Non-invasive transcutaneous influenza immunization using vaccine-loaded vaterite particles.

Transcutaneous immunization receives much attention due to the recognition of a complex network of immunoregulatory cells in various layers of the skin. The elaboration of non-invasive needle-free approaches towards antigen delivery holds especially great potential here while searching for a hygienically optimal vaccination strategy. Here, we report on a novel protocol for transfollicular immunization aiming at delivery of an inactivated influenza vaccine to perifollicular antigen presenting cells without disrupting the stratum corneum integrity. Porous calcium carbonate (vaterite) submicron carriers and sonophoresis were utilized for this purpose. Transportation of the vaccine-loaded particles into hair follicles of mice was assessed in vivo via optical coherence tomography monitoring. The effectiveness of the designed immunization protocol was further demonstrated in an animal model by means of micro-neutralization and enzyme-linked immunosorbent assays. The titers of secreted virus-specific IgGs were compared to those obtained in response to intramuscular immunization using conventional influenza vaccine formulation demonstrating no statistically significant differences in antibody levels between the groups. The findings of our pilot study render the intra-follicular delivery of the inactivated influenza vaccine by means of vaterite carriers a promising alternative to invasive immunization.

Immersion optical clearing of adipose tissue in rats: ex vivo and in vivo studies.

Optical clearing (OC) of adipose tissue has not been studied enough, although it can be promising in medical applications, including surgery and cosmetology, for example, to visualize blood vessels or increase the permeability of tissues to laser beams. The main objective of this work is to develop technology for OC of abdominal adipose tissue in vivo using hyperosmotic optical clearing agents (OCAs). The maximum OC effect (77%) was observed for ex vivo rat adipose tissue samples exposed to OCA on fructose basis for 90 minutes. For in vivo studies, the maximum effect of OC (65%) was observed when using OCA based on diatrizoic acid and dimethylsulfoxide for 120 minutes. Histological analysis showed that in vivo application of OCAs may induce a limited local necrosis of fat cells. The efficiency of OC correlated with local tissue damage through cell necrosis due to accompanied cell lipolysis.

Boosting transfection efficiency: A systematic study using layer-by-layer based gene delivery platform.

Nowadays, the nanoparticle-based delivery approach is becoming more and more attractive in gene therapy due to its low toxicity and immunogenicity, sufficient packaging capacity, targeting, and straightforward, low-cost, large-scale good manufacturing practice (GMP) production. A number of research works focusing on multilayer structures have explored different factors and parameters that can affect the delivery efficiency of pDNA. However, there are no systematic studies on the performance of these structures for enhanced gene delivery regarding the gene loading methods, the use of additional organic components and cell/particle incubation conditions. Here, we conducted a detailed analysis of different parameters such as (i) strategy for loading pDNA into carriers, (ii) incorporating both pDNA and organic additives within one carrier and (iii) variation of cell/particle incubation conditions, to evaluate their influence on the efficiency of pDNA delivery with multilayer structures consisting of inorganic cores and polymer layers. Our results reveal that an appropriate combination of all these parameters leads to the development of optimized protocols for high transfection efficiency, compared to the non-optimized process (> 70% vs. < 7%), and shows a good safety profile. In conclusion, we provide the proof-of-principle that these multilayer structures with the developed parameters are a promising non-viral platform for an efficient delivery of nucleic acids.

Calcium Carbonate Core-Shell Particles for Incorporation of 225Ac and Their Application in Local α-Radionuclide Therapy.

Actinium-225 (225Ac) radiolabeled submicrometric core-shell particles (SPs) made of calcium carbonate (CaCO3) coated with biocompatible polymers [tannic acid-human serum albumin (TA/HSA)] have been developed to improve the efficiency of local α-radionuclide therapy in melanoma models (B16-F10 tumor-bearing mice). The developed 225Ac-SPs possess radiochemical stability and demonstrate effective retention of 225Ac and its daughter isotopes. The SPs have been additionally labeled with zirconium-89 (89Zr) to perform the biodistribution studies using positron emission tomography-computerized tomography (PET/CT) imaging for 14 days after intratumoral injection. According to the PET/CT analysis, a significant accumulation of 89Zr-SPs in the tumor area is revealed for the whole investigation period, which correlates with the direct radiometry analysis after intratumoral administration of 225Ac-SPs. The histological analysis has revealed no abnormal changes in healthy tissue organs after treatment with 225Ac-SPs (e.g., no acute pathologic findings are detected in the liver and kidneys). At the same time, the inhibition of tumor growth has been observed as compared with control samples [nonradiolabeled SPs and phosphate-buffered saline (PBS)]. The treatment of mice with 225Ac-SPs has resulted in prolonged survival compared to the control samples. Thus, our study validates the application of 225Ac-doped core-shell submicron CaCO3 particles for local α-radionuclide therapy.

Layer-by-Layer technique as a versatile tool for gene delivery applications.

Introduction: Gene therapy is a breakthrough medical field which focuses on the therapeutic delivery of recombinant nucleic acids in order to treat or prevent a broad spectrum of diseases. However, a number of important obstacles remain before its wide introduction into clinical practice can be envisaged. One of the biggest bottlenecks is the lack of efficient and safe delivery technologies, particularly, for in vivo distribution. Above and beyond standard requirements for carriers, the delivery systems for gene therapy ideally use a hit-and-run principle (to minimize off-target effect and display of immunogenic moieties). None of the currently used viral vectors fulfills all of these requirements. Therefore, the growing variety of non-viral delivery platforms represents a promising alternative.Areas covered: This review summarizes the Layer-by-Layer (LbL) approaches that can be effectively used for the gene delivery, considering various examples with the transfer of pDNA, mRNA, siRNA as well as genome-editing tools. Ex vivo gene modification of clinically relevant cells and clinical aspects for possible application of LbL systems in gene therapy are also underlined.Expert opinion: The LbL technique provides broad opportunities for the delivery of genetic material for various purposes and offers promise for future clinical application in gene therapy.

An investigation of calcium carbonate core-shell particles for incorporation of 225Ac and sequester of daughter radionuclides: in vitro and in vivo studies.

Alpha therapy provides an outstanding prospect in the treatment of recalcitrant and micrometastatic cancers. However, side effects on the normal tissues and organs (especially, kidneys) due to the release of daughter isotopes from α-emitters remain a bottleneck. In this work, calcium carbonate core-shell particles of different sizes were considered as isotope carriers for encapsulation of 225Ac (highly powerful alpha-emitter that generates 4 net alpha particle isotopes in a short decay chain) in order to achieve in vitro and in vivo retention of 225Ac and its daughter isotopes. According to the in vitro studies, the developed calcium carbonate core-shell particles were able to retain 225Ac and its daughter isotopes (221Fr and 213Bi) exhibited good stability in biological media and dose-dependent biocompatibility (over 30 d). The SPECT imaging demonstrated the size-dependent distribution of 225Ac-doped core-shell particles. Further, in vivo studies confirmed the high retention efficiency of calcium carbonate core-shell particles, which was demonstrated in normal Wistar rats (up to 10 d). Interestingly, the radioactivity accumulation in kidney and urine was significantly less for encapsulated 225Ac than in case of non-encapsulated form of 225Ac (225Ac conjugated with albumin), indicating the absence of radioisotope leakage from the developed particles. Thus, our study validates the application of 225Ac-doped core-shell particles to sequester α-emitter (225Ac) and its decay products in order to reduce their systemic toxicity during alpha therapy.

A highly efficient and safe gene delivery platform based on polyelectrolyte core-shell nanoparticles for hard-to-transfect clinically relevant cell types.

While DNA and messenger RNA (mRNA) based therapies are currently changing the biomedical field, the delivery of genetic materials remains the key problem preventing the wide introduction of these methods into clinical practice. Therefore, the creation of new methods for intracellular gene delivery, particularly to hard-to-transfect, clinically relevant cell populations is a pressing issue. Here, we report on the design of a novel approach to format 50-150 nm calcium carbonate particles in the vaterite state and using them as a template for polymeric core-shell nanoparticles. We apply such core-shell nanoparticles as safe and efficient carriers for mRNA and pDNA. We prove that such nanocarriers are actively internalized by up to 99% of primary T-lymphocytes and exert minimal toxicity with the viability of >90%. We demonstrate that these nanocarriers mediate more efficient transfection compared with the standard electroporation method (90% vs. 51% for mRNA and 62% vs. 39% for plasmid DNA) in primary human T-lymphocytes as a model of the hard to transfect type that is widely used in gene and cell therapy approaches. Importantly, these polymeric nanocarriers can be used in serum containing basic culture medium without special conditions and equipment, thus having potential for being introduced in clinical development. As a result, we have provided proof-of-principle that our nanosized containers represent a promising universal non-viral platform for efficient and safe gene delivery.

Overcoming the delivery problem for therapeutic genome editing: Current status and perspective of non-viral methods.

Besides its broad application in research and biotechnology, genome editing (GE) has great potential for clinical gene therapy, but delivery of GE tools remains a bottleneck. Whereas significant progress has been made in ex vivo GE delivery (e.g., by electroporation), establishment of efficient and safe in vivo delivery systems is still a challenge. Above and beyond standard vector requirements (safety, minimal/absent toxicity and immunogenicity, sufficient packaging capacity, targeting, straight and low-cost large-scale good manufacturing practice (GMP) production), GE delivery systems ideally use a hit-and-run principle to minimize off-targets as well as display of immunogenic peptides. Since currently used viral vectors do not fulfil all of these requirements, the broad variety of non-viral delivery platforms represents a promising alternative. This review provides a comprehensive analysis of the most relevant aspects of non-viral physical and chemical delivery methods in non-clinical studies and clinical trials, ranging from classic electroporation to advanced drug carriers that can transport GE tools in form of plasmid DNAs (pDNAs), mRNAs, and ribonucleoproteins (RNPs). For comparison, advantages and shortcomings of viral delivery systems are shortly discussed. In summary, we review various delivery approaches and discuss the future perspectives to use drug carriers for in vivo GE in clinical trials.

Radiolabeling Strategies of Micron- and Submicron-Sized Core-Shell Carriers for In Vivo Studies.

Core-shell particles made of calcium carbonate and coated with biocompatible polymers using the Layer-by-Layer technique can be considered as a unique drug-delivery platform that enables us to load different therapeutic compounds, exhibits a high biocompatibility, and can integrate several stimuli-responsive mechanisms for drug release. However, before implementation for diagnostic or therapeutic purposes, such core-shell particles require a comprehensive in vivo evaluation in terms of physicochemical and pharmacokinetic properties. Positron emission tomography (PET) is an advanced imaging technique for the evaluation of in vivo biodistribution of drug carriers; nevertheless, an incorporation of positron emitters in these carriers is needed. Here, for the first time, we demonstrate the radiolabeling approaches of calcium carbonate core-shell particles with different sizes (CaCO3 micron-sized core-shell particles (MicCSPs) and CaCO3 submicron-sized core-shell particles (SubCSPs)) to precisely determine their in vivo biodistribution after intravenous administration in rats. For this, several methods of radiolabeling have been developed, where the positron emitter (68Ga) was incorporated into the particle's core (co-precipitation approach) or onto the surface of the shell (either layer coating or adsorption approaches). According to the obtained data, radiochemical bounding and stability of 68Ga strongly depend on the used radiolabeling approach, and the co-precipitation method has shown the best radiochemical stability in human serum (96-98.5% for both types of core-shell particles). Finally, we demonstrate the size-dependent effect of core-shell particles' distribution on the specific organ uptake, using a combination of imaging techniques, PET, and computerized tomography (CT), as well as radiometry of separate organs. Thus, our findings open up new perspectives of CaCO3-radiolabeled core-shell particles for their further implementation into clinical practice.

Endovascular addressing improves the effectiveness of magnetic targeting of drug carrier. Comparison with the conventional administration method.

Many nanomedicine approaches are struggling to reach high enough effectiveness in delivery if applied systemically. The perspective is sought to explore the clinical practices currently used for localized treatment. In this study, we combine in vivo targeting of carriers sensitive to the external magnetic field with clinically used endovascular delivery to specific site. Fluorescent micron-size capsules made of biodegradable polymers and containing magnetite nanoparticles incorporated in the capsule wall were explored in vivo using Near-Infrared Fluorescence Live Imaging for Real-Time. Comparison of systemic (intravenous) and directed (intra-arterial) administration of the magnetic microcapsule targeting in the hindpaw vessels demonstrated that using femoral artery injection in combination with magnetic field exposure is 4 times more efficient than tail vein injection. Thus, endovascular targeting significantly improves the capabilities of nanoengineered drug delivery systems reducing the systemic side effects of therapy.

Development of Optimized Strategies for Growth Factor Incorporation onto Electrospun Fibrous Scaffolds To Promote Prolonged Release.

Growth factor incorporation in biomedical constructs for their local delivery enables specific pharmacological effects such as the induction of cell growth and differentiation. This has enabled a promising way to improve the tissue regeneration process. However, it remains challenging to identify an appropriate approach that provides effective growth factor loading into biomedical constructs with their following release kinetics in a prolonged manner. In the present work, we performed a systematic study, which explores the optimal strategy of growth factor incorporation into sub-micrometric-sized CaCO3 core-shell particles (CSPs) and hollow silica particles (SiPs). These carriers were immobilized onto the surface of the polymer scaffolds based on polyhydroxybutyrate (PHB) with and without reduced graphene oxide (rGO) in its structure to examine the functionality of incorporated growth factors. Bone morphogenetic protein-2 (BMP-2) and ErythroPOietin (EPO) as growth factor models were included into CSPs and SiPs using different entrapping strategies, namely, physical adsorption, coprecipitation technique, and freezing-induced loading method. It was shown that the loading efficiency, release characteristics, and bioactivity of incorporated growth factors strongly depend on the chosen strategy of their incorporation into delivery systems. Overall, we demonstrated that the combination of scaffolds with drug delivery systems containing growth factors has great potential in the field of tissue regeneration compared with individual scaffolds.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

Impact of Nanocapsules on Red Blood Cells Interplay Jointly Assessed by Optical Tweezers and Microscopy.

In the framework of novel medical paradigm the red blood cells (RBCs) have a great potential to be used as drug delivery carriers. This approach requires an ultimate understanding of the peculiarities of mutual interaction of RBC influenced by nano-materials composed the drugs. Optical tweezers (OT) is widely used to explore mechanisms of cells' interaction with the ability to trap non-invasively, manipulate and displace living cells with a notably high accuracy. In the current study, the mutual interaction of RBC with polymeric nano-capsules (NCs) is investigated utilizing a two-channel OT system. The obtained results suggest that, in the presence of NCs, the RBC aggregation in plasma satisfies the 'cross-bridges' model. Complementarily, the allocation of NCs on the RBC membrane was observed by scanning electron microscopy (SEM), while for assessment of NCs-induced morphological changes the tests with the human mesenchymal stem cells (hMSC) was performed. The combined application of OT and advanced microscopy approaches brings new insights into the conception of direct observation of cells interaction influenced by NCs for the estimation of possible cytotoxic effects.

Safe and Effective Delivery of Antitumor Drug Using Mesenchymal Stem Cells Impregnated with Submicron Carriers.

An important area in modern malignant tumor therapy is the optimization of antitumor drugs pharmacokinetics. The use of some antitumor drugs is limited in clinical practice due to their high toxicity. Therefore, the strategy for optimizing the drug pharmacokinetics focuses on the generation of high local concentrations of these drugs in the tumor area with minimal systemic and tissue-specific toxicity. This can be achieved by encapsulation of highly toxic antitumor drug (vincristine (VCR) that is 20-50 times more toxic than widely used the antitumor drug doxorubicin) into nano- and microcarriers with their further association into therapeutically relevant cells that possess the ability to migrate to sites of tumor. Here, we fundamentally examine the effect of drug carrier size on the behavior of human mesenchymal stem cells (hMSCs), including internalization efficiency, cytotoxicity, cell movement, to optimize the conditions for the development of carrier-hMSCs drug delivery platform. Using the malignant tumors derived from patients, we evaluated the capability of hMSCs associated with VCR-loaded carriers to target tumors using a three-dimensional spheroid model in collagen gel. Compared to free VCR, the developed hMSC-based drug delivery platform showed enhanced antitumor activity regarding those tumors that express CXCL12 (stromal cell-derived factor-1 (SDF-1)) gene, inducing directed migration of hMSCs via CXCL12 (SDF-1)/CXCR4 pathway. These results show that the combination of encapsulated antitumor drugs and hMSCs, which possess the properties of active migration into tumors, is therapeutically beneficial and demonstrated high efficiency and low systematic toxicity, revealing novel strategies for chemotherapy in the future.

Inhibition of influenza A virus by mixed siRNAs, targeting the PA, NP, and NS genes, delivered by hybrid microcarriers.

In the present study, a highly effective carrier system has been developed for the delivery of antiviral siRNA mixtures. The developed hybrid microcarriers, made of biodegradable polymers and SiO2 nanostructures, more efficiently mediate cellular uptake of siRNA than commercially available liposome-based reagents and polyethyleneimine (PEI); they also demonstrate low in vitro toxicity and protection of siRNA from RNase degradation. A series of siRNA designs (targeting the most conserved regions of three influenza A virus (IAV) genes: NP, NS, and PA) were screened in vitro using RT-qPCR, ELISA analysis, and hemagglutination assay. Based on the results of screening, the three most effective siRNAs (PA-1630, NP-717, and NS-777) were selected for in situ encapsulation into hybrid microcarriers. It was revealed that pre-treatment of cells with a mixture of PA-1630, NP-717, and NS-777 siRNAs, delivered by hybrid microcarriers, provided stronger inhibition of viral M1 mRNA expression and control of NP protein level, after viral infection, than single pre-treatment by any of three encapsulated siRNAs used in the study. Moreover, the effective inhibition of replication in several IAV subtypes (H1N1, H1N1pdm, H5N2, and H7N9) using a cocktail of the three selected siRNAs, delivered by our hybrid capsules to the cells, was achieved. In conclusion, we have developed a proof-of-principle which shows that our hybrid microcarrier technology (utilizing a therapeutic siRNA cocktail) may represent a promising approach in anti-influenza therapy.