CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.

In this study, bone scaffolds composed of polycaprolactone (PCL), piezoelectric poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and silicate containing hydroxyapatite (PHBV-SiHA) were successfully fabricated by a conventional electrospinning process. The morphological, chemical, wetting and biological properties of the scaffolds were examined. All fabricated scaffolds are composed of randomly oriented fibres with diameters from 800nm to 12μm. Fibre size increased with the addition of SiHA to PHBV scaffolds. Moreover, fibre surface roughness in the case of hybrid scaffolds was also increased. XRD, FTIR and Raman spectroscopy were used to analyse the chemical composition of the scaffolds, and contact angle measurements were performed to reveal the wetting behaviour of the synthesized materials. To determine the influence of the piezoelectric nature of PHBV in combination with SiHA nanoparticles on cell attachment and proliferation, PCL (non-piezoelectric), pure PHBV, and PHBV-SiHA scaffolds were seeded with human mesenchymal stem cells (hMSCs). In vitro study on hMSC adhesion, viability, spreading and osteogenic differentiation showed that the PHBV-SiHA scaffolds had the largest adhesion and differentiation abilities compared with other scaffolds. Moreover, the piezoelectric PHBV scaffolds have demonstrated better calcium deposition potential compared with non-piezoelectric PCL. The results of the study revealed pronounced advantages of hybrid PHBV-SiHA scaffolds to be used in bone tissue engineering.

Carefully designed micro-and nanocarriers can provide significant advantages over conventional macroscopic counterparts in biomedical applications. The set of requirements including a high loading capacity, triggered release mechanisms, biocompatibility, and biodegradability should be considered for the successful delivery realization. Porous calcium carbonate (CaCO3) is one of the most promising platforms, which can encompass all the beforehand mentioned requirements. Here, we study both the particles formation and biological applicability of CaCO3. In particular, anisotropic differently shaped CaCO3 particles were synthesized using green sustainable approach based on co-precipitation of calcium chloride and sodium carbonate/bicarbonate at different ratios in the presence of organic additives. The impact of salts concentrations, reaction time, as well as organic additives was systematically researched to achieve controllable and reliable design of CaCO3 particles. It has been demonstrated that the crystallinity (vaterite or calcite phase) of particles depends on the initial salts' concentrations. The loading capacity of prepared CaCO3 particles is determined by their surface properties such as specific surface area, pore size and zetapotential. Differently shaped CaCO3 particles (spheroids, ellipsoids, toroids) were used to evaluate their uptake efficiency on the example of C6 glioma cells. The results show that the ellipsoidal particles possess a higher probability for internalization by cancer cells. All tested particles were also found to have a good biocompatibility. The capability to design physicochemical properties of CaCO3 particles has a significant impact on drug delivery applications, since the particles geometry substantially affects cell behavior (internalization, toxicity) and allows outperforming standard spherical counterparts.

The incorporation of bioactive compounds onto polymer fibrous scaffolds with further control of drug release kinetics is essential to improve the functionality of scaffolds for personalized drug therapy and regenerative medicine. In this study, polymer and hybrid microcapsules were prepared and used as drug carriers, which are further deposited onto polymer microfiber scaffolds [polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB), and PHB doping with the conductive polyaniline (PANi) of 2 wt % (PHB-PANi)]. The number of immobilized microcapsules decreased with increase in their ζ-potential due to electrostatic repulsion with the negatively charged fiber surface, depending on the polymer used for the scaffold's fabrication. Additionally, the immobilization of the capsules in dynamic mechanical conditions at a frequency of 10 Hz resulted in an increase in the number of the capsules on the fibers with increase in the scaffold piezoelectric response in the order PCL < PHB < PHB-PANi, depending on the chemical composition of the capsules. The immobilization of microcapsules loaded with different bioactive molecules onto the scaffold surface enabled multimodal triggering by physical (ultrasound, laser radiation) and biological (enzymatic treatment) stimuli, providing controllable release of the cargo from scaffolds. Importantly, the microcapsules immobilized onto the surface of the scaffolds did not influence the cell growth, viability, and cell proliferation on the scaffolds. Moreover, the attachment of human mesenchymal stem cells (hMSCs) on the scaffolds revealed that the PHB and PHB-PANi scaffolds promoted adhesion of hMSCs compared to that of the PCL scaffolds. Two bioactive compounds, antibiotic ceftriaxone sodium (CS) and osteogenic factor dexamethasone (DEXA), were chosen to load the microcapsules and demonstrate the antimicrobial properties and osteogenesis of the scaffolds. The modified scaffolds had prolonged release of CS or DEXA, which provided an improved antimicrobial effect, as well as enhanced osteogenic differentiation and mineralization of the scaffolds modified with capsules compared to that of individual scaffolds soaked in CS solution or incubated in an osteogenic medium. Thus, the immobilization of microcapsules provides a simple, convenient way to incorporate bioactive compounds onto polymer scaffolds, which makes these multimodal materials suitable for personalized drug therapy and bone tissue engineering.

The implementation of RNAi technology into the clinical practice has been significantly postponing due to the issues regarding to the delivery of naked siRNA predominantly to target cells. Here we report the approach to enhance the efficiency of siRNA delivery by encapsulating the siRNA into new carrier systems which are obtained via the combination of widely used layer-by-layer technique and in situ modification by sol-gel chemistry. We used three types of siRNAs (NP-717, NP-1155 and NP-1496) in encapsulated form as new therapeutic agents against H1N1 influenza virus infection. By employing the hybrid microcontainers for the siRNA encapsulation we demonstrate the reduction of viral nucleoprotein (NP) level and inhibition of influenza virus production in infected cell lines (MDCK and A549). The obtained hybrid carriers based on assembled biodegradable polyelectrolytes and sol-gel coating possess several advantages such as a high cell uptake efficiency, low toxicity, efficient intracellular delivery of siRNAs and the protection of siRNAs from premature degradation before reaching the target cells. These findings underpin a great potential of versatile microencapsulation technology for the development of anti-viral RNAi delivery systems against influenza virus infection.

Radiopharmaceuticals have proven to be effective agents, since they can be successfully applied for both diagnostics and therapy. Effective application of relevant radionuclides in pre-clinical and clinical studies depends on the choice of a sufficient delivery platform. Herein, we provide a comprehensive review on the most relevant aspects in radionuclide delivery using the most employed carrier systems, including, (i) monoclonal antibodies and their fragments, (ii) organic and (iii) inorganic nanoparticles, and (iv) microspheres. This review offers an extensive analysis of radionuclide delivery systems, the approaches of their modification and radiolabeling strategies with the further prospects of their implementation in multimodal imaging and disease curing. Finally, the comparative outlook on the carriers and radionuclide choice, as well as on the targeting efficiency of the developed systems is discussed.

Mesenchymal stem cells (MSCs) are widely used in cell therapy due to their convenience, multiline differentiation potential, reproducible protocols, and biological properties. The potential of MSCs to impregnate magnetic microcapsules and their possible influence on cell function and ability to response to magnetic field have been explored. Interestingly, the cells suspended in media show much higher ability in internalization of microcapsules, then MSCs adhere into the surface. There is no significant effect of microcapsules on cell toxicity compared with other cell line-capsule internalization reported in literature. Due to internalization of magnetic capsules by the cells, such cell engineering platform is responsive to external magnetic field, which allows to manipulate MSC migration. Magnetically sorted MSCs are capable to differentiation as confirmed by their conversion to adipogenic and osteogenic cells using standard protocols. There is a minor effect of capsule internalization on cell adhesion, though MSCs are still able to form spheroid made by dozen of thousand MSCs. This work demonstrates the potential of use of microcapsule impregnated MSCs to carry internalized micron-sized vesicles and being navigated with external magnetic signaling.

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.