Existing literature has also supported this correlation of toxicity and a positive surface modification, and nanoparticles with this modification have the ability to change the serum protein composition and protein corona. Additionally, hydrophobicity can also be another surface characteristic that can affect the uptake of particles.

The scientists concluded that the rate of uptake of particles may also be dependent on the type of cell, with intestinal cells being more accessible to particles with amine modifications which decrease their hydrophobicity;however, neutral polystyrene particles were taken up by liver cells more easily.

The significance of this research was in carrying out an investigation about different factors and characteristics of polystyrene particles, both micro- and nanosized plastics, which cause uptake by biological systems.

The in vitro data of these researchers suggested microplastics toxicity was due to their dispersant property, while nanoplastic toxicity could be attributed to their size.

However, the surface chemistry is also a significant characteristic for both micro- and nanosized plastic particles, aiding their cellular uptake and the overall impactwithin the biological system if they were ingested.

This research can aid in understanding the properties of micro- and nanosized plastics, which can assist in possibly creating a defense system.

This could potentially include advanced water treatments which can reduce their cellular uptake if ingested through tap and mineral water, as well as reduce the concentration of plastic particles being allowed into drinking water systems.

Additionally, the field of medicine and health could also be expanded into the detoxification of ingested materials, with further research providing an insight into how plastic toxicity can affect patients and what kind of treatments can be developed against it.

The implications of this research as well as further investigations, will be useful for reducing and sustaining human and environmental health.

Continue reading: How Polymers Analyze Nanoplastics in the Environment

Stock, V., Bhmert, L., Coban, G., Tyra, G., Vollbrecht, M., Voss, L., Paul, M., Braeuning, A. and Sieg, H., (2022)Microplastics and nanoplastics: Size, surface and dispersant What causes the effect?. Toxicology in Vitro, p.105314. Available at: https://doi.org/10.1016/j.tiv.2022.105314

Society, N., (2022)Microplastics. [online] National Geographic Society. Available at: https://www.nationalgeographic.org/encyclopedia/microplastics/

Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.

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Surface Modification and Size Contribute to Nanoplastic Toxicity - AZoNano

Introduction

Over the last years, nanotechnology has been introduced in our daily routine. This revolutionary technology has been applied in multiple fields through an integrated approach. An increasing number of applications and products containing nanomaterials or at least with nano-based claims have become available. This also happens in pharmaceutical research. The use of nanotechnology in the development of new medicines is now part of our research and in the European Union (EU) it has been recognized as a Key Enabling Technology, capable of providing new and innovative medical solution to address unmet medical needs (Bleeker et al., 2013; Ossa, 2014; Tinkle et al., 2014; Pita et al., 2016).

The application of nanotechnology for medical purposes has been termed nanomedicine and is defined as the use of nanomaterials for diagnosis, monitoring, control, prevention and treatment of diseases (Tinkle et al., 2014). However, the definition of nanomaterial has been controversial among the various scientific and international regulatory corporations. Some efforts have been made in order to find a consensual definition due to the fact that nanomaterials possess novel physicochemical properties, different from those of their conventional bulk chemical equivalents, due to their small size. These properties greatly increase a set of opportunities in the drug development; however, some concerns about safety issues have emerged. The physicochemical properties of the nanoformulation which can lead to the alteration of the pharmacokinetics, namely the absorption, distribution, elimination, and metabolism, the potential for more easily cross biological barriers, toxic properties and their persistence in the environment and human body are some examples of the concerns over the application of the nanomaterials (Bleeker et al., 2013; Tinkle et al., 2014).

To avoid any concern, it is necessary establishing an unambiguous definition to identify the presence of nanomaterials. The European Commission (EC) created a definition based on the European Commission Joint Research Center and on the Scientific Committee on Emerging and Newly Identified Health Risks. This definition is only used as a reference to determine whether a material is considered a nanomaterial or not; however, it is not classified as hazardous or safe. The EC claims that it should be used as a reference for additional regulatory and policy frameworks related to quality, safety, efficacy, and risks assessment (Bleeker et al., 2013; Boverhof et al., 2015).

According to the EC recommendation, nanomaterial refers to a natural, incidental, or manufactured material comprising particles, either in an unbound state or as an aggregate wherein one or more external dimensions is in the size range of 1100 nm for 50% of the particles, according to the number size distribution. In cases of environment, health, safety or competitiveness concern, the number size distribution threshold of 50% may be substituted by a threshold between 1 and 50%. Structures with one or more external dimensions below 1 nm, such as fullerenes, graphene flakes, and single wall carbon nanotubes, should be considered as nanomaterials. Materials with surface area by volume in excess of 60 m2/cm3 are also included (Commission Recommendation., 2011). This defines a nanomaterial in terms of legislation and policy in the European Union. Based on this definition, the regulatory bodies have released their own guidances to support drug product development.

The EMA working group introduces nanomedicines as purposely designed systems for clinical applications, with at least one component at the nanoscale, resulting in reproducible properties and characteristics, related to the specific nanotechnology application and characteristics for the intended use (route of administration, dose), associated with the expected clinical advantages of nano-engineering (e.g., preferential organ/tissue distribution; Ossa, 2014).

Food and Drug Administration (FDA) has not established its own definition for nanotechnology, nanomaterial, nanoscale, or other related terms, instead adopting the meanings commonly employed in relation to the engineering of materials that have at least one dimension in the size range of approximately 1 nanometer (nm) to 100 nm. Based on the current scientific and technical understanding of nanomaterials and their characteristics, FDA advises that evaluations of safety, effectiveness, public health impact, or regulatory status of nanotechnology products should consider any unique properties and behaviors that the application of nanotechnology may impart (Guidance for Industry, FDA, 2014).

According to the former definition, there are three fundamental aspects to identify the presence of a nanomaterial, which are size, particle size distribution (PSD) and surface area (Commission Recommendation., 2011; Bleeker et al., 2013; Boverhof et al., 2015).

The most important feature to take into account is size, because it is applicable to a huge range of materials. The conventional range is from 1 to 100 nm. However, there is no bright line to set this limit. The maximum size that a material can have to be considered nanomaterial is an arbitrary value because the psychochemical and biological characteristics of the materials do not change abruptly at 100 nm. To this extent, it is assumed that other properties should be taken in account (Lvestam et al., 2010; Commission Recommendation., 2011; Bleeker et al., 2013; Boverhof et al., 2015).

The pharmaceutical manufacturing of nanomaterials involves two different approaches: top down and bottom down. The top down process involves the breakdown of a bulk material into a smaller one or smaller pieces by mechanical or chemical energy. Conversely, the bottom down process starts with atomic or molecular species allowing the precursor particles to increase in size through chemical reaction (Luther, 2004; Oberdrster, 2010; Boverhof et al., 2015). These two processes of manufacturing are in the origin of different forms of particles termed primary particle, aggregate and agglomerate (Figure 1). The respective definition is (sic):

Figure 1. Schematic representation of the different forms of particles: primary particle, aggregate, and agglomerate (reproduced with permission from Oberdrster, 2010).

particle is a minute piece of matter with defined physical boundaries (Oberdrster, 2010; Commission Recommendation., 2011);

aggregate denotes a particle comprising strongly bound or fused particlesand the external surface can be smaller than the sum of the surface areas of the individual particles (Oberdrster, 2010; Commission Recommendation., 2011);

agglomerate means a collection of weakly bound particles or aggregates where the resulting external surface area are similar to the sum of the surface areas of the individual components (Oberdrster, 2010; Commission Recommendation., 2011).

Considering the definition, it is understandable why aggregates and agglomerates are included. They may still preserve the properties of the unbound particles and have the potential to break down in to nanoscale (Lvestam et al., 2010; Boverhof et al., 2015). The lower size limit is used to distinguish atoms and molecules from particles (Lvestam et al., 2010).

The PSD is a parameter widely used in the nanomaterial identification, reflecting the range of variation of sizes. It is important to set the PSD, because a nanomaterial is usually polydisperse, which means, it is commonly composed by particles with different sizes (Commission Recommendation., 2011; Bleeker et al., 2013; Boverhof et al., 2015).

The determination of the surface area by volume is a relational parameter, which is necessary when requested by additional legislation. The material is under the definition if the surface area by volume is larger than 60 m2/cm3, as pointed out. However, the PSD shall prevail, and for example, a material is classified as a nanomaterial based on the particle size distribution, even if the surface area by volume is lower than the specified 60 m2/cm3 (Commission Recommendation., 2011; Bleeker et al., 2013; Boverhof et al., 2015).

Nanomaterials can be applied in nanomedicine for medical purposes in three different areas: diagnosis (nanodiagnosis), controlled drug delivery (nanotherapy), and regenerative medicine. A new area which combines diagnostics and therapy termed theranostics is emerging and is a promising approach which holds in the same system both the diagnosis/imaging agent and the medicine. Nanomedicine is holding promising changes in clinical practice by the introduction of novel medicines for both diagnosis and treatment, having enabled to address unmet medical needs, by (i) integrating effective molecules that otherwise could not be used because of their high toxicity (e.g., Mepact), (ii) exploiting multiple mechanisms of action (e.g., Nanomag, multifunctional gels), (iii) maximizing efficacy (e.g., by increasing bioavailability) and reducing dose and toxicity, (iv) providing drug targeting, controlled and site specific release, favoring a preferential distribution within the body (e.g., in areas with cancer lesions) and improved transport across biological barriers (Chan, 2006; Mndez-Rojas et al., 2009; Zhang et al., 2012; Ossa, 2014).

This is a result of intrinsic properties of nanomaterials that have brought many advantages in the pharmaceutical development. Due to their small size, nanomaterials have a high specific surface area in relation to the volume. Consequently, the particle surface energy is increased, making the nanomaterials much more reactive. Nanomaterials have a tendency to adsorb biomolecules, e.g., proteins, lipids, among others, when in contact with the biological fluids. One of the most important interactions with the living matter relies on the plasma/serum biomoleculeadsorption layer, known as corona, that forms on the surface of colloidal nanoparticles (Pino et al., 2014). Its composition is dependent on the portal of entry into the body and on the particular fluid that the nanoparticles come across with (e.g., blood, lung fluid, gastro-intestinal fluid, etc.). Additional dynamic changes can influence the corona constitution as the nanoparticle crosses from one biological compartment to another one (Pearson et al., 2014; Louro, 2018).

Furthermore, optical, electrical and magnetic properties can change and be tunable through electron confinement in nanomaterials. In addition, nanomaterials can be engineered to have different size, shape, chemical composition and surface, making them able to interact with specific biological targets (Oberdrster et al., 2005; Kim et al., 2010). A successful biological outcome can only be obtained resorting to careful particle design. As such, a comprehensive knowledge of how the nanomaterials interact with biological systems are required for two main reasons.

The first one is related to the physiopathological nature of the diseases. The biological processes behind diseases occur at the nanoscale and can rely, for example, on mutated genes, misfolded proteins, infection by virus or bacteria. A better understanding of the molecular processes will provide the rational design on engineered nanomaterials to target the specific site of action desired in the body (Kim et al., 2010; Albanese et al., 2012). The other concern is the interaction between nanomaterial surface and the environment in biological fluids. In this context, characterization of the biomolecules corona is of utmost importance for understanding the mutual interaction nanoparticle-cell affects the biological responses. This interface comprises dynamic mechanisms involving the exchange between nanomaterial surfaces and the surfaces of biological components (proteins, membranes, phospholipids, vesicles, and organelles). This interaction stems from the composition of the nanomaterial and the suspending media. Size, shape, surface area, surface charge and chemistry, energy, roughness, porosity, valence and conductance states, the presence of ligands, or the hydrophobic/ hydrophilic character are some of the material characteristics that influence the respective surface properties. In turn, the presence of water molecules, acids and bases, salts and multivalent ions, surfactants are some of the factors related to the medium that will influence the interaction. All these aspects will govern the characteristics of the interface between the nanomaterial and biological components and, consequently, promote different cellular fates (Nel et al., 2009; Kim et al., 2010; Albanese et al., 2012; Monopoli et al., 2012).

A deeper knowledge about how the physicochemical properties of the biointerface influence the cellular signaling pathway, kinetics and transport will thus provide critical rules to the design of nanomaterials (Nel et al., 2009; Kim et al., 2010; Albanese et al., 2012; Monopoli et al., 2012).

The translation of nanotechnology form the bench to the market imposed several challenges. General issues to consider during the development of nanomedicine products including physicochemical characterization, biocompatibility, and nanotoxicology evaluation, pharmacokinetics and pharmacodynamics assessment, process control, and scale-reproducibility (Figure 2) are discussed in the sections that follow.

Figure 2. Schematic representation of the several barriers found throughout the development of a nanomedicine product.

The characterization of a nanomedicine is necessary to understand its behavior in the human body, and to provide guidance for the process control and safety assessment. This characterization is not consensual in the number of parameters required for a correct and complete characterization. Internationally standardized methodologies and the use of reference nanomaterials are the key to harmonize all the different opinions about this topic (Lin et al., 2014; Zhao and Chen, 2016).

Ideally, the characterization of a nanomaterial should be carried out at different stages throughout its life cycle, from the design to the evaluation of its in vitro and in vivo performance. The interaction with the biological system or even the sample preparation or extraction procedures may modify some properties and interfere with some measurements. In addition, the determination of the in vivo and in vitro physicochemical properties is important for the understanding of the potential risk of nanomaterials (Lin et al., 2014; Zhao and Chen, 2016).

The Organization for Economic Co-operation and Development started a Working Party on Manufactured Nanomaterials with the International Organization for Standardization to provide scientific advice for the safety use of nanomaterials that include the respective physicochemical characterization and the metrology. However, there is not an effective list of minimum parameters. The following characteristics should be a starting point to the characterization: particle size, shape and size distribution, aggregation and agglomeration state, crystal structure, specific surface area, porosity, chemical composition, surface chemistry, charge, photocatalytic activity, zeta potential, water solubility, dissolution rate/kinetics, and dustiness (McCall et al., 2013; Lin et al., 2014).

Concerning the chemical composition, nanomaterials can be classified as organic, inorganic, crystalline or amorphous particles and can be organized as single particles, aggregates, agglomerate powders or dispersed in a matrix which give rise to suspensions, emulsions, nanolayers, or films (Luther, 2004).

Regarding dimension, if a nanomaterial has three dimensions below 100 nm, it can be for example a particle, a quantum dot or hollow sphere. If it has two dimensions below 100 nm it can be a tube, fiber or wire and if it has one dimension below 100 nm it can be a film, a coating or a multilayer (Luther, 2004).

Different techniques are available for the analysis of these parameters. They can be grouped in different categories, involving counting, ensemble, separation and integral methods, among others (Linsinger et al., 2012; Contado, 2015).

Counting methods make possible the individualization of the different particles that compose a nanomaterial, the measurement of their different sizes and visualization of their morphology. The particles visualization is preferentially performed using microscopy methods, which include several variations of these techniques. Transmission Electron Microscopy (TEM), High-Resolution TEM, Scanning Electron Microscopy (SEM), cryo-SEM, Atomic Force Microscopy and Particle Tracking Analysis are just some of the examples. The main disadvantage of these methods is the operation under high-vacuum, although recently with the development of cryo-SEM sample dehydration has been prevented under high-vacuum conditions (Linsinger et al., 2012; Contado, 2015; Hodoroaba and Mielke, 2015).

These methods involve two steps of sample treatment: the separation of the particles into a monodisperse fraction, followed by the detection of each fraction. Field-Flow Fractionation (FFF), Analytical Centrifugation (AC) and Differential Electrical Mobility Analysis are some of the techniques that can be applied. The FFF techniques include different methods which separate the particles according to the force field applied. AC separates the particles through centrifugal sedimentation (Linsinger et al., 2012; Contado, 2015; Hodoroaba and Mielke, 2015).

Ensemble methods allow the report of intensity-weighted particle sizes. The variation of the measured signal over time give the size distribution of the particles extracted from a combined signal. Dynamic Light Scattering (DLS), Small-angle X-ray Scattering (SAXS) and X-ray Diffraction (XRD) are some of the examples. DLS and QELS are based on the Brownian motion of the sample. XRD is a good technique to obtain information about the chemical composition, crystal structure and physical properties (Linsinger et al., 2012; Contado, 2015; Hodoroaba and Mielke, 2015).

The integral methods only measure an integral property of the particle and they are mostly used to determine the specific surface area. Brunauer Emmet Teller is the principal method used and is based on the adsorption of an inert gas on the surface of the nanomaterial (Linsinger et al., 2012; Contado, 2015; Hodoroaba and Mielke, 2015).

Other relevant technique is the electrophoretic light scattering (ELS) used to determine zeta potential, which is a parameter related to the overall charge a particle acquires in a particular medium. ELS measures the electrophoretic mobility of particles in dispersion, based on the principle of electrophoresis (Linsinger et al., 2012).

The Table 1 shows some of principal methods for the characterization of the nanomaterials including the operational principle, physicochemical parameters analyzed and respective limitations.

Another challenge in the pharmaceutical development is the control of the manufacturing process by the identification of the critical parameters and technologies required to analyse them (Gaspar, 2010; Gaspar et al., 2014; Sainz et al., 2015).

New approaches have arisen from the pharmaceutical innovation and the concern about the quality and safety of new medicines by regulatory agencies (Gaspar, 2010; Gaspar et al., 2014; Sainz et al., 2015).

Quality-by-Design (QbD), supported by Process Analytical Technologies (PAT) is one of the pharmaceutical development approaches that were recognized for the systematic evaluation and control of nanomedicines (FDA, 2004; Gaspar, 2010; Gaspar et al., 2014; Sainz et al., 2015; European Medicines Agency, 2017).

Note that some of the physicochemical characteristics of nanomaterials can change during the manufacturing process, which compromises the quality and safety of the final nanomedicine. The basis of QbD relies on the identification of the Quality Attributes (QA), which refers to the chemical, physical or biological properties or another relevant characteristic of the nanomaterial. Some of them may be modified by the manufacturing and should be within a specific range for quality control purposes. In this situation, these characteristics are considered Critical Quality Attributes (CQA). The variability of the CQA can be caused by the critical material attributes and process parameters (Verma et al., 2009; Riley and Li, 2011; Bastogne, 2017; European Medicines Agency, 2017).

The quality should not be tested in nanomedicine, but built on it instead, by the understanding of the therapeutic purpose, pharmacological, pharmacokinetic, toxicological, chemical and physical properties of the medicine, process formulation, packaging, and the design of the manufacturing process. This new approach allows better focus on the relevant relationships between the characteristics, parameters of the formulation and process in order to develop effective processes to ensure the quality of the nanomedicines (FDA, 2014).

According to the FDA definition PAT is a system for designing, analzsing, and controlling manufacturing through timely measurements (i.e., during processing) of critical quality and performance attributes of raw and in-process materials and processes, with the goal of ensuring final product quality (FDA, 2014). The PAT tools analyse the critical quality and performance attributes. The main point of the PAT is to assure and enhance the understanding of the manufacturing concept (Verma et al., 2009; Riley and Li, 2011; FDA, 2014; Bastogne, 2017; European Medicines Agency, 2017).

Biocompatibility is another essential property in the design of drug delivery systems. One very general and brief definition of a biocompatible surface is that it cannot trigger an undesired' response from the organism. Biocompatibility is alternatively defined as the ability of a material to perform with an appropriate response in a specific application (Williams, 2003; Keck and Mller, 2013).

Pre-clinical assessment of nanomaterials involve a thorough biocompatibility testing program, which typically comprises in vivo studies complemented by selected in vitro assays to prove safety. If the biocompatibility of nanomaterials cannot be warranted, potentially advantageous properties of nanosystems may raise toxicological concerns.

Regulatory agencies, pharmaceutical industry, government, and academia are making efforts to accomplish specific and appropriate guidelines for risk assessment of nanomaterials (Hussain et al., 2015).

In spite of efforts to harmonize the procedures for safety evaluation, nanoscale materials are still mostly treated as conventional chemicals, thus lacking clear specific guidelines for establishing regulations and appropriate standard protocols. However, several initiatives, including scientific opinions, guidelines and specific European regulations and OECD guidelines such as those for cosmetics, food contact materials, medical devices, FDA regulations, as well as European Commission scientific projects (NanoTEST project, http://www.nanotest-fp7.eu) specifically address nanomaterials safety (Juillerat-Jeanneret et al., 2015).

In this context, it is important to identify the properties, to understand the mechanisms by which nanomaterials interact with living systems and thus to understand exposure, hazards and their possible risks.

Note that the pharmacokinetics and distribution of nanoparticles in the body depends on their surface physicochemical characteristics, shape and size. For example, nanoparticles with 10 nm in size were preferentially found in blood, liver, spleen, kidney, testis, thymus, heart, lung, and brain, while larger particles are detected only in spleen, liver, and blood (De Jong et al., 2008; Adabi et al., 2017).

In turn, the surface of nanoparticles also impacts upon their distribution in these organs, since their combination with serum proteins available in systemic circulation, influencing their cellular uptake. It should be recalled that a biocompatible material generates no immune response. One of the cause for an immune response can rely on the adsorption pattern of body proteins. An assessment of the in vivo protein profile is therefore crucial to address these interactions and to establish biocompatibility (Keck et al., 2013).

Finally, the clearance of nanoparticles is also size and surface dependent. Small nanoparticles, bellow 2030 nm, are rapidly cleared by renal excretion, while 200 nm or larger particles are more efficiently taken up by mononuclear phagocytic system (reticuloendothelial system) located in the liver, spleen, and bone marrow (Moghimi et al., 2001; Adabi et al., 2017).

Studies are required to address how nanomaterials penetrate cells and tissues, and the respective biodistribution, degradation, and excretion.

Due to all these issues, a new field in toxicology termed nanotoxicology has emerged, which aims at studying the nanomaterial effects deriving from their interaction with biological systems (Donaldson et al., 2004; Oberdrster, 2010; Fadeel, 2013).

The evaluation of possible toxic effects of the nanomaterials can be ascribed to the presence of well-known molecular responses in the cell. Nanomaterials are able to disrupt the balance of the redox systems and, consequently, lead to the production of reactive species of oxygen (ROS). ROS comprise hydroxyl radicals, superoxide anion and hydrogen peroxide. Under normal conditions, the cells produce these reactive species as a result of the metabolism. However, when exposed to nanomaterials the production of ROS increases. Cells have the capacity to defend itself through reduced glutathione, superoxide dismutase, glutathione peroxidase and catalase mechanisms. The superoxide dismutase converts superoxide anion into hydrogen peroxide and catalase, in contrast, converts it into water and molecular oxygen (Nel et al., 2006; Arora et al., 2012; Azhdarzadeh et al., 2015). Glutathione peroxidase uses glutathione to reduce some of the hydroperoxides. Under normal conditions, the glutathione is almost totally reduced. Nevertheless, an increase in ROS lead to the depletion of the glutathione and the capacity to neutralize the free radicals is decreased. The free radicals will induce oxidative stress and interact with the fatty acids in the membranes of the cell (Nel et al., 2006; Arora et al., 2012; Azhdarzadeh et al., 2015).

Consequently, the viability of the cell will be compromised by the disruption of cell membranes, inflammation responses caused by the upregulation of transcription factors like the nuclear factor kappa , activator protein, extracellular signal regulated kinases c-Jun, N-terminal kinases and others. All these biological responses can result on cell apoptosis or necrosis. Distinct physiological outcomes are possible due to the different pathways for cell injury after the interaction between nanomaterials and cells and tissues (Nel et al., 2006; Arora et al., 2012; Azhdarzadeh et al., 2015).

Over the last years, the number of scientific publications regarding toxicological effects of nanomaterials have increased exponentially. However, there is a big concern about the results of the experiments, because they were not performed following standard and harmonized protocols. The nanomaterial characterization can be considered weak once there are not standard nanomaterials to use as reference and the doses used in the experiences sometimes cannot be applied in the biological system. Therefore, the results are not comparable. For a correct comparison, it is necessary to perform a precise and thorough physicochemical characterization to define risk assessment guidelines. This is the first step for the comparison between data from biological and toxicological experiments (Warheit, 2008; Fadeel et al., 2015; Costa and Fadeel, 2016).

Although nanomaterials may have an identical composition, slight differences e.g., in the surface charge, size, or shape could impact on their respective activity and, consequently, on their cellular fate and accumulation in the human body, leading to different biological responses (Sayes and Warheit, 2009).

Sayes and Warheit (2009) proposed a three phases model for a comprehensive characterization of nanomaterials. Accordingly, the primary phase is achieved in the native state of the nanomaterial, specifically, in its dry state. The secondary characterization is performed with the nanomaterials in the wet phase, e.g., as solution or suspension. The tertiary characterization includes in vitro and in vivo interactions with biological systems. The tertiary characterization is the most difficult from the technical point of view, especially in vivo, because of all the ethical questions concerning the use of animals in experiments (Sayes and Warheit, 2009).

Traditional toxicology uses of animals to conduct tests. These types of experiments using nanomaterials can be considered impracticable and unethical. In addition, it is time-consuming, expensive and sometimes the end points achieved are not enough to correctly correlate with what happens in the biological systems of animals and the translation to the human body (Collins et al., 2017).

In vitro studies are the first assays used for the evaluation of cytotoxicity. This approach usually uses cell lines, primary cells from the tissues, and/or a mixture of different cells in a culture to assess the toxicity of the nanomaterials. Different in vitro cytotoxicity assays to the analysis of the cell viability, stress, and inflammatory responses are available. There are several cellular processes to determine the cell viability, which consequently results in different assays with distinct endpoints. The evaluation of mitochondrial activity, the lactate dehydrogenase release from the cytosol by tretazolium salts and the detection of the biological marker Caspase-3 are some of the examples that imposes experimental variability in this analysis. The stress response is another example which can be analyzed by probes in the evaluation of the inflammatory response via enzyme linked immunosorbent assay are used (Kroll et al., 2009).

As a first approach, in vitro assays can predict the interaction of the nanomaterials with the body. However, the human body possesses compensation mechanisms when exposed to toxics and a huge disadvantage of this model is not to considered them. Moreover, they are less time consuming, more cost-effective, simpler and provide an easier control of the experimental conditions (Kroll et al., 2009; Fadeel et al., 2013b).

Their main drawback is the difficulty to reproduce all the complex interactions in the human body between sub-cellular levels, cells, organs, tissues and membranes. They use specific cells to achieve specific endpoints. In addition, in vitro assays cannot predict the physiopathological response of the human body when exposed to nanomaterials (Kroll et al., 2009; Fadeel et al., 2013b).

Another issue regarding the use of this approach is the possibility of interaction between nanomaterials and the reagents of the assay. It is likely that the reagents used in the in vitro assays interfere with the nanomaterial properties. High adsorption capacity, optical and magnetic properties, catalytic activity, dissolution, and acidity or alkalinity of the nanomaterials are some of the examples of properties that may promote this interaction (Kroll et al., 2009).

Many questions have been raised by the regulators related to the lack of consistency of the data produced by cytotoxicity assays. New assays for a correct evaluation of the nanomaterial toxicity are, thus, needed. In this context, new approaches have arisen, such as the in silico nanotoxicology approach. In silico methods are the combination of toxicology with computational tools and bio-statistical methods for the evaluation and prediction of toxicity. By using computational tools is possible to analyse more nanomaterials, combine different endpoints and pathways of nanotoxicity, being less time-consuming and avoiding all the ethical questions (Warheit, 2008; Raunio, 2011).

Quantitative structure-activity relationship models (QSAR) were one the first applications of computational tools applied in toxicology. QSAR models are based on the hypothesis that the toxicity of nanomaterials and their cellular fate in the body can be predicted by their characteristics, and different biological reactions are the result of physicochemical characteristics, such as size, shape, zeta potential, or surface charge, etc., gathered as a set of descriptors. QSAR aims at identifying the physicochemical characteristics which lead to toxicity, so as to provide alterations to reduce toxicology. A mathematical model is created, which allows liking descriptors and the biological activity (Rusyn and Daston, 2010; Winkler et al., 2013; Oksel et al., 2015).

Currently, toxigenomics is a new area of nanotoxicology, which includes a combination between genomics and nanotoxicology to find alterations in the gene, protein and in the expressions of metabolites (Rusyn et al., 2012; Fadeel et al., 2013a).

Hitherto, different risk assessment approaches have been reported. One of them is the DF4nanoGrouping framework, which concerns a functionality driven scheme for grouping nanomaterials based on their intrinsic properties, system dependent properties and toxicological effects (Arts et al., 2014, 2016). Accordingly, nanomaterials are categorized in four groups, including possible subgroups. The four main groups encompass (1) soluble, (2) biopersistent high aspect ratio, (3) passive, that is, nanomaterials without obvious biological effects and (4) active nanomaterials, that is, those demonstrating surface-related specific toxic properties. The DF4nanoGrouping foresees a stepwise evaluation of nanomaterial properties and effects with increasing biological complexity. In case studies that includes carbonaceous nanomaterials, metal oxide, and metal sulfate nanomaterials, amorphous silica and organic pigments (all nanomaterials having primary particle sizes smaller than 100 nm), the usefulness of the DF4nanoGrouping for nanomaterial hazard assessment has already been established. It facilitates grouping and targeted testing of nanomaterials, also ensuring that enough data for the risk assessment of a nanomaterial are available, and fostering the use of non-animal methods (Landsiedel et al., 2017). More recently, DF4nanoGrouping developed three structure-activity relationship classification, decision tree, models by identifying structural features of nanomaterials mainly responsible for the surface activity (size, specific surface area, and the quantum-mechanical calculated property lowest unoccupied molecular orbital), based on a reduced number of descriptors: one for intrinsic oxidative potential, two for protein carbonylation, and three for no observed adverse effect concentration (Gajewicz et al., 2018)

Keck and Mller also proposed a nanotoxicological classification system (NCS) (Figure 3) that ranks the nanomaterials into four classes according to the respective size and biodegradability (Mller et al., 2011; Keck and Mller, 2013).

Due to the size effects, this parameter is assumed as truly necessary, because when nanomaterials are getting smaller and smaller there is an increase in solubility, which is more evident in poorly soluble nanomaterials than in soluble ones. The adherence to the surface of membranes increases with the decrease of the size. Another important aspect related to size that must be considered is the phagocytosis by macrophages. Above 100 nm, nanomaterials can only be internalized by macrophages, a specific cell population, while nanomaterials below 100 nm can be internalized by any cell due to endocytosis. Thus, nanomaterials below 100 nm are associated to higher toxicity risks in comparison with nanomaterials above 100 nm (Mller et al., 2011; Keck and Mller, 2013).

In turn, biodegradability was considered a required parameter in almost all pharmaceutical formulations. The term biodegradability applies to the biodegradable nature of the nanomaterial in the human body. Biodegradable nanomaterials will be eliminated from the human body. Even if they cause some inflammation or irritation the immune system will return to the regular function after elimination. Conversely, non-biodegradable nanomaterials will stay forever in the body and change the normal function of the immune system (Mller et al., 2011; Keck and Mller, 2013).

There are two more factors that must be taken into account in addition to the NCS, namely the route of administration and the biocompatibility surface. When a particle is classified by the NCS, toxicity depends on the route of administration. For example, the same nanomaterials applied dermally or intravenously can pose different risks to the immune system.

In turn, a non-biocompatibility surface (NB) can activate the immune system by adsorption to proteins like opsonins, even if the particle belongs to the class I of the NCS (Figure 3). The biocompatibility (B) is dictated by the physicochemical surface properties, irrespective of the size and/or biodegradability. This can lead to further subdivision in eight classes from I-B, I-NB, to IV-B and IV-NB (Mller et al., 2011; Keck and Mller, 2013).

NCS is a simple guide to the evaluation of the risk of nanoparticles, but there are many other parameters playing a relevant role in nanotoxicity determination (Mller et al., 2011; Keck and Mller, 2013). Other suggestions encompass more general approaches, combining elements of toxicology, risk assessment modeling, and tools developed in the field of multicriteria decision analysis (Rycroft et al., 2018).

A forthcoming challenge in the pharmaceutical development is the scale-up and reproducibility of the nanomedicines. A considerable number of nanomedicines fail these requirements and, consequently, they are not introduced on the pharmaceutical market (Agrahari and Hiremath, 2017).

The traditional manufacturing processes do not create three dimensional medicines in the nanometer scale. Nanomedicine manufacturing processes, as already mentioned above, compromise top-down and bottom-down approaches, which include multiple steps, like homogenization, sonication, milling, emulsification, and sometimes, the use of organic solvents and further evaporation. In a small-scale, it is easy to control and achieve the optimization of the formulation. However, at a large scale it becomes very challenging, because slight variations during the manufacturing process can originate critical changes in the physicochemical characteristics and compromise the quality and safety of the nanomedicines, or even the therapeutic outcomes. A detailed definition of the acceptable limits for the CQA is very important, and these parameters must be identified and analyzed at the small-scale, in order to understand how the manufacturing process can change them: this will help the implementation of the larger scale. Thus, a deep process of understanding the critical steps and the analytical tools established for the small-scale will be a greatly help for the introduction of the large scale (Desai, 2012; Kaur et al., 2014; Agrahari and Hiremath, 2017).

Another requirement for the introduction of medicines in the pharmaceutical market is the reproducibility of every batch produced. The reproducibility is achieved in terms of physicochemical characterization and therapeutic purpose. There are specific ranges for the variations between different batches. Slight changes in the manufacturing process can compromise the CQA and, therefore, they may not be within a specific range and create an inter-batch variation (Desai, 2012; Kaur et al., 2014; Agrahari and Hiremath, 2017).

Over the last decades, nanomedicines have been successfully introduced in the clinical practice and the continuous development in pharmaceutical research is creating more sophisticated ones which are entering in clinic trials. In the European Union, the nanomedicine market is composed by nanoparticles, liposomes, nanocrystals, nanoemulsions, polymeric-protein conjugates, and nanocomplexes (Hafner et al., 2014). Table 2 shows some examples of commercially available nanomedicines in the EU (Hafner et al., 2014; Choi and Han, 2018).

In the process of approval, nanomedicines were introduced under the traditional framework of the benefit/risk analysis. Another related challenge is the development of a framework for the evaluation of the follow-on nanomedicines at the time of reference medicine patent expiration (Ehmann et al., 2013; Tinkle et al., 2014).

Nanomedicine comprises both biological and non-biological medical products. The biological nanomedicines are obtained from biological sources, while non-biological are mentioned as non-biological complex drugs (NBCD), where the active principle consists of different synthetic structures (Tinkle et al., 2014; Hussaarts et al., 2017; Mhlebach, 2018).

In order to introduce a generic medicine in the pharmaceutical market, several parameters need to be demonstrated, as described elsewhere. For both biological and non-biological nanomedicines, a more complete analysis is needed, that goes beyond the plasma concentration measurement. A stepwise comparison of bioequivalence, safety, quality, and efficacy, in relation to the reference medicine, which leads to therapeutic equivalence and consequently interchangeability, is required (Astier et al., 2017).

For regulatory purposes, the biological nanomedicines are under the framework set by European Medicines Agency (EMA) This framework is a regulatory approach for the follow-on biological nanomedicines, which include recommendations for comparative quality, non-clinical and clinical studies (Mhlebach et al., 2015).

The regulatory approach for the follow-on NBCDs is still ongoing. The industry frequently asks for scientific advice and a case-by-case is analyzed by the EMA. Sometimes, the biological framework is the base for the regulation of the NBCDs, because they have some features in common: the structure cannot be fully characterized and the in vivo activity is dependent on the manufacturing process and, consequently, the comparability needs to establish throughout the life cycle, as happens to the biological nanomedicines. Moreover, for some NBCDs groups like liposomes, glatiramoids, and iron carbohydrate complexes, there are draft regulatory approaches, which help the regulatory bodies to create a final framework for the different NBCDs families (Schellekens et al., 2014).

EMA already released some reflection papers regarding nanomedicines with surface coating, intravenous liposomal, block copolymer micelle, and iron-based nano-colloidal nanomedicines (European Medicines Agency, 2011, 2013a,b,c). These papers are applied to both new nanomedicines and nanosimilars, in order to provide guidance to developers in the preparation of marketing authorization applications.The principles outlined in these documents address general issues regarding the complexity of the nanosystems and provide basic information for the pharmaceutical development, non-clinical and early clinical studies of block-copolymer micelle, liposome-like, and nanoparticle iron (NPI) medicinal products drug products created to affect pharmacokinetic, stability and distribution of incorporated or conjugated active substances in vivo. Important factors related to the exact nature of the particle characteristics, that can influence the kinetic parameters and consequently the toxicity, such as the physicochemical nature of the coating, the respective uniformity and stability (both in terms of attachment and susceptibility to degradation), the bio-distribution of the product and its intracellular fate are specifically detailed.

After a nanomedicine obtains the marketing authorization, there is a long way up to the introduction of the nanomedicine in the clinical practice in all EU countries. This occurs because the pricing and reimbursement decisions for medicines are taken at an individual level in each member state of the EU (Sainz et al., 2015).

In order to provide patient access to medicines, the multidisciplinary process of Health Technology Assessment (HTA), is being developed. Through HTA, information about medicine safety, effectiveness and cost-effectiveness is generated so as support health and political decision-makers (Sainz et al., 2015).

Currently, pharmacoeconomics studies assume a crucial role previous to the commercialization of nanomedicines. They assess both the social and economic importance through the added therapeutic value, using indicators such as quality-adjusted life expectancy years and hospitalization (Sainz et al., 2015).

The EUnetHTA was created to harmonize and enhance the entry of new medicines in the clinical practice, so as to provide patients with novel medicines. The main goal of EUnetHTA is to develop decisive, appropriate and transparent information to help the HTAs in EU countries.

Currently, EUnetHTA is developing the Joint Action 3 until 2020 and the main aim is to define and implement a sustainable model for the scientific and technical cooperation on Health Technology Assessment (HTA) in Europe.

The reformulation of pre-existing medicines or the development of new ones has been largely boosted by the increasing research in nanomedicine. Changes in toxicity, solubility and bioavailability profile are some of the modifications that nanotechnology introduces in medicines.

In the last decades, we have assisted to the translation of several applications of nanomedicine in the clinical practice, ranging from medical devices to nanopharmaceuticals. However, there is still a long way toward the complete regulation of nanomedicines, from the creation of harmonized definitions in all Europe to the development of protocols for the characterization, evaluation and process control of nanomedicines. A universally accepted definition for nanomedicines still does not exist, and may even not be feasible at all or useful. The medicinal products span a large range in terms of type and structure, and have been used in a multitude of indications for acute and chronic diseases. Also, ongoing research is rapidly leading to the emergence of more sophisticated nanostructured designs that requires careful understanding of pharmacokinetic and pharmacodynamic properties of nanomedicines, determined by the respective chemical composition and physicochemical properties, which thus poses additional challenges in regulatory terms.

EMA has recognized the importance of the establishment of recommendations for nanomedicines to guide their development and approval. In turn, the nanotechnology methods for the development of nanomedicines bring new challenges for the current regulatory framework used.

EMA have already created an expert group on nanomedicines, gathering members from academia and European regulatory network. The main goal of this group is to provide scientific information about nanomedicines in order to develop or review guidelines. The expert group also helps EMA in discussions with international partners about nanomedicines. For the developer an early advice provided from the regulators for the required data is highly recommended.

The equivalence of complex drug products is another topic that brings scientific and regulatory challenges. Evidence for sufficient similarity must be gathered using a careful stepwise, hopefully consensual, procedure. In the coming years, through all the innovation in science and technology, it is expected an increasingly higher number of medicines based on nanotechnology. For a common understanding among different stakeholders the development of guidelines for the development and evaluation of nanomedicines is mandatory, in order to approve new and innovative nanomedicines in the pharmaceutical market. This process must be also carried out along with interagency harmonization efforts, to support rational decisions pertaining to scientific and regulatory aspects, financing and market access.

CV conceived the original idea and directed the work. SS took the lead in writing the manuscript. AP and JS helped supervise the manuscript. All authors provided critical feedback and helped shape the research, analysis and revision of the manuscript.

This work was financially supported by Fundao para a Cincia e a Tecnologia (FCT) through the Research Project POCI-01-0145-FEDER-016648, the project PEst-UID/NEU/04539/2013, and COMPETE (Ref. POCI-01-0145-FEDER-007440). The Coimbra Chemistry Center is supported by FCT, through the Project PEst-OE/QUI/UI0313/2014 and POCI-01-0145-FEDER-007630. This paper was also supported by the project UID/QUI/50006/2013LAQV/REQUIMTE.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Adabi, M., Naghibzadeh, M., Adabi, M., Zarrinfard, M. A., Esnaashari, S., Seifalian, A. M., et al. (2017). Biocompatibility and nanostructured materials: applications in nanomedicine. Artif. Cells Nanomed. Biotechnol. 45, 833842. doi: 10.1080/21691401.2016.1178134

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Frontiers | Nanomedicine: Principles, Properties, and ...

Nanotechnology, the manipulation of matter at the atomic and molecular scale to create materials with remarkably varied and new properties, is a rapidly expanding area of research with huge potential in many sectors, ranging from healthcare to construction and electronics. In medicine, it promises to revolutionize drug delivery, gene therapy, diagnostics, and many areas of research, development and clinical application.

This article does not attempt to cover the whole field, but offers, by means of some examples, a few insights into how nanotechnology has the potential to change medicine, both in the research lab and clinically, while touching on some of the challenges and concerns that it raises.

The prefix nano stems from the ancient Greek for dwarf. In science it means one billionth (10 to the minus 9) of something, thus a nanometer (nm) is is one billionth of a meter, or 0.000000001 meters. A nanometer is about three to five atoms wide, or some 40,000 times smaller than the thickness of human hair. A virus is typically 100 nm in size.

The ability to manipulate structures and properties at the nanoscale in medicine is like having a sub-microscopic lab bench on which you can handle cell components, viruses or pieces of DNA, using a range of tiny tools, robots and tubes.

Therapies that involve the manipulation of individual genes, or the molecular pathways that influence their expression, are increasingly being investigated as an option for treating diseases. One highly sought goal in this field is the ability to tailor treatments according to the genetic make-up of individual patients.

This creates a need for tools that help scientists experiment and develop such treatments.

Imagine, for example, being able to stretch out a section of DNA like a strand of spaghetti, so you can examine or operate on it, or building nanorobots that can walk and carry out repairs inside cell components. Nanotechnology is bringing that scientific dream closer to reality.

For instance, scientists at the Australian National University have managed to attach coated latex beads to the ends of modified DNA, and then using an optical trap comprising a focused beam of light to hold the beads in place, they have stretched out the DNA strand in order to study the interactions of specific binding proteins.

Meanwhile chemists at New York University (NYU) have created a nanoscale robot from DNA fragments that walks on two legs just 10 nm long. In a 2004 paper published in the journal Nano Letters, they describe how their nanowalker, with the help of psoralen molecules attached to the ends of its feet, takes its first baby steps: two forward and two back.

One of the researchers, Ned Seeman, said he envisages it will be possible to create a molecule-scale production line, where you move a molecule along till the right location is reached, and a nanobot does a bit chemisty on it, rather like spot-welding on a car assembly line. Seemans lab at NYU is also looking to use DNA nanotechnology to make a biochip computer, and to find out how biological molecules crystallize, an area that is currently fraught with challenges.

The work that Seeman and colleagues are doing is a good example of biomimetics, where with nanotechnology they can imitate some of the biological processes in nature, such as the behavior of DNA, to engineer new methods and perhaps even improve them.

DNA-based nanobots are also being created to target cancer cells. For instance, researchers at Harvard Medical School in the US reported recently in Science how they made an origami nanorobot out of DNA to transport a molecular payload. The barrel-shaped nanobot can carry molecules containing instructions that make cells behave in a particular way. In their study, the team successfully demonstrates how it delivered molecules that trigger cell suicide in leukemia and lymphoma cells.

Nanobots made from other materials are also in development. For instance, gold is the material scientists at Northwestern University use to make nanostars, simple, specialized, star-shaped nanoparticles that can href=http://www.medicalnewstoday.com/articles/243856.php>deliver drugs directly to the nuclei of cancer cells. In a recent paper in the journal ACS Nano, they describe how drug-loaded nanostars behave like tiny hitchhikers, that after being attracted to an over-expressed protein on the surface of human cervical and ovarian cancer cells, deposit their payload right into the nuclei of those cells.

The researchers found giving their nanobot the shape of a star helped to overcome one of the challenges of using nanoparticles to deliver drugs: how to release the drugs precisely. They say the shape helps to concentrate the light pulses used to release the drugs precisely at the points of the star.

Scientists are discovering that protein-based drugs are very useful because they can be programmed to deliver specific signals to cells. But the problem with conventional delivery of such drugs is that the body breaks most of them down before they reach their destination.

But what if it were possible to produce such drugs in situ, right at the target site? Well, in a recent issue of Nano Letters, researchers at Massachusetts Institute of Technology (MIT) in the US show how it may be possible to do just that. In their proof of principle study, they demonstrate the feasibility of self-assembling nanofactories that make protein compounds, on demand, at target sites. So far they have tested the idea in mice, by creating nanoparticles programmed to produce either green fluorescent protein (GFP) or luciferase exposed to UV light.

The MIT team came up with the idea while trying to find a way to attack metastatic tumors, those that grow from cancer cells that have migrated from the original site to other parts of the body. Over 90% of cancer deaths are due to metastatic cancer. They are now working on nanoparticles that can synthesize potential cancer drugs, and also on other ways to switch them on.

Nanofibers are fibers with diameters of less than 1,000 nm. Medical applications include special materials for wound dressings and surgical textiles, materials used in implants, tissue engineering and artificial organ components.

Nanofibers made of carbon also hold promise for medical imaging and precise scientific measurement tools. But there are huge challenges to overcome, one of the main ones being how to make them consistently of the correct size. Historically, this has been costly and time-consuming.

But last year, researchers from North Carolina State University, revealed how they had developed a new method for making carbon nanofibers of specific sizes. Writing in ACS Applied Materials & Interfaces in March 2011, they describe how they managed to grow carbon nanofibers uniform in diameter, by using nickel nanoparticles coated with a shell made of ligands, small organic molecules with functional parts that bond directly to metals.

Nickel nanoparticles are particularly interesting because at high temperatures they help grow carbon nanofibers. The researchers also found there was another benefit in using these nanoparticles, they could define where the nanofibers grew and by correct placement of the nanoparticles they could grow the nanofibers in a desired specific pattern: an important feature for useful nanoscale materials.

Lead is another substance that is finding use as a nanofiber, so much so that neurosurgeon-to-be Matthew MacEwan, who is studying at Washington University School of Medicine in St. Louis, started his own nanomedicine company aimed at revolutionizing the surgical mesh that is used in operating theatres worldwide.

The lead product is a synthetic polymer comprising individual strands of nanofibers, and was developed to repair brain and spinal cord injuries, but MacEwan thinks it could also be used to mend hernias, fistulas and other injuries.

Currently, the surgical meshes used to repair the protective membrane that covers the brain and spinal cord are made of thick and stiff material, which is difficult to work with. The lead nanofiber mesh is thinner, more flexible and more likely to integrate with the bodys own tissues, says MacEwan. Every thread of the nanofiber mesh is thousands of times smaller than the diameter of a single cell. The idea is to use the nanofiber material not only to make operations easier for surgeons to carry out, but also so there are fewer post-op complications for patients, because it breaks down naturally over time.

Researchers at the Polytechnic Institute of New York University (NYU-Poly) have recently demonstrated a new way to make nanofibers out of proteins. Writing recently in the journal Advanced Functional Materials, the researchers say they came across their finding almost by chance: they were studying certain cylinder-shaped proteins derived from cartilage, when they noticed that in high concentrations, some of the proteins spontaneously came together and self-assembled into nanofibers.

They carried out further experiments, such as adding metal-recognizing amino acids and different metals, and found they could control fiber formation, alter its shape, and how it bound to small molecules. For instance, adding nickel transformed the fibers into clumped mats, which could be used to trigger the release of an attached drug molecule.

The researchers hope this new method will greatly improve the delivery of drugs to treat cancer, heart disorders and Alzheimers disease. They can also see applications in regeneration of human tissue, bone and cartilage, and even as a way to develop tinier and more powerful microprocessors for use in computers and consumer electronics.

Recent years have seen an explosion in the number of studies showing the variety of medical applications of nanotechnology and nanomaterials. In this article we have glimpsed just a small cross-section of this vast field. However, across the range, there exist considerable challenges, the greatest of which appear to be how to scale up production of materials and tools, and how to bring down costs and timescales.

But another challenge is how to quickly secure public confidence that this rapidly expanding technology is safe. And so far, it is not clear whether that is being done.

There are those who suggest concerns about nanotechnology may be over-exaggerated. They point to the fact that just because a material is nanosized, it does not mean it is dangerous, indeed nanoparticles have been around since the Earth was born, occurring naturally in volcanic ash and sea-spray, for example. As byproducts of human activity, they have been present since the Stone Age, in smoke and soot.

Of attempts to investigate the safety of nanomaterials, the National Cancer Institute in the US says there are so many nanoparticles naturally present in the environment that they are often at order-of-magnitude higher levels than the engineered particles being evaluated. In many respects, they point out, most engineered nanoparticles are far less toxic than household cleaning products, insecticides used on family pets, and over-the-counter dandruff remedies, and that for instance, in their use as carriers of chemotherapeutics in cancer treatment, they are much less toxic than the drugs they carry.

It is perhaps more in the food sector that we have seen some of the greatest expansion of nanomaterials on a commercial level. Although the number of foods that contain nanomaterials is still small, it appears set to change over the next few years as the technology develops. Nanomaterials are already used to lower levels of fat and sugar without altering taste, or to improve packaging to keep food fresher for longer, or to tell consumers if the food is spoiled. They are also being used to increase the bioavailablity of nutrients (for instance in food supplements).

But, there are also concerned parties, who highlight that while the pace of research quickens, and the market for nanomaterials expands, it appears not enough is being done to discover their toxicological consequences.

This was the view of a science and technology committee of the House of Lords of the British Parliament, who in a recent report on nanotechnology and food, raise several concerns about nanomaterials and human health, particularly the risk posed by ingested nanomaterials.

For instance, one area that concerns the committee is the size and exceptional mobility of nanoparticles: they are small enough, if ingested, to penetrate cell membranes of the lining of the gut, with the potential to access the brain and other parts of the body, and even inside the nuclei of cells.

Another is the solubility and persistence of nanomaterials. What happens, for instance, to insoluble nanoparticles? If they cant be broken down and digested or degraded, is there a danger they will accumulate and damage organs? Nanomaterials comprising inorganic metal oxides and metals are thought to be the ones most likely to pose a risk in this area.

Also, because of their high surface area to mass ratio, nanoparticles are highly reactive, and may for instance, trigger as yet unknown chemical reactions, or by bonding with toxins, allow them to enter cells that they would otherwise have no access to.

For instance, with their large surface area, reactivity and electrical charge, nanomaterials create the conditions for what is described as particle aggregation due to physical forces and particle agglomoration due to chemical forces, so that individual nanoparticles come together to form larger ones. This may lead not only to dramatically larger particles, for instance in the gut and inside cells, but could also result in disaggregation of clumps of nanoparticles, which could radically alter their physicochemical properties and chemical reactivity.

Such reversible phenomena add to the difficulty in understanding the behaviour and toxicology of nanomaterials, says the committee, whose overall conclusion is that neither Government nor the Research Councils are giving enough priority to researching the safety of nanotechnology, especially considering the timescale within which products containing nanomaterials may be developed.

They recommend much more research is needed to ensure that regulatory agencies can effectively assess the safety of products before they are allowed onto the market.

It would appear, therefore, whether actual or perceived, the potential risk that nanotechnology poses to human health must be investigated, and be seen to be investigated. Most nanomaterials, as the NCI suggests, will likely prove to be harmless.

But when a technology advances rapidly, knowledge and communication about its safety needs to keep pace in order for it to benefit, especially if it is also to secure public confidence. We only have to look at what happened, and to some extent is still happening, with genetically modified food to see how that can go badly wrong.

Written by Catharine Paddock PhD

Original post:
Nanotechnology In Medicine: Huge Potential, But What Are ...

Introduction

Given the multi-lineage differentiation abilities of mesenchymal stem cells (MSCs) isolated from different tissues and organs, MSCs have been widely used in various medical fields, particularly regenerative medicine.13 The representative sources of MSCs are bone marrow, adipose, periodontal, muscle, and umbilical cord blood.410 Interestingly, slight differences have been reported in the characteristics of MSCs depending on the different sources, including their population in source tissues, immunosuppressive activities, proliferation, and resistance to cellular aging.11 Bone marrow-derived MSCs (BM-MSCs) are the most intensively studied and show clinically promising results for cartilage and bone regeneration.11 However, the isolation procedures for BM-MSCs are complicated because bone marrow contains a relatively small fraction of MSCs (0.0010.01% of the cells in bone marrow).12 Furthermore, bone marrow aspiration to harvest MSCs in human bones is a painful procedure and the slower proliferation rate of BM-MSCs is a clinical limitation.13 In comparison with BM-MSCs, adipose-derived MSCs (AD-MSCs) are relatively easy to collect and can produce up to 500 times the cell population of BM-MSCs.14 AD-MSCs showed a greater ability to regenerate damaged cartilage and bone tissues with increased immunosuppressive ability.14,15 Umbilical cord blood-derived MSCs (UC-MSCs) proliferate faster than BM-MSCs and are resistant to significant cellular aging.11

MSCs have been investigated and gained worldwide attention as potential therapeutic candidates for incurable diseases such as arthritis, spinal cord injury, and cardiac disease.3,1623 In particular, the inherent tropism of MSCs to inflammatory sites has been thoroughly studied.24 This inherent tropism, also known as homing ability, originates from the recognition of various chemokine sources in inflamed tissues, where profiled chemokines are continuously secreted and the MSCs migrate to the chemokines in a concentration-dependent manner.24 Rheumatoid arthritis (RA) is a representative inflammatory disease that primarily causes inflammation in the joints, and this long-term autoimmune disorder causes worsening pain and stiffness following rest. RA affects approximately 24.5 million people as of 2015, but only symptomatic treatments such as pain medications, steroids, and nonsteroidal anti-inflammatory drugs (NSAIDs), or slow-acting drugs that inhibit the rapid progression of RA, such as disease-modifying antirheumatic drugs (DMARDs) are currently available. However, RA drugs have adverse side effects, including hepatitis, osteoporosis, skeletal fracture, steroid-induced arthroplasty, Cushings syndrome, gastrointestinal (GI) intolerance, and bleeding.2527 Thus, MSCs are rapidly emerging as the next generation of arthritis treatment because they not only recognize and migrate toward chemokines secreted in the inflamed joints but also regulate inflammatory progress and repair damaged cells.28

However, MSCs are associated with many challenges that need to be overcome before they can be used in clinical settings.2931 One of the main challenges is the selective accumulation of systemically administered MSCs in the lungs and liver when they are administered intravenously, leading to insufficient concentrations of MSCs in the target tissues.32,33 In addition, most of the administered MSCs are typically initially captured by macrophages in the lungs, liver, and spleen.3234 Importantly, the viability and migration ability of MSCs injected in vivo differed from results previously reported as favorable therapeutic effects and migration efficiency in vitro.35

To improve the delivery of MSCs, researchers have focused on chemokines, which are responsible for MSCs ability to move.36 The chemokine receptors are the key proteins on MSCs that recognize chemokines, and genetic engineering of MSCs to overexpress the chemokine receptor can improve the homing ability, thus enhancing their therapeutic efficacy.37 Genetic engineering is a convenient tool for modifying native or non-native genes, and several technologies for genetic engineering exist, including genome editing, gene knockdown, and replacement with various vectors.38,39 However, safety issues that prevent clinical use persist, for example, genome integration, off-target effects, and induction of immune response.40 In this regard, MSC mimicking nanoencapsulations can be an alternative strategy for maintaining the homing ability of MSCs and overcoming the current safety issues.4143 Nanoencapsulation involves entrapping the core nanoparticles of solids or liquids within nanometer-sized capsules of secondary materials.44

MSC mimicking nanoencapsulation uses the MSC membrane fraction as the capsule and targeting molecules, that is chemokine receptors, with several types of nanoparticles, as the core.45,46 MSC mimicking nanoencapsulation consists of MSC membrane-coated nanoparticles, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes. Nano drug delivery is an emerging field that has attracted significant interest due to its unique characteristics and paved the way for several unique applications that might solve many problems in medicine. In particular, the nanoscale size of nanoparticles (NPs) enhances cellular uptake and can optimize intracellular pathways due to their intrinsic physicochemical properties, and can therefore increase drug delivery to target tissues.47,48 However, the inherent targeting ability resulting from the physicochemical properties of NPs is not enough to target specific tissues or damaged tissues, and additional studies on additional ligands that can bind to surface receptors on target cells or tissues have been performed to improve the targeting ability of NPs.49 Likewise, nanoencapsulation with cell membranes with targeting molecules and encapsulation of the core NPs with cell membranes confer the targeting ability of the source cell to the NPs.50,51 Thus, MSC mimicking nanoencapsulation can mimic the superior targeting ability of MSCs and confer the advantages of each core NP. In addition, MSC mimicking nanoencapsulations have improved circulation time and camouflaging from phagocytes.52

This review discusses the mechanism of MSC migration to inflammatory sites, addresses the potential strategy for improving the tropism of MSCs using genetic engineering, and discusses the promising therapeutic agent, MSC mimicking nanoencapsulations.

The MSC migration mechanism can be exploited for diverse clinical applications.53 The MSC migration mechanism can be divided into five stages: rolling by selectin, activation of MSCs by chemokines, stopping cell rolling by integrin, transcellular migration, and migration to the damaged site (Figure 1).54,55 Chemokines are secreted naturally by various cells such as tumor cells, stromal cells, and inflammatory cells, maintaining high chemokine concentrations in target cells at the target tissue and inducing signal cascades.5658 Likewise, MSCs express a variety of chemokine receptors, allowing them to migrate and be used as new targeting vectors.5961 MSC migration accelerates depending on the concentration of chemokines, which are the most important factors in the stem cell homing mechanism.62,63 Chemokines consist of various cytokine subfamilies that are closely associated with the migration of immune cells. Chemokines are divided into four classes based on the locations of the two cysteine (C) residues: CC-chemokines, CXC-chemokine, C-chemokine, and CX3 Chemokine.64,65 Each chemokine binds to various MSC receptors and the binding induces a chemokine signaling cascade (Table 1).56,66

Table 1 Chemokine and Chemokine Receptors for Different Chemokine Families

Figure 1 Representation of stem cell homing mechanism.

The mechanisms underlying MSC and leukocyte migration are similar in terms of their migratory dynamics.55 P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are major proteins involved in leukocyte migration that interact with P-selectin and E-selectin present in vascular endothelial cells. However, these promoters are not present in MSCs (Figure 2).53,67

Figure 2 Differences in adhesion protein molecules between leukocytes and mesenchymal stem cells during rolling stages and rolling arrest stage of MSC. (A) The rolling stage of leukocytes starts with adhesion to endothelium with ESL-1 and PSGL-1 on leukocytes. (B) The rolling stage of MSC starts with the adhesion to endothelium with Galectin-1 and CD24 on MSC, and the rolling arrest stage was caused by chemokines that were encountered in the rolling stage and VLA-4 with a high affinity for VACM present in endothelial cells.

Abbreviations: ESL-1, E-selectin ligand-1; PSGL-1, P-selectin glycoprotein ligand-1 VLA-4, very late antigen-4; VCAM, vascular cell adhesion molecule-1.

The initial rolling is facilitated by selectins expressed on the surface of endothelial cells. Various glycoproteins on the surface of MSCs can bind to the selectins and continue the rolling process.68 However, the mechanism of binding of the glycoprotein on MSCs to the selectins is still unclear.69,70 P-selectins and E-selectins, major cell-cell adhesion molecules expressed by endothelial cells, adhere to migrated cells adjacent to endothelial cells and can trigger the rolling process.71 For leukocyte migration, P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) expressed on the membranes of leukocytes interact with P-selectins and E-selectins on the endothelial cells, initiating the process.72,73 As already mentioned, MSCs express neither PSGL-1 nor ESL-1. Instead, they express galectin-1 and CD24 on their surfaces, and these bind to E-selectin or P-selectin (Figure 2).7476

In the migratory activation step, MSC receptors are activated in response to inflammatory cytokines, including CXCL12, CXCL8, CXCL4, CCL2, and CCL7.77 The corresponding activation of chemokine receptors of MSCs in response to inflammatory cytokines results in an accumulation of MSCs.58,78 For example, inflamed tissues release inflammatory cytokines,79 and specifically, fibroblasts release CXCL12, which further induces the accumulation of MSCs through ligandreceptor interaction after exposure to hypoxia and cytokine-rich environments in the rat model of inflammation.7982 Previous studies have reported that overexpressing CXCR4, which is a receptor to recognize CXCL12, in MSCs improves the homing ability of MSCs toward inflamed sites.83,84 In short, cytokines are significantly involved in the homing mechanism of MSCs.53

The rolling arrest stage is facilitated by integrin 41 (VLA-4) on MSC.85 VLA-4 is expressed by MSCs which are first activated by CXCL-12 and TNF- chemokines, and activated VLA-4 binds to VCAM-1 expressed on endothelial cells to stop the rotational movement (Figure 2).86,87

Karp et al categorized the migration of MSCs as either systemic homing or non-systemic homing. Systemic homing refers to the process of migration through blood vessels and then across the vascular endothelium near the inflamed site.67,88 The process of migration after passing through the vessels or local injection is called non-systemic homing. In non-systemic migration, stem cells migrate through a chemokine concentration gradient (Figure 3).89 MSCs secrete matrix metalloproteinases (MMPs) during migration. The mechanism underlying MSC migration is currently undefined but MSC migration can be advanced by remodeling the matrix through the secretion of various enzymes.9093 The migration of MSCs to the damaged area is induced by chemokines released from the injured site, such as IL-8, TNF-, insulin-like growth factor (IGF-1), and platelet-derived growth factors (PDGF).9496 MSCs migrate toward the damaged area following a chemokine concentration gradient.87

Figure 3 Differences between systemic and non-systemic homing mechanisms. Both systemic and non-systemic homing to the extracellular matrix and stem cells to their destination, MSCs secrete MMPs and remodel the extracellular matrix.

Abbreviation: MMP, matrix metalloproteinase.

RA is a chronic inflammatory autoimmune disease characterized by distinct painful stiff joints and movement disorders.97 RA affects approximately 1% of the worlds population.98 RA is primarily induced by macrophages, which are involved in the innate immune response and are also involved in adaptive immune responses, together with B cells and T cells.99 Inflammatory diseases are caused by high levels of inflammatory cytokines and a hypoxic low-pH environment in the joints.100,101 Fibroblast-like synoviocytes (FLSs) and accumulated macrophages and neutrophils in the synovium of inflamed joints also express various chemokines.102,103 Chemokines from inflammatory reactions can induce migration of white blood cells and stem cells, which are involved in angiogenesis around joints.101,104,105 More than 50 chemokines are present in the rheumatoid synovial membrane (Table 2). Of the chemokines in the synovium, CXCL12, MIP1-a, CXCL8, and PDGF are the main ones that attract MSCs.106 In the RA environment, CXCL12, a ligand for CXCR4 on MSCs, had 10.71 times higher levels of chemokines than in the normal synovial cell environment. MIP-1a, a chemokine that gathers inflammatory cells, is a ligand for CCR1, which is normally expressed on MSC.107,108 CXCL8 is a ligand for CXCR1 and CXCR2 on MSCs and induces the migration of neutrophils and macrophages, leading to ROS in synovial cells.59 PDGF is a regulatory peptide that is upregulated in the synovial tissue of RA patients.109 PDGF induces greater MSC migration than CXCL12.110 Importantly, stem cells not only have the homing ability to inflamed joints but also have potential as cell therapy with the anti-apoptotic, anti-catabolic, and anti-fibrotic effect of MSC.111 In preclinical trials, MSC treatment has been extensively investigated in collagen-induced arthritis (CIA), a common autoimmune animal model used to study RA. In the RA model, MSCs downregulated inflammatory cytokines such as IFN-, TNF-, IL-4, IL-12, and IL1, and antibodies against collagen, while anti-inflammatory cytokines, such as tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2), transforming growth factor-beta (TGF-), IL-10, and IL-6, were upregulated.112116

Table 2 Rheumatoid Arthritis (RA) Chemokines Present in the Pathological Environment and Chemokine Receptors Present in Mesenchymal Stem Cells

Genetic engineering can improve the therapeutic potential of MSCs, including long-term survival, angiogenesis, differentiation into specific lineages, anti- and pro-inflammatory activity, and migratory properties (Figure 4).117,118 Although MSCs already have an intrinsic homing ability, the targeting ability of MSCs and their derivatives, such as membrane vesicles, which are utilized to produce MSC mimicking nanoencapsulation, can be enhanced.118 The therapeutic potential of MSCs can be magnified by reprogramming MSCs via upregulation or downregulation of their native genes, resulting in controlled production of the target protein, or by introducing foreign genes that enable MSCs to express native or non-native products, for example, non-native soluble tumor necrosis factor (TNF) receptor 2 can inhibit TNF-alpha signaling in RA therapies.28

Figure 4 Genetic engineering of mesenchymal stem cells to enhance therapeutic efficacy.

Abbreviations: Sfrp2, secreted frizzled-related protein 2; IGF1, insulin-like growth factor 1; IL-2, interleukin-2; IL-12, interleukin-12; IFN-, interferon-beta; CX3CL1, C-X3-C motif chemokine ligand 1; VEGF, vascular endothelial growth factor; HGF, human growth factor; FGF, fibroblast growth factor; IL-10, interleukin-10; IL-4, interleukin-4; IL18BP, interleukin-18-binding protein; IFN-, interferon-alpha; SDF1, stromal cell-derived factor 1; CXCR4, C-X-C motif chemokine receptor 4; CCR1, C-C motif chemokine receptor 1; BMP2, bone morphogenetic protein 2; mHCN2, mouse hyperpolarization-activated cyclic nucleotide-gated.

MSCs can be genetically engineered using different techniques, including by introducing particular genes into the nucleus of MSCs or editing the genome of MSCs (Figure 5).119 Foreign genes can be transferred into MSCs using liposomes (chemical method), electroporation (physical method), or viral delivery (biological method). Cationic liposomes, also known as lipoplexes, can stably compact negatively charged nucleic acids, leading to the formation of nanomeric vesicular structure.120 Cationic liposomes are commonly produced with a combination of a cationic lipid such as DOTAP, DOTMA, DOGS, DOSPA, and neutral lipids, such as DOPE and cholesterol.121 These liposomes are stable enough to protect their bound nucleic acids from degradation and are competent to enter cells via endocytosis.120 Electroporation briefly creates holes in the cell membrane using an electric field of 1020 kV/cm, and the holes are then rapidly closed by the cells membrane repair mechanism.122 Even though the electric shock induces irreversible cell damage and non-specific transport into the cytoplasm leads to cell death, electroporation ensures successful gene delivery regardless of the target cell or organism. Viral vectors, which are derived from adenovirus, adeno-associated virus (AAV), or lentivirus (LV), have been used to introduce specific genes into MSCs. Recombinant lentiviral vectors are the most widely used systems due to their high tropism to dividing and non-dividing cells, transduction efficiency, and stable expression of transgenes in MSCs, but the random genome integration of transgenes can be an obstacle in clinical applications.123 Adenovirus and AAV systems are appropriate alternative strategies because currently available strains do not have broad genome integration and a strong immune response, unlike LV, thus increasing success and safety in clinical trials.124 As a representative, the Oxford-AstraZeneca COVID-19 vaccine, which has been authorized in 71 countries as a vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread globally and led to the current pandemic, transfers the spike protein gene using an adenovirus-based viral vector.125 Furthermore, there are two AAV-based gene therapies: Luxturna for rare inherited retinal dystrophy and Zolgensma for spinal muscular atrophy.126

Figure 5 Genetic engineering techniques used in the production of bioengineered mesenchymal stem cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 were recently used for genome editing and modification because of their simpler design and higher efficiency for genome editing, however, there are safety issues such as off-target effects that induce mutations at sites other than the intended target site.127 The foreign gene is then commonly transferred into non-integrating forms such as plasmid DNA and messenger RNA (mRNA).128

The gene expression machinery can also be manipulated at the cytoplasmic level through RNA interference (RNAi) technology, inhibition of gene expression, or translation using neutralizing targeted mRNA molecules with sequence-specific small RNA molecules such as small interfering RNA (siRNA) or microRNA (miRNA).129 These small RNAs can form enzyme complexes that degrade mRNA molecules and thus decrease their activity by inhibiting translation. Moreover, the pre-transcriptional silencing mechanism of RNAi can induce DNA methylation at genomic positions complementary to siRNA or miRNA with enzyme complexes.

CXC chemokine receptor 4 (CXCR4) is one of the most potent chemokine receptors that is genetically engineered to enhance the migratory properties of MSCs.130 CXCR4 is a chemokine receptor specific for stromal-derived factor-1 (SDF-1), also known as CXC motif chemokine 12 (CXCL12), which is produced by damaged tissues, such as the area of inflammatory bone destruction.131 Several studies on engineering MSCs to increase the expression of the CXCR4 gene have reported a higher density of the CXCR4 receptor on their outer cell membrane and effectively increased the migration of MSCs toward SDF-1.83,132,133 CXC chemokine receptor 7 (CXCR7) also had a high affinity for SDF-1, thus the SDF-1/CXCR7 signaling axis was used to engineer the MSCs.134 CXCR7-overexpressing MSCs in a cerebral ischemia-reperfusion rat hippocampus model promoted migration based on an SDF-1 gradient, cooperating with the SDF-1/CXCR4 signaling axis (Figure 6).37

Figure 6 Engineered mesenchymal stem cells with enhanced migratory abilities.

Abbreviations: CXCR4, C-X-C motif chemokine receptor 4; CXCR7, C-X-C motif chemokine receptor 7; SDF1, stromal cell-derived factor 1; CXCR1, C-X-C motif chemokine receptor 1; IL-8, interleukin-8; Aqp1, aquaporin 1; FAK, focal adhesion kinase.

CXC chemokine receptor 1 (CXCR1) enhances MSC migratory properties.59 CXCR1 is a receptor for IL-8, which is the primary cytokine involved in the recruitment of neutrophils to the site of damage or infection.135 In particular, the IL-8/CXCR1 axis is a key factor for the migration of MSCs toward human glioma cell lines, such as U-87 MG, LN18, U138, and U251, and CXCR1-overexpressing MSCs showed a superior capacity to migrate toward glioma cells and tumors in mice bearing intracranial human gliomas.136

The migratory properties of MSCs were also controlled via aquaporin-1 (Aqp1), which is a water channel molecule that transports water across the cell membrane and regulates endothelial cell migration.137 Aqp1-overexpressing MSCs showed enhanced migration to fracture gap of a rat fracture model with upregulated focal adhesion kinase (FAK) and -catenin, which are important regulators of cell migration.138

Nur77, also known as nerve growth factor IB or NR4A1, and nuclear receptor-related 1 (Nurr1), can play a role in improving the migratory capabilities of MSCs.139,140 The migrating MSCs expressed higher levels of Nur77 and Nurr1 than the non-migrating MSCs, and overexpression of these two nuclear receptors functioning as transcription factors enhanced the migration of MSCs toward SDF-1. The migration of cells is closely related to the cell cycle, and normally, cells in the late S or G2/M phase do not migrate.141 The overexpression of Nur77 and Nurr1 increased the proportion of MSCs in the G0/G1-phase similar to the results of migrating MSCs had more cells in the G1-phase.

MSC mimicking nanoencapsulations are nanoparticles combined with MSC membrane vesicles and these NPs have the greatest advantages as drug delivery systems due to the sustained homing ability of MSCs as well as the advantages of NPs. Particles sized 10150 nm have great advantages in drug delivery systems because they can pass more freely through the cell membrane by the interaction with biomolecules, such as clathrin and caveolin, to facilitate uptake across the cell membrane compared with micron-sized materials.142,143 Various materials have been used to formulate NPs, including silica, polymers, metals, and lipids.144,145 NPs have an inherent ability, called passive targeting, to accumulate at specific sites based on their physicochemical properties such as size, surface charge, surface hydrophilicity, and geometry.146148 However, physicochemical properties are not enough to target specific tissues or damaged tissues, and thus active targeting is a clinically approved strategy involving the addition of ligands that can bind to surface receptors on target cells or tissues.149,150 MSC mimicking nanoencapsulation uses natural or genetically engineered MSC membranes to coat synthetic NPs, producing artificial ectosomes and fusing them with liposomes to increase their targeting ability (Figure 7).151 Especially, MSCs have been studied for targeting inflammation and regenerative drugs, and the mechanism and efficacy of migration toward inflamed tissues have been actively investigated.152 MSC mimicking nanoencapsulation can mimic the well-known migration ability of MSCs and can be equally utilized without safety issues from the direct application of using MSCs. Furthermore, cell membrane encapsulations have a wide range of functions, including prolonged blood circulation time and increased active targeting efficacy from the source cells.153,154 MSC mimicking encapsulations enter recipient cells using multiple pathways.155 MSC mimicking encapsulations can fuse directly with the plasma membrane and can also be taken up through phagocytosis, micropinocytosis, and endocytosis mediated by caveolin or clathrin.156 MSC mimicking encapsulations can be internalized in a highly cell type-specific manner that depends on the recognition of membrane surface molecules by the cell or tissue.157 For example, endothelial colony-forming cell (ECFC)-derived exosomes were shown CXCR4/SDF-1 interaction and enhanced delivery toward the ischemic kidney, and Tspan8-alpha4 complex on lymph node stroma derived extracellular vesicles induced selective uptake by endothelial cells or pancreatic cells with CD54, serving as a major ligand.158,159 Therefore, different source cells may contain protein signals that serve as ligands for other cells, and these receptorligand interactions maximized targeted delivery of NPs.160 This natural mechanism inspired the application of MSC membranes to confer active targeting to NPs.

Figure 7 Mesenchymal stem cell mimicking nanoencapsulation.

Cell membrane-coated NPs (CMCNPs) are biomimetic strategies developed to mimic the properties of cell membranes derived from natural cells such as erythrocytes, white blood cells, cancer cells, stem cells, platelets, or bacterial cells with an NP core.161 Core NPs made of polymer, silica, and metal have been evaluated in attempts to overcome the limitations of conventional drug delivery systems but there are also issues of toxicity and reduced biocompatibility associated with the surface properties of NPs.162,163 Therefore, only a small number of NPs have been approved for medical application by the FDA.164 Coating with cell membrane can enhance the biocompatibility of NPs by improving immune evasion, enhancing circulation time, reducing RES clearance, preventing serum protein adsorption by mimicking cell glycocalyx, which are chemical determinants of self at the surfaces of cells.151,165 Furthermore, the migratory properties of MSCs can also be transferred to NPs by coating them with the cell membrane.45 Coating NPs with MSC membranes not only enhances biocompatibility but also maximizes the therapeutic effect of NPs by mimicking the targeting ability of MSCs.166 Cell membrane-coated NPs are prepared in three steps: extraction of cell membrane vesicles from the source cells, synthesis of the core NPs, and fusion of the membrane vesicles and core NPs to produce cell membrane-coated NPs (Figure 8).167 Cell membrane vesicles, including extracellular vesicles (EVs), can be harvested through cell lysis, mechanical disruption, and centrifugation to isolate, purify the cell membrane vesicles, and remove intracellular components.168 All the processes must be conducted under cold conditions, with protease inhibitors to minimize the denaturation of integral membrane proteins. Cell lysis, which is classically performed using mechanical lysis, including homogenization, sonication, or extrusion followed by differential velocity centrifugation, is necessary to remove intracellular components. Cytochalasin B (CB), a drug that affects cytoskeletonmembrane interactions, induces secretion of membrane vesicles from source cells and has been used to extract the cell membrane.169 The membrane functions of the source cells are preserved in CB-induced vesicles, forming biologically active surface receptors and ion pumps.170 Furthermore, CB-induced vesicles can encapsulate drugs and NPs successfully, and the vesicles can be harvested by centrifugation without a purification step to remove nuclei and cytoplasm.171 Clinically translatable membrane vesicles require scalable production of high volumes of homogeneous vesicles within a short period. Although mechanical methods (eg, shear stress, ultrasonication, or extrusion) are utilized, CB-induced vesicles have shown potential for generating membrane encapsulation for nano-vectors.168 The advantages of CB-induced vesicles versus other methods are compared in Table 3.

Table 3 Comparison of Membrane Vesicle Production Methods

Figure 8 MSC membrane-coated nanoparticles.

Abbreviations: EVs, extracellular vesicles; NPs, nanoparticles.

After extracting cell membrane vesicles, synthesized core NPs are coated with cell membranes, including surface proteins.172 Polymer NPs and inorganic NPs are adopted as materials for the core NPs of CMCNPs, and generally, polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), chitosan, and gelatin are used. PLGA has been approved by FDA is the most common polymer of NPs.173 Biodegradable polymer NPs have gained considerable attention in nanomedicine due to their biocompatibility, nontoxic properties, and the ability to modify their surface as a drug carrier.174 Inorganic NPs are composed of gold, iron, copper, and silicon, which have hydrophilic, biocompatible, and highly stable properties compared with organic materials.175 Furthermore, some photosensitive inorganic NPs have the potential for use in photothermal therapy (PTT) and photodynamic therapy (PDT).176 The fusion of cell membrane vesicles and core NPs is primarily achieved via extrusion or sonication.165 Cell membrane coating of NPs using mechanical extrusion is based on a different-sized porous membrane where core NPs and vesicles are forced to generate vesicle-particle fusion.177 Ultrasonic waves are applied to induce the fusion of vesicles and NPs. However, ultrasonic frequencies need to be optimized to improve fusion efficiency and minimize drug loss and protein degradation.178

CMCNPs have extensively employed to target and treat cancer using the membranes obtained from red blood cell (RBC), platelet and cancer cell.165 In addition, membrane from MSC also utilized to target tumor and ischemia with various types of core NPs, such as MSC membrane coated PLGA NPs targeting liver tumors, MSC membrane coated gelatin nanogels targeting HeLa cell, MSC membrane coated silica NPs targeting HeLa cell, MSC membrane coated PLGA NPs targeting hindlimb ischemia, and MSC membrane coated iron oxide NPs for targeting the ischemic brain.179183 However, there are few studies on CMCNPs using stem cells for the treatment of arthritis. Increased targeting ability to arthritis was introduced using MSC-derived EVs and NPs.184,185 MSC membrane-coated NPs are proming strategy for clearing raised concerns from direct use of MSC (with or without NPs) in terms of toxicity, reduced biocompatibility, and poor targeting ability of NPs for the treatment of arthritis.

Exosomes are natural NPs that range in size from 40 nm to 120 nm and are derived from the multivesicular body (MVB), which is an endosome defined by intraluminal vesicles (ILVs) that bud inward into the endosomal lumen, fuse with the cell surface, and are then released as exosomes.186 Because of their ability to express receptors on their surfaces, MSC-derived exosomes are also considered potential candidates for targeting.187 Exosomes are commonly referred to as intracellular communication molecules that transfer various compounds through physiological mechanisms such as immune response, neural communication, and antigen presentation in diseases such as cancer, cardiovascular disease, diabetes, and inflammation.188

However, there are several limitations to the application of exosomes as targeted therapeutic carriers. First, the limited reproducibility of exosomes is a major challenge. In this field, the standardized techniques for isolation and purification of exosomes are lacking, and conventional methods containing multi-step ultracentrifugation often lead to contamination of other types of EVs. Furthermore, exosomes extracted from cell cultures can vary and display inconsistent properties even when the same type of donor cells were used.189 Second, precise characterization studies of exosomes are needed. Unknown properties of exosomes can hinder therapeutic efficiencies, for example, when using exosomes as cancer therapeutics, the use of cancer cell-derived exosomes should be avoided because cancer cell-derived exosomes may contain oncogenic factors that may contribute to cancer progression.190 Finally, cost-effective methods for the large-scale production of exosomes are needed for clinical application. The yield of exosomes is much lower than EVs. Depending on the exosome secretion capacity of donor cells, the yield of exosomes is restricted, and large-scale cell culture technology for the production of exosomes is high difficulty and costly and isolation of exosomes is the time-consuming and low-efficient method.156

Ectosome is an EV generated by outward budding from the plasma membrane followed by pinching off and release to the extracellular parts. Recently, artificially produced ectosome utilized as an alternative to exosomes in targeted therapeutics due to stable productivity regardless of cell type compared with conventional exosome. Artificial ectosomes, containing modified cargo and targeting molecules have recently been introduced for specific purposes (Figure 9).191,192 Artificial ectosomes are typically prepared by breaking bigger cells or cell membrane fractions into smaller ectosomes, similar size to natural exosomes, containing modified cargo such as RNA molecules, which control specific genes, and chemical drugs such as anticancer drugs.193 Naturally secreted exosomes in conditioned media from modified source cells can be harvested by differential ultracentrifugation, density gradients, precipitation, filtration, and size exclusion chromatography for exosome separation.194 Even though there are several commercial kits for isolating exosomes simply and easily, challenges in compliant scalable production on a large scale, including purity, homogeneity, and reproducibility, have made it difficult to use naturally secreted exosomes in clinical settings.195 Therefore, artificially produced ectosomes are appropriate for use in clinical applications, with novel production methods that can meet clinical production criteria. Production of artificially produced ectosomes begins by breaking the cell membrane fraction of cultured cells and then using them to produce cell membrane vesicles to form ectosomes. As mentioned above, cell membrane vesicles are extracted from source cells in several ways, and cell membrane vesicles are extracted through polycarbonate membrane filters to reduce the mean size to a size similar to that of natural exosomes.196 Furthermore, specific microfluidic devices mounted on microblades (fabricated in silicon nitride) enable direct slicing of living cells as they flow through the hydrophilic microchannels of the device.197 The sliced cell fraction reassembles and forms ectosomes. There are several strategies for loading exogenous therapeutic cargos such as drugs, DNA, RNA, lipids, metabolites, and proteins, into exosomes or artificial ectosomes in vitro: electroporation, incubation for passive loading of cargo or active loading with membrane permeabilizer, freeze and thaw cycles, sonication, and extrusion.198 In addition, protein or RNA molecules can be loaded by co-expressing them in source cells via bio-engineering, and proteins designed to interact with the protein inside the cell membrane can be loaded actively into exosomes or artificial ectosomes.157 Targeting molecules at the surface of exosomes or artificial ectosomes can also be engineered in a manner similar to the genetic engineering of MSCs.

Figure 9 Mesenchymal stem cell-derived exosomes and artificial ectosomes. (A) Wound healing effect of MSC-derived exosomes and artificial ectosomes,231 (B) treatment of organ injuries by MSC-derived exosomes and artificial ectosomes,42,232234 (C) anti-cancer activity of MSC-derived exosomes and artificial ectosomes.200,202,235

Most of the exosomes derived from MSCs for drug delivery have employed miRNAs or siRNAs, inhibiting translation of specific mRNA, with anticancer activity, for example, miR-146b, miR-122, and miR-379, which are used for cancer targeting by membrane surface molecules on MSC-derived exosomes.199201 Drugs such as doxorubicin, paclitaxel, and curcumin were also loaded into MSC-derived exosomes to target cancer.202204 However, artificial ectosomes derived from MSCs as arthritis therapeutics remains largely unexplored area, while EVs, mixtures of natural ectosomes and exosomes, derived from MSCs have studied in the treatment of arthritis.184 Artificial ectosomes with intrinsic tropism from MSCs plus additional targeting ability with engineering increase the chances of ectosomes reaching target tissues with ligandreceptor interactions before being taken up by macrophages.205 Eventually, this will decrease off-target binding and side effects, leading to lower therapeutic dosages while maintaining therapeutic efficacy.206,207

Liposomes are spherical vesicles that are artificially synthesized through the hydration of dry phospholipids.208 The clinically available liposome is a lipid bilayer surrounding a hollow core with a diameter of 50150 nm. Therapeutic molecules, such as anticancer drugs (doxorubicin and daunorubicin citrate) or nucleic acids, can be loaded into this hollow core for delivery.209 Due to their amphipathic nature, liposomes can load both hydrophilic (polar) molecules in an aqueous interior and hydrophobic (nonpolar) molecules in the lipid membrane. They are well-established biomedical applications and are the most common nanostructures used in advanced drug delivery.210 Furthermore, liposomes have several advantages, including versatile structure, biocompatibility, low toxicity, non-immunogenicity, biodegradability, and synergy with drugs: targeted drug delivery, reduction of the toxic effect of drugs, protection against drug degradation, and enhanced circulation half-life.211 Moreover, surfaces can be modified by either coating them with a functionalized polymer or PEG chains to improve targeted delivery and increase their circulation time in biological systems.212 Liposomes have been investigated for use in a wide variety of therapeutic applications, including cancer diagnostics and therapy, vaccines, brain-targeted drug delivery, and anti-microbial therapy. A new approach was recently proposed for providing targeting features to liposomes by fusing them with cell membrane vesicles, generating molecules called membrane-fused liposomes (Figure 10).213 Cell membrane vesicles retain the surface membrane molecules from source cells, which are responsible for efficient tissue targeting and cellular uptake by target cells.214 However, the immunogenicity of cell membrane vesicles leads to their rapid clearance by macrophages in the body and their low drug loading efficiencies present challenges for their use as drug delivery systems.156 However, membrane-fused liposomes have advantages of stability, long half-life in circulation, and low immunogenicity due to the liposome, and the targeting feature of cell membrane vesicles is completely transferred to the liposome.215 Furthermore, the encapsulation efficiencies of doxorubicin were similar when liposomes and membrane-fused liposomes were used, indicating that the relatively high drug encapsulation capacity of liposomes was maintained during the fusion process.216 Combining membrane-fused liposomes with macrophage-derived membrane vesicles showed differential targeting and cytotoxicity against normal and cancerous cells.217 Although only a few studies have been conducted, these results corroborate that membrane-fused liposomes are a potentially promising future drug delivery system with increased targeting ability. MSCs show intrinsic tropism toward arthritis, and further engineering and modification to enhance their targeting ability make them attractive candidates for the development of drug delivery systems. Fusing MSC exosomes with liposomes, taking advantage of both membrane vesicles and liposomes, is a promising technique for future drug delivery systems.

Figure 10 Mesenchymal stem cell membrane-fused liposomes.

MSCs have great potential as targeted therapies due to their greater ability to home to targeted pathophysiological sites. The intrinsic ability to home to wounds or to the tumor microenvironment secreting inflammatory mediators make MSCs and their derivatives targeting strategies for cancer and inflammatory disease.218,219 Contrary to the well-known homing mechanisms of various blood cells, it is still not clear how homing occurs in MSCs. So far, the mechanism of MSC tethering, which connects long, thin cell membrane cylinders called tethers to the adherent area for migration, has not been clarified. Recent studies have shown that galectin-1, VCAM-1, and ICAM are associated with MSC tethering,53,220 but more research is needed to accurately elucidate the tethering mechanism of MSCs. MSC chemotaxis is well defined and there is strong evidence relating it to the homing ability of MSCs.53 Chemotaxis involves recognizing chemokines through chemokine receptors on MSCs and migrating to chemokines in a gradient-dependent manner.221 RA, a representative inflammatory disease, is associated with well-profiled chemokines such as CXCR1, CXCR4, and CXCR7, which are recognized by chemokine receptors on MSCs. In addition, damaged joints in RA continuously secrete cytokines until they are treated, giving MSCs an advantage as future therapeutic agents for RA.222 However, there are several obstacles to utilizing MSCs as RA therapeutics. In clinical settings, the functional capability of MSCs is significantly affected by the health status of the donor patient.223 MSC yield is significantly reduced in patients undergoing steroid-based treatment and the quality of MSCs is dependent on the donors age and environment.35 In addition, when MSCs are used clinically, cryopreservation and defrosting are necessary, but these procedures shorten the life span of MSCs.224 Therefore, NPs mimicking MSCs are an alternative strategy for overcoming the limitations of MSCs. Additionally, further engineering and modification of MSCs can enhance the therapeutic effect by changing the targeting molecules and loaded drugs. In particular, upregulation of receptors associated with chemotaxis through genetic engineering can confer the additional ability of MSCs to home to specific sites, while the increase in engraftment maximizes the therapeutic effect of MSCs.36,225

Furthermore, there are several methods that can be used to exploit the targeting ability of MSCs as drug delivery systems. MSCs mimicking nanoencapsulation, which consists of MSC membrane-coated NPs, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes, can mimic the targeting ability of MSCs while retaining the advantages of NPs. MSC-membrane-coated NPs are synthesized using inorganic or polymer NPs and membranes from MSCs to coat inner nanosized structures. Because they mimic the biological characteristics of MSC membranes, MSC-membrane-coated NPs can not only escape from immune surveillance but also effectively improve targeting ability, with combined functions of the unique properties of core NPs and MSC membranes.226 Exosomes are also an appropriate candidate for use in MSC membranes, utilizing these targeting abilities. However, natural exosomes lack reproducibility and stable productivity, thus artificial ectosomes with targeting ability produced via synthetic routes can increase the local concentration of ectosomes at the targeted site, thereby reducing toxicity and side effects and maximizing therapeutic efficacy.156 MSC membrane-fused liposomes, a novel system, can also transfer the targeting molecules on the surface of MSCs to liposomes; thus, the advantages of liposomes are retained, but with targeting ability. With advancements in nanotechnology of drug delivery systems, the research in cell-mimicking nanoencapsulation will be very useful. Efficient drug delivery systems fundamentally improve the quality of life of patients with a low dose of medication, low side effects, and subsequent treatment of diseases.227 However, research on cell-mimicking nanoencapsulation is at an early stage, and several problems need to be addressed. To predict the nanotoxicity of artificially synthesized MSC mimicking nanoencapsulations, interactions between lipids and drugs, drug release mechanisms near the targeted site, in vivo compatibility, and immunological physiological studies must be conducted before clinical application.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2019M3A9H1103690), by the Gachon University Gil Medical Center (FRD2021-03), and by the Gachon University research fund of 2020 (GGU-202008430004).

The authors report no conflicts of interest in this work.

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Stem Cell Mimicking Nanoencapsulation for Targeting Arthrit | IJN - Dove Medical Press

The advent and advancement of nanotechnology in medicine can create a more straightforward treatment regimen with lower dose frequency and much less maintenance.

Fremont, CA:Nanomedicine employs nanoscale technology for medical applications, which may include the use of particles ranging in size from 1 to 100 nm. In recent years, the novel use of nanotechnology in medicine has become an exciting development, with innovative Food and Drug Administration (FDA)-approved concepts that can revolutionize disease detection and treatment.

Infections are a significant burden throughout the world, with high mortality rates associated with conditions such as lower respiratory infections, tuberculosis, diarrhea, malaria, HIV, and others. These infectious diseases have a greater impact in the developing world, where mortality rates associated with these conditions are the highest because of a scarcity of vaccines and anti-infectives.

Observations from temporal trends in mortality show that, while overall mortality rates are decreasing, there is still a large disparity in death rates between high and low socio-demographic index (SDI) countries. Furthermore, clinical trials for contagious diseases are lower than trials for other disorders such as cardiovascular disease and even cancer, demonstrating the need for advanced infectious disease treatment through nanotechnology.

Nanotechnology in Medicine

The advent and advancement of nanotechnology in medicine can create a more straightforward treatment regimen with lower dose frequency and much less maintenance.

The use of injectable nanocarriers capable of transporting, delivering, or releasing drugs over long periods of time would be revolutionary for the challenges that low SDI countries face. Nanocarriers, which control drug release by utilizing an inactive vehicle such as a lipid or polymer, or a slow breakdown of a drug-using poorly soluble mediums such as nano-drug crystals, are examples of drug delivery systems.

Low SID Challenges

Low socio-demographic index countries frequently face a slew of challenges. This includes patients who do not adhere to therapies, the need for ongoing patient monitoring, and other factors such as the inability to pay for drugs or maintaining drug stability in high or humid temperatures.

These factors can make it difficult for low-income countries to effectively treat patients with infections, ranging from hospitals being unable to adequately support their patients to patients being unable to access drugs.

Long-term care for conditions such as HIV, tuberculosis, or malaria can significantly burden patients and healthcare systems. HIV would necessitate lifelong treatment, whereas tuberculosis would necessitate a combination of oral medications administered over several months to years. Prolonged treatment plans can lead to a lack of patient adherence, reducing treatment efficacy and resulting in failure because of a lack of optimal drug levels.

Continued here:
How to Improve Infectious Diseases with Nanotechnology - CIO Applications

A video through a post is being widely shared on social media in the context of the present COVID-19 vaccination across the world. A doctor named Rima Laibow had claimed in the video that the World Health Organisation (WHO) has been working on vaccines to create permanent sterility. Let us fact-check the claim made in the post.

Claim: 2009 video of Dr Rima Laibow confirming that WHO had been working on COVID-19 vaccines to create permanent sterility.

Fact: There is no evidence to show that WHO has been preparing vaccines to create complete sterility. The video is from a show named Conspiracy Theory with Jesse Ventura where Jesse Ventura, the host talks with people on various conspiracy theories. The video had been created in the context of Swine Flu that was spreading in 2008-09 and has nothing to do with COVID-19. There is no evidence to show that the Swine Flu vaccine had created permanent sterility. In fact, there is no evidence that the present COVID-19 vaccines will cause complete sterility. The COVID-19 vaccines do not contain adjuvants based on squalene, the substance mentioned in the video. Dr Rima Laibow is a conspiracy theorist whose organisation had been selling unlicensed drugs in the US at various times. Hence, the claim made in the post is FALSE.

When reverse image search is done on the screenshots of the video, a Facebook video was found with similar visuals saying that the video is from a TruTV show named Conspiracy Theory with Jesse Ventura. The viral video has been taken from Season 1, Episode 5 of the show, Secret Societies, where Jesse Ventura, the host talks to the doctor named Rima Laibow who talks about a pandemic on the horizon that would cause forced vaccinations with the goal of decreasing worlds population. The video was aired in December 2009.

However, the video had been created in the context of the Swine Flu that was spreading in 2008-09 and has nothing to do with COVID-19. There is no evidence to show that the Swine Flu vaccine had created permanent sterility.

Squalene, a substance mentioned in the video, is naturally found in humans, animals, and plants. Squalene is an essential building block to make certain hormones and other substances in the body, manufactured in the livers, and circulates in the bloodstream with the highest amount found in the skin. Squalene-based adjuvants (to enhance the immunogenicity of antibodies) can be found in Swine Flu vaccines. However, the COVID-19 vaccines reportedly do not contain adjuvants based on squalene.

There is no evidence to show that WHO has been preparing vaccines to create complete sterility. In fact, there is no evidence that the present COVID-19 vaccines will cause complete sterility.

Jesse Ventura is a former governor of Minnesota and an American TV host. In 2009-2012, he hosted a show for TruTV based on various conspiracy theories, named Conspiracy Theory with Jesse Ventura. Dr. Rima Laibows organisation, The Natural Solutions Foundation has been selling unlicensed drugs in the United States at various times.

In 2014, Dr. Rima Laibow distributed the drug Nano-Silver by a fraudulent scheme, claiming that the drug could cure the Ebola virus. In early 2020, she began selling the medicine for coronavirus. On 13 November 2020, at the Food and Drug Administrations request, the US Department of Justice filed a civil complaint against her and the organisation. Subsequently, in December 2021, a federal court had ordered Dr. Rima Laibow and the organisation to stop distributing unapproved Nano Silver products touted as COVID-19 treatment.

To sum it up, a 2009 video from a show based on conspiracy theories is shared to say that the WHO had been working on COVID-19 vaccines to create permanent sterility.

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2009 show based on conspiracy theories is shared as proof of WHO working on COVID-19 vaccines to create permanent sterility - Factly

Introduction

Over the last decades, personalized medicine has greatly evolved with the development of imaging tools that improve the management of several diseases, especially cancer.1 Among all the non-invasive imaging techniques, the nuclear imaging modalities Single Photon Emission Computed Tomography (SPECT) and Positron Emission Tomography (PET) stand out mainly due to their high sensitivity prospect of obtaining quantitative information. In fact, PET and SPECT imaging can provide detailed information about the in vivo behavior and pharmacokinetics of several compounds, such as nanomedicines, and can facilitate their translation to clinics.2

On the other hand, nanomedicine has emerged as a promising strategy to improve diagnosis and treatment of prevalent diseases including cancer.3,4 One of the most promising advantages of nanomedicine is the possibility to combine therapeutic molecules with diagnostic agents into single multifunctional nanoparticles, known as nanotheranostics, opening an entirely new field of development towards the implementation of personalized medicine.5,6 In recent years, the combination of nanoparticles with radionuclides is rapidly growing and there are a great number of submissions for the Food and Drug Administration approval.7 Different types of nanoparticles are investigated for nuclear medicine applications and multimodal imaging.8 For example, inorganic nanoparticles have been widely studied due to their intrinsic physical properties, that convert them into materials with a high potential for multimodal imaging.9,10 Nevertheless, organic nanoparticles are still the most in demand for the development of imaging probes by virtue of their biodegradable and biocompatible composition, preventing a long-term accumulation in the body and undesirable toxic side effects.11 Indeed, liposomes are the most extended type of organic nanoparticles for nuclear imaging applications. Liposomes can be radiolabeled by different methodologies, which can be adapted to different kinds of nanoparticles, such as micelles, solid lipid nanoparticles, and nanoemulsions.12 Chelator-based radiolabeling strategies offer a high versatility for the incorporation of radionuclides with different properties, suitable for complementary imaging techniques and nanotheranostics.13

Nanoemulsions are defined as nanoscale droplets in which two immiscible liquids are mixed to form a single phase. Their biocompatible composition, easy production by soft and scalable methodologies and improved drug loading capacity compared to liposomes, are relevant advantages that have prompted their use in biomedicine.14,15

Nanoemulsions have been widely studied for fluorescence, MRI and ultrasounds imaging.16,17 However, their use in nuclear imaging is still recent and there are only few reports describing radiolabeled nanoemulsions.1820 Our group has recently reported the development and characterization of sphingomyelin nanoemulsions that incorporate sphingomyelin, one of the main lipids in cell membranes (SNs), and claimed their potential in drug delivery.21 The principal advantages of SNs relate to their safe and simple composition, long-term colloidal stability, and capacity for accommodation of different types of functionalities and therapeutic payloads.2123 Previous attempts by our research group have proved that SNs can be radiolabeled with Fluorine-18 for PET imaging following a maleimide reaction. However, radiochemical yields (RCY) were found to be highly dependent on the crosslinking efficacy.24 The aim of this work was to provide an optimized composition and straightforward methodology for the radiolabeling of SNs with 68Ga and 67Ga radioisotopes using a chelator-based strategy. Radiolabeled formulations can be used for indistinct application in PET and SPECT imaging, and therefore adaptable to specific needs and biomedical applications.

Octadecylamine (stearylamine, >99%, Merck Group, Darmstadt, Germany) was conjugated to 2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA, NOTA, >94%, Macrocyclics, Dallas, TX, USA) to obtain a lipid derivative chelator for further inclusion into the nanoemulsions. Details about the reaction protocol and the product characterization are included in the Supporting Information.

SNs were prepared following a method previously reported by our group with minor modifications.21 Briefly, oleic acid (5 mg, 6588%, Merck Group, Darmstadt, Germany), egg sphingomyelin (0.5 mg, 98%, Lipoid GmbH, Ludwigshafen, Germany) and the surfactant C16/C18-COO-C9H9O3 (0.5 mg, 96%, GalChimia S.L, A Corua, Spain) with a lipid ratio 1:0.1:0.1 w/w were dissolved in 100 L of absolute ethanol (99.7%, Cienytech S.L., A Corua, Spain). All the additional lipid derivatives used to functionalize SNs, such us 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (DTPA, 0.05 mg, >99%, Avanti Polar Lipids, Alabama, Al, USA), NOTA (0.05 mg) or an oleic acid modified polyethylene glycol (PEG, 2 kDa, 0.125 mg, Nanocs, New York, NY, USA) were included in the organic phase. Then, this organic phase was injected in 1 mL of MilliQ water (Millipore Milli-Q system) under magnetic stirring using an insulin syringe (0.5 mL, 0.3312 mm ICO.C.1) and nanoemulsions (SNs, DTPA-SNs, NOTA-SNs or NOTA-PEG-SNs) were spontaneously formed. To prepare SNs coated with hyaluronic acid (NOTA-HA-SNs), the organic phase containing the lipids and NOTA was injected under stirring in 1 mL of an aqueous solution of sodium hyaluronate (HA, 170 kDa, 2 mg mL1, >95%, Bioiberica, S.A.U, Barcelona, Spain).

All the nanoemulsions were physiochemically characterized using a Nanosizer 2000 (Malvern Instruments, Malvern, UK). The mean size and its distribution, defined by the polydispersity index (PDI), were measured by Dynamic Light Scattering (DLS). Measurements were performed on disposable microcuvettes (ZEN0040, Malvern Instruments) upon dilution of the SNs in MilliQ water, reaching a final lipid concentration of 0.5 mg mL1. The zeta potential (ZP) was analyzed by Laser Doppler Anemometry (LDA) diluting SNs in MilliQ water (lipid concentration 0.12 mg mL1) with Folded capillary cuvettes (DTS1070, Malvern Instruments). The stability of SNs, DTPA-SNs and NOTA-SNs was tested under storage conditions at 4 C up to one month and also after incubation with human serum at 37 C for 72 h. The colloidal properties were measured by DLS maintaining the conditions mentioned before. Parameters such as the medium (water) and the temperature (25C) were fixed for all the measurements.

The morphology of SNs was observed by Field Emission Scanning Electron Microscopy (FESEM) using a ZEISS FESEM ULTRA Plus, microscope (Carl Zeiss Micro Imaging, GmbH, Germany). Before the measurement, 20 L of sphingomyelin nanoemulsions (0.5 mg mL1) were stained with 20 L phosphotungstic acid (2% w/v). Then, 20 L of the mixture was placed on a carbon coated grid and left for 2 minutes. The excess was removed using a filter paper and the grid was allowed to dry. The grid was washed 5 times with 100 L of filtered MilliQ water and it was dried overnight.

A549 (ATCC CCL-185), MDA-MB-231 (ATCC HTB-26) were cultured in Dulbeccos modified Eagles medium high glucose (DMEM, Merck Group, Darmstadt, Germany) and OMM-2.5 (kindly provided by Martine J. Jager from Leiden University Medical Center, Leiden, The Netherlands) were grown in RPMI (Gibco, Thermo Scientific S.L., Waltham, MA, United States). Both media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin:streptomycin (Gibco, Thermo Scientific S.L., Waltham, MA, United States). Cells were maintained at 37 C with 95% relative humidity and 5% CO2.

Cellular uptake of SNs was studied by confocal microscopy. 8104 cells/well were seeded in an 8-well -chamber (SPL Life Sciences Co., Ltd., Gyeonggi-do, Korea). After 24 h, cells were treated with SNs (0.13 mg mL1 per well) labeled with C11-TopFluor sphingomyelin (>99%, Avanti Polar Lipids, Alabama, Al, USA). After 4 h of incubation at 37 C, cells were washed with 1x phosphate buffer saline (PBS) twice and fixed with 4% paraformaldehyde for 15 min. Cells were then washed twice with 1x PBS and the cellular nuclei were counterstained with Hoechst 33,342 (Thermo Scientific S.L., Waltham, MA, United States) for 5 min. After washing, the slide was mounted with Mowiol (Merck Group, Darmstadt, Germany) and a coverslip. The samples were left to dry in the dark overnight at RT, following their storage at 20 C, until taken for observation under a confocal microscope (Confocal Laser Microscope Leica SP8).

68Ga (t = 68 min, + = 89% and EC = 11%) was obtained from a 68Ge/68Ga generator system (ITG Isotope Technologies Garching GmbH, Germany) in which 68Ge (t = 270 d) was attached to a column based on an organic matrix generator. The 68Ga was eluted with 4 mL of 0.05 M hydrochloric acid. Then, 500 L of DTPA-SNs or NOTA-SNs (13 mg mL1) were mixed with 500 L of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES, 0.5 M, pH 5.05). The mixture was incubated with 1.5 mL of 68Ga (300 MBq) at 30 C for 30 min and purified by PD-10 columns. The incorporated radioactivity was measured in an activimeter (AtomLabTM500, Biodex).

67Ga-citrate (t = 78.3 h, 100% EC = 39% 93 keV, 21% 185 keV, 17% 300 keV) was obtained from CURIUM (France) as sterile solution with a pH between 5 and 8 and a radiochemical purity at least equal to 95%. 67Ga-citrate solution was converted to 67GaCl3 using a method previously described.25 In brief, 2 mL of 67Ga-citrate (37 MBq) diluted in distilled water was filtered with a SEP-PAK Plus silica cartridge (ABX, Advanced Biochemical Compounds, Germany) using a 5 mL plastic syringe. Afterwards, the silica cartridge was washed three times with 5 mL of distilled water to remove the free citrate ions. The 67Ga3+ ions were eluted with 3 mL of HCl 0.1 M, obtaining a solution of 67GaCl3 which was concentrated on a rotary vacuum evaporator to get a final volume of 500 L. The pH was adjusted to 45 with NaOH 0.1 M and the solution was incubated with 500 L of NOTA-SNs, NOTA-HA-SNs or NOTA-PEG-SNs (10 mg mL1) diluted in HEPES buffer (1.5 M, pH 5.05) for 1 h at 37 C. The labeled nanoemulsions were eventually purified with PD-10 columns and the radioactivity was measured in the activimeter.

The radiochemical yield (RCY) was calculated as a percentage of decay corrected activity found in the post-purification solution compared to the starting activity. The radiochemical stability (RCS) of 68Ga-labeled nanoemulsions was assessed by incubating the emulsions with animal serum at 37 C for 4 hours. In case of 67Ga-labeled nanoemulsions, the stability was measured after incubation with animal serum for 0, 24, 48 and 72 h, according to the acquisition time points. In both cases, after the incubation time the mixture was purified by a PD-10 column and the activity of the elution was measured and decay corrected. Radiochemical purity (RCP) was analyzed by instant thin-layer chromatography (ITLC), and details regarding experimental protocols are included in the Supporting Information.

In vivo PET/CT imaging was performed in healthy mice (C57BL/6) with a nanoPET/CT small-animal imaging system (Mediso Medical Imaging Systems, Budapest, Hungary). List-mode PET data acquisition commenced 2 hours post bolus injection of ~12 MBq of 68Ga-DTPA-SNs or 68Ga-NOTA-SNs (12 MBq, n = 5) through the tail vein and continued for 30 minutes. At the end of PET, microCT was performed for attenuation correction and anatomic reference. The dynamic PET images in a 105105 matrix (frame rates: 310 min, 130 min, 160 min) were reconstructed using a Tera-Tomo 3D iterative algorithm. Acquisition and reconstruction were performed with proprietary Nucline software (Mediso, Budapest, Hungary). Qualitative Image analysis in mice was performed using Osirix software (Pixmeo, Switzerland). Animal experiments were conducted according to the ethical and animal welfare committee at CNIC and the Spanish and UE legislation. Experimental protocols have been approved by Madrid regional government (PROEX16/277).

SPECT studies were carried out on male SpragueDawley rats with an average weight of 299.5 23.45 g supplied by the animal facility at the University of Santiago de Compostela (Spain). Planar dynamic SPECT images were acquired with a single-head clinical Siemens Orbiter gamma camera (Siemens Medical Solutions, Inc., USA) using a parallel collimator specifically designed for low-energy photons and high spatial resolution. Data were acquired in list-mode format in order to apply energy and spatial linearity, and uniformity corrections. In vivo NOTA-SNs and free 67Ga biodistribution were studied after the intravenous injection (17.70 8.5 MBq, n = 5) in healthy rats at different time-points: 24, 48 and 72 h. In order to compare the differences in biodistribution between NOTA-SNs, HA-SNs and PEG-SNs, healthy rats were intravenously injected (13.2 0.3 MBq, n = 3) and the images were acquired dynamically during the first 60 min after injection (30 frames/2min). All images were analyzed using AMIDE software (amide.sourceforge.net). Quantitative analysis was carried out in a dynamic study by using circularly delineated Regions of Interest (ROIs) in the heart and liver, with 9 mm in diameter. The mean uptake was calculated over time in every region averaged over each of the 3 frames (6 min) and the results were reported as heart-to-liver ratio.

Ex vivo biodistribution of 68Ga-labeled nanoemulsions was conducted 4 h post-injection. In case of 67Ga-labeled nanoemulsions, biodistribution studies were performed 72 h post-injection. Animals were sacrificed in a CO2 chamber, organs were extracted and counted with a Wizard 1470 gamma counter (Perkin Elmer) for 1 min each (n=5 per experiment). Radioactivity decay was corrected, and a biodistribution was presented as the percentage of injected dose per gram (% ID/g).

All the experiments were performed at least in triplicate. Data are expressed as mean standard deviation (SD). Statistical analyses were calculated using GraphPad Prism software (version 8.0). Students t-test was used to compare significant differences between the two groups. * (p0.05), ** (p0.01), ***(p0.001)was considered statistically significant.

Here, we describe the radiolabeling of SNs with Gallium-68 and Gallium-67 for their application in PET and SPECT imaging. SNs were prepared by ethanol injection, a one-step mild technique that allows obtaining colloidal nanoemulsions within seconds (Figure 1A, left). The reproducibility of the preparation method (Figure 1A, right) was obtained after measuring 24 independent batches by DLS (raw data are showed in Table S1). SNs showed spherical morphology, as observed in FESEM images (Figure 1B). Additionally, SNs were efficiently internalized in cancer cells (Figure 1C), which is a relevant factor to take into account in order to determine the potential of a formulation for biomedicine applications. Stability determinations in cell culture media were also performed and are shown in Figure S1, Supporting Information. To convert SNs into suitable probes for PET and SPECT imaging, we followed a chelator-mediated approach, which is one of the most used methods to radiolabel nanoparticles with radionuclides, such as 64Cu, 68Ga, 99mTc or 111In.13,26 Labeling organic nanoparticles, and especially lipid nanoparticles, can be done by the use of lipid-derivative chelators. These conjugates can be inserted into the membrane of the lipid particles at the time of their preparation.2729 In this study, we used two different lipid-derivative chelators to determine the best candidate for in vivo imaging. First, we selected the acyclic chelator diethylenetriaminepentaacetic acid modified with a dimyristoyl-sn-glycero-3-phosphoethanolamine chain (DTPA). Second, we synthesized the amphiphilic derivative of the macrocyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as previously described.3032 In brief, a stearylamine was reacted with the isothiocyanate macrocycle p-SCN-Bn-NOTA (1, Figure S2A, Supporting Information). The nucleophilic substitution in N,N-dimethylformamide afforded the corresponding thiourea derivative (2, NOTA-stearylamine, Figure S2A, Supporting Information) after recrystallization at moderate yield (23%). The NOTA-stearylamine derivative 2 was characterized by high-resolution mass spectrometry (Figure S2B Supporting Information) and NMR, confirming its structure (Figures S3 and S4 Supporting Information). Both lipid-modified chelators were spontaneously incorporated into the lipidic layer of SNs. According to results shown in Table 1, a slight increase in size was observed for DTPA-SNs and NOTA-SNs with respect to the control SNs, which could be indicative of the efficient incorporation of the chelators. In all cases, we observed a narrow distribution of the particles with a PDI 0.2.

Table 1 Physicochemical Characterization of SNs, DTPA-SNs and NOTA-SNs Measured by DLS and LDA (Results are Expressed as Mean Standard Deviation, n = 3)

Figure 1 (A) Scheme of the one-step method used for the preparation of SNs (left) and the method reproducibility after measuring the hydrodynamic size of 24 independent batches by DLS (right); horizontal bars represent size mean and standard deviation (127 9 nm). (B) Representative Field Emission Scanning Electron Microscopy (FESEM) images of SNs acquired with STEM (top) and InLens (bottom) detectors. (C) Confocal microscopy images showing the internalization of SNs in different cancer cell lines. SNs are labeled in green (TopFluor-SM) and cell nuclei are labeled in blue (Hoechst).

Stability studies under storage conditions at 4 C showed that all the formulations were highly stable during the tested period (Figure 2A), indicating that the incorporation of the lipid-derivative chelators does not compromise the colloidal properties of SNs. In addition, they showed high stability in the human serum for 72 h at 37 C, as shown in Figure 2B, demonstrating their potential for in vivo applications. Although organic nanoparticles offer relevant advantages with respect to inorganic nanoparticles, in general, their preparation is still complex, as dendrimers, liposomes or nanogels tend to require multi-step preparation methods and/or typically the use of high-energy techniques. On the contrary, this methodology provides long-term stable DTPA-SNs and NOTA-SNs particles in few minutes through a one-step protocol. Moreover, the preparation of DTPA-SNs and NOTA-SNs avoids the use of high-energy techniques and uses low-cost and conventional starting materials. In fact, compared with previously reported organic nanosystems, we describe here the simplest and easiest methodology for the gallium radiolabeling through a chelator-based strategy.3335

Figure 2 (A) Storage stability of SNs, DTPA-SNs and NOTA-SNs at 4 C measuring the evolution of the average size by DLS for one month (n=3). (B) Stability in human serum at 37 C during 72 h measured by DLS (n=3). (C) Radiochemical yield of 68Ga-DTPA-SNs and 68Ga-NOTA-SNs after incubation with the radioisotope for 30 min at 30 C (n=3). (D) Radiochemical stability of 68Ga-SNs, 68Ga-DTPA-SNs and 68Ga-NOTA-SNs after incubation with serum 4 h at 37 C (n=3).

The combination of nanomaterials with 68Ga for PET imaging has attracted a great deal of attention in recent years with several works devoted to the radiolabeling of inorganic nanoparticles.36 However, only a few studies with organic nanoparticles, specifically PEGylated DTPA and NODAGA liposomes, PAMAM dendrimer-DOTA conjugates, NODAGA and DOTA nanogels, NODAGA polymeric nanoparticles and PSMA-DOTA microemulsions, have been reported so far.33,3741

DTPA-SNs, NOTA-SNs and non-chelator SNs (control) were radiolabeled by incubation with 68Ga3+ at 30 C for 30 min, and then purified by gel filtration in PD-10 columns. Figure 2C reveals that DTPA-SNs and NOTA-SNs were efficiently labeled with 68Ga, reaching RCY of 82 4% for DTPA-SNs and 92 2% for NOTA-SNs. Differences in RCY might be related to some release of 68Ga-DTPA-PE from the nanoemulsions in the purification process and/or to a better incorporation of the NOTA-SA derivative with SNs. With respect to the control formulation, nonspecific radiolabeling (RCY, 30 12%) was observed. This might be due to some entrapment of the radioisotope into the lipid membrane of the nanoemulsions mediated by electrostatic interactions. Radiochemical yields were in line with other works in the field, such as liposomes, nanogels, biopolymer nanoparticles and microemulsions.33,3941 The radiochemical purity was evaluated by ITLC using a sodium citrate solution as mobile phase. Under these conditions, free 68Ga showed a retention factor of 0.75 (Figure S5A, Supporting Information). In case of 68Ga-nanoemulsions, we could not detect the presence of free 68Ga, showing an RCP higher than 99% (Figure S5B, Supporting Information).

The RCS of the nanoemulsions upon incubation with serum at 37 C was also determined (Figure 2D). As expected, radiolabeled control SNs (without a chelator) were not able to retain the gallium. In the case of DTPA-SNs, only 50 3% of 68Ga were retained, while NOTA-SNs showed the highest stability, retaining 90 2% of activity, again in line with cyclic chelators as NODAGA or DOTA.37,39,40,42 On the other hand, although DTPA is commonly used to form complexes with gallium and other radioisotopes, the formation of less stable complexes can be a consequence of its acyclic structure.43 This was confirmed after intravenous injection of DTPA-SNs and NOTA-SNs (12 MBq, n = 5) to healthy mice. 3D PET/CT images were acquired 2 h post-administration and showed major accumulation in the reticuloendothelial system (RES) organs and heart (Figure 3A). As expected, DTPA-SNs showed a higher circulation in the bloodstream due to the premature release of the radionuclide from the nanoemulsion. Ex vivo biodistribution results were conducted 4 h post-injection (Figure 3B) and corroborate major liver and spleen accumulation. This is in concordance with the in vivo pattern observed for most of the nanoparticles, especially lipid nanoparticles, such as liposomes and nanoemulsions.19,24,37,44 Remarkably, NOTA-SNs have a relatively long circulation time, showing a 10% of the injected dose in the bloodstream 4 h after intravenous injection. This in vivo pattern differs from other 68Ga-labeled emulsions recently reported, with a shorter circulation half-life, mainly due to differences in size and composition.41 These results indicate that the pharmacokinetics of NOTA-SNs can be better studied after the radiolabeling with longer half-life radioisotopes, such as 67Ga. Nevertheless, NOTA-SNs could be surface decorated with specific biomolecules in order to reduce their circulation time and to perform suitable probes for targeted 68Ga molecular imaging.45

Figure 3 (A) Representative PET/CT whole-body coronal images of 68Ga-DTPA-SNs and 68Ga-NOTA-SNs biodistribution in healthy mice 2 h after intravenous injection (n=5). (B) Ex vivo biodistribution of both radiolabeled nanoemulsions 4 h post-injection (n=5). **(p0.01), ***(p0.001) was considered statistically significant.

67Ga, compared with 68Ga (t = 68 minutes), allowed a long-term biodistribution study of NOTA-SNs by SPECT imaging. For NOTA-SNs radiolabeling, the clinical formulation 67Ga-citrate was initially converted into the chloride form (GaCl3) as previously described.25 Briefly, 67Ga-citrate was trapped in a silica cartridge, washed with distilled water and finally eluted with HCl 0.1 M, rendering a 90% yield. To ensure a successful radiolabeling and taking into account that with the half-life of 67Ga there are no strong limitations for increasing the incubation time, we optimized the process (the solution was incubated with NOTA-SNs for 1 h at 37 C). Then, the free radionuclide was removed by filtration in PD-10 columns, obtaining a 80 2% of RCY (Figure 4A, before incubation with serum), between 10% and 20% higher than polymer and protein-based nanoparticles previously reported.34,46 The measured RCP was 98.9%, conducted by ITLC (Table S2, Supporting Information). In addition, RCS studies proved that the labeling was highly stable in serum over 72 h (Figure 4A). Then, due to the longer half-time of this radioisotope, SPECT studies were designed to evaluate the pharmacokinetics of NOTA-SNs at prolonged time periods. In vivo SPECT images were acquired 24, 48 and 72 h after intravenous injection of the radiolabeled NOTA-SNs. In parallel, we evaluated the in vivo uptake of the free radioisotope as a control. Animals injected with free 67Ga (control) showed uptake in the bloodstream, liver, lacrimal and salivary glands (Figure 4B), according previous reports.34,47 We can also observe free 67Ga in the bladder and kidneys, a consequence of renal clearance. In comparison, NOTA-SNs showed similar biodistribution than observed in PET/CT images, with main accumulation in liver and RES organs, and its intensity decreases over time. After 72 h, we measured the ex vivo biodistribution and observed that the radioactivity remained only in liver and spleen with less than 5% ID/g in both organs (Figure S6, Supporting Information). This could be related to the biodegradation of the particles and/or their excretion through the urine, in line with our previous report in which we described cationic fluorine-labeled nanoemulsions.24 However, further studies must be carried out to determine the excretion routes of NOTA-SNs.

Figure 4 (A) Radiochemical yield of 67Ga-NOTA-SNs (control) and radiochemical stability after incubation with serum 37 C at different points (0, 24, 48 and 72 h, n=3). (B) Whole-body SPECT images showing the biodistribution of 67Ga-NOTA-SNs compared with free 67Ga during 72 h (n=5).

We finally investigated the possibility of surface-modification of NOTA-SNs without interfering with the radiolabeling procedure, to determine if it is possible to modulate the in vivo behavior.48 Among the different strategies for surface modification that have been reported to date, PEGylation is the most established approach.49 We coated NOTA-SNs with PEG (NOTA-PEG-SNs), using for this purpose a lipid-PEG derivative. However, it is well known that PEGylation might also lead to relevant drawbacks, such as the development of an immunological response, antibody generation and toxic side effects caused by the oxidative side products.50 Bearing in mind these limitations, we have also investigated the in vivo effect of an alternative coating, hyaluronic acid (NOTA-HA-SNs). HA is a biocompatible polysaccharide widely used in biomedical research that has been reported to increase the circulating time of lipid nanoparticles.51,52 Surface-modified nanoemulsions (NOTA-PEG-SNs and NOTA-HA-SNs) presented a similar size than the reference formulation (NOTA-SNs) (Figure 5A). With respect to the zeta potential, relevant modifications were only noticed in the case of NOTA-HA-SNs, which rendered more negative values (Figure 5B). Radiolabeling with 67Ga was successfully done, leading to RCY of 80 8% and 76 1% for NOTA-HA-SNs and NOTA-PEG-SNs, respectively (Figure 5C), indicating that the coatings do not significantly interfere in the interaction between the chelator and the radioisotope, and that coated NOTA-HA-SNs and NOTA-PEG-SNs could be tracked by SPECT in a comparable fashion to the reference formulation (NOTA-SNs) (Figure S7, Supporting Information).

Figure 5 (A) NOTA-HA-SNs and NOTA-PEG-SNs hydrodynamic size distribution measured by DLS (171 5 nm and 138 8 nm, respectively, results are expressed as mean standard deviation, n=3), (B) Zeta potential of NOTA-HA-SNs and NOTA-PEG-SNs measured by LDA ( 64 2 mV and 50 1 mV respectively, results are expressed as mean standard deviation, n=3). (C) Radiochemical yield of 67Ga-NOTA-HA-SNs and 67Ga-NOTA-PEG-SNs (n=3). (D) Quantitative analysis expressed as heart to liver ratio showing the differences in biodistribution between intravenously injected 67Ga-nanoemulsions.

A dynamic SPECT study was then carried out, and the tracer uptake ratio between heart and liver was calculated to evaluate the circulation/elimination pharmacokinetic profile. As shown in Figures 5E, 67Ga-PEG-NOTA-SNs showed significantly higher circulation in the bloodstream in line with previous reports referring to PEGylated nanoemulsions.53 Noteworthy, the PEGylation effect is observed even at a very low density (3 mol%), in concordance to other reports of nanoemulsions in which PEG densities vary between 0.5 and 50 mol% with respect to the total amount of surfactant.53,54 In the case of NOTA-HA-SNs, the results were comparable to the reference formulation of NOTA-SNs, a fact that could be explained by the influence of the HA molecular weight and density of the coating. These factors will therefore need further optimization and will be the subject of future work intended for the development of applications in cancer nanotheranostics where HA coating could be particularly relevant to improve accumulation in the tumor.55,56 Altogether, these results confirm that it is possible to modulate the composition and in vivo behavior of NOTA-SNs, to open up their application in specific indications in the biomedical field.

We described here a simple and highly efficient preparation method for chelator-functionalized biocompatible SNs and the subsequent radiolabeling with 68Ga and 67Ga. The radiolabeled formulations showed great radiochemical properties for in vivo applications and were efficiently followed-up by PET and SPECT imaging. Importantly, we have also proved that the biodistribution of the SNs can be modulated by modifying the surface properties. The capacity to modulate the radiolabeling, modality of imaging and tracking period, as well as the biodistribution properties, highlight the interest of SNs, which have the potential to be easily adapted to the requirements of different and specific biomedical applications. In summary, we believe that SNs have the potential for the development of advanced probes for nuclear imaging and nanotheranostics. In particular, future experiments could involve the evaluation of 68/67Ga-SNs in tumor bearing animal models to further determine the real potential of this formulation in cancer nanotheranostics, taking into account the anticancer properties of 67Ga.

All data generated or analyzed during this study are included in this published article and its Supporting Information file.

Animal experiments were conducted according to the ethical and animal welfare committee at CNIC and the Spanish and EU legislation. Experimental protocols were approved by Madrid regional government (PROEX16/277).

All authors contributed to data analysis, drafting or revising the article, have agreed on the journal to which the article will be submitted, gave final approval for the version to be published, and agreed to be accountable for all aspects of the work.

Authors thank the financial support given by Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund (FEDER) (PI15/00828, PI18/00176 and DTS18/00133), by ERA-NET EURONANOMED III project METASTARG (AC18/00045) and by Asociacin Espaola Contra el Cncer (AECC, IDEAS18153DELA). The first author also acknowledges the financial support from Axencia Galega de Innovacin (GAIN) and Xunta de Galicia (IN848C_20170721_7) and ISCII (FI19/00206).

The author reports no conflicts of interest in this work.

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Biodistribution of sphingolipid nanoemulsions with 68Ga | IJN - Dove Medical Press

COLUMBUS, Ohio, July 20, 2021 /PRNewswire/ --Matrix Meats, Inc., the leading developer of nanofiber scaffolds manufactured to support the production of clean cultivated meat products,is entering a new phase of growth with the addition of five new team members. The hiring surge marks an important milestone for Matrix Meats, which achieved record success in its oversubscribed seed stage round late last year.

The new employees are spread across various company divisions, but each play a critical role in advancing Matrix Meats' innovative manufacturing and strategic partnership goals.

Devan Ohst is heading Matrix Meats' laboratory as the new Director of Research and Development. Transitioning from his background in regenerative medicine and medical devices, he boasts an impressive rsum, with over eight years in advanced material engineering. Ohst will oversee Matrix Meats' technical operations, research activity, and act as a liaison for the company's partners.

Matrix Meats also welcomed Nichole Brown as the company's inaugural Director of Operations. Brown will be spearheading the company's growth strategy and structure as the cultivated meat sector continues to soar. Following 12 years in food and restaurant management, Brown has worked with both international franchises, such as Taco Bell, as well as burgeoning startups. She is a crucial component to furthering Matrix Meats' leadership in groundbreaking, ethical meat production, which is already solidified by 14 active development partnerships with cultivated meat producers in seven countries.

Kevin Doand Mitch Kahn join Matrix Meats as Project Engineers. Do brings a precise eye for production to the company, using his passion for sustainability and climate change to scale up Matrix Meats' scaffolding development. He was an obvious choice for Matrix Meats following his five-month internship under the company's Director of Research and Development, Devan Ohst. Bringing six years of hands-on medical and laboratory experience to Matrix Meats, Kahn harnesses his background in biomedical engineering to put nanofiber scaffolds through intensive, iterative processes. His expertise in materials science and polymer replacement have equipped him with the skills to assess viable candidates for scaffold creation.

Hardy Castada, PhDwas inspired by the burgeoning plant-based meat movement when he joined Matrix Meats as a Senior Food Scientist. Castada will be utilizing his experience in food science and chemistry to facilitate high quality, safety, and nutrition standards. In his previous role at the Ohio State University Department of Food Science and Technology, Castada spearheaded several innovative food and biomedical projects that were widely published in peer-reviewed journals.

"As with any startup, Matrix Meats has been operating with a lean team since our launch in 2019. We were overwhelmed by the fervent interest and positive attention received during last year's seed stage round, and it gave us the confidence to grow our team and support our position in cultivated meat production," said Matrix Meats CEO Eric Jenkusky. "In the short time they've been on board, Devan, Nichole, Kevin, Mitch and Hardy have already been tremendous assets to the company and ardent believers in the global impact of cellular agriculture."

In conjunction with Matrix Meat's numerous team additions, the company also promoted Zac Graber from Operations Manager to Director of Business Development. Graber has been with Matrix Meats since the beginning of 2020, becoming the third employee to join the rapidly growing startup.

Matrix Meats completed its seed stage round in December of 2020 and achieved oversubscribed investment from companies such as Unovis Asset Management, CPT Capital, Siddhi Capital and Clear Current Capital.

About Matrix Meats, Inc.

Matrix Meats, Inc., based in Columbus, Ohio, is a designer and manufacturer of the foremost nano-fiber scaffolds to enable the production of clean, healthy, and environmentally friendly cultured meat to ethically feed the world. Further information: http://www.matrixmeats.com

Press ContactErin MandzikJConnelly862-246-9911[emailprotected]

SOURCE Matrix Meats

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Matrix Meats Adds to Team as Interest in Cultivated Meat Grows - PRNewswire