Allocation of rhodamine-loaded nanocapsules from blood circulatory system to adjacent tissues assessed in vivo by fluorescence spectroscopy

Modern fluorescent modalities play an important role in the functional diagnostic of various physiological processes in living tissues. Utilizing the fluorescence spectroscopy approach we observe the circulation of fluorescent-labelled nanocapsules with rhodamine tetramethylrhodamine in a microcirculatory blood system. The measurements were conducted transcutaneously on the surface of healthy Wistar rat thighs in vivo. The administration of the preparation capsule suspension with a rhodamine concentration of 5 mg kg−1 of the animal weight resulted in a two-fold increase of fluorescence intensity relative to the baseline level. The dissemination of nanocapsules in the adjacent tissues via the circulatory system was observed and assessed quantitatively. The approach can be used for the transdermal assessment of rhodamine-loaded capsules 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

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

Multilayered polyelectrolyte assemblies as delivery system for biomedical applications

Abstract Gene therapy is a rapidly developing medical field, which focuses on the utilization of therapeutic delivery of recombinant nucleic acids into a patient’s cells to treat or prevent a broad spectrum of diseases. However, several important obstacles remain before its wide introduction into clinical application can be implemented. One of the biggest bottlenecks is a lack of efficient and safe delivery technologies, particularly, for in vivo distribution. Additionally, standard requirements for carriers are still an open question (safety, minimal/absent toxicity and immunogenicity, sufficient packaging capacity, targeting, straight and low-cost large-scale Good Manufacturing Practice (GMP) production). Therefore, a growing variety of non-viral delivery platforms represent a promising alternative. Nanotechnology opens new possibilities for resolving biomedical issues. Polymer and hybrid micro- and core–shell nanoparticles are currently under development as a platform for safe and efficient gene delivery. The present thesis describes the development of new safety gene delivery system based on polymer nanoparticles. The results show that nucleic acids (DNA/RNA) can be successfully imbedded into the nanoparticle structures and delivered to various types of cells. For the characterization of the biocompatibility of nanoparticles in vitro, two optical methods were considered. Compatibility with red blood cells (important for intravenous delivery) was assessed using optical tweezers. Capsule biodistribution in vivo was studied with fluorescence spectroscopy and a radiolabeling technique. The data and experience gained from this research open new prospects in the fields of delivery systems areas, gene therapy, and diagnostics in vivo and new possibilities for future clinical applications.Tiivistelmä Geeniterapia on nopeasti kehittyvä lääketieteellinen ala, joka keskittyy rekombinanttisten nukleiinihappojen terapeuttisen annon hyödyntämiseen potilaan soluihin laajan kirjon tautien hoitamiseksi tai ehkäisemiseksi. On kuitenkin olemassa useita tärkeitä esteitä, ennen kuin sen laajaa käyttöönottoa kliinisessä sovelluksessa voidaan toteuttaa. Yksi suurimmista pullonkauloista on tehokkaiden ja turvallisten jakelutekniikoiden puute etenkin in vivo -jakelussa. Myös kiistanalainen vakiovaatimukset operaattoreille ovat edelleen avoin ongelma (turvallisuus, vähäinen / puuttuva myrkyllisyys ja immunogeenisuus, riittävä pakkauskapasiteetti, kohdennus, suora ja edullinen laajamittainen GMP-tuotanto). Siksi kasvava valikoima ei-viraalisia jakelualustoja on lupaava vaihtoehto. Nanoteknologia avaa uuden mahdollisuuden ratkaista biolääketieteelliset kysymykset. Polymeerisiä ja hybridimikro- ja ydin-kuori-nanohiukkasia kehitetään parhaillaan turvallisen ja tehokkaan geeninsiirron alustana. Tässä opinnäytetyössä kuvataan polymeerisiin nanohiukkasiin perustuvan uuden turvallisuusgeenin kuljetusjärjestelmän kehittäminen. Tulokset osoittivat, että nukleiinihapot (DNA / RNA) voidaan upottaa onnistuneesti nanohiukkasten rakenteeseen ja toimittaa erityyppisiin soluihin. Nanohiukkasten biologisen yhteensopivuuden in vitro karakterisoimiseksi otettiin huomioon kaksi optista menetelmää. Yhteensopivuus punasolujen kanssa (tärkeä laskimoon annettaessa) arvioitiin optisilla pinseteillä. Kapselien biologinen jakautuminen in vivo mitattiin ja tutkittiin fluoresenssispektroskopialla ja radioleimaustekniikalla. Tästä tutkimuksesta saadut tiedot ja kokemukset avaavat uusia näkymiä jakelujärjestelmiin, geeniterapiaan ja diagnostiikkaan in vivo ja avaavat uusia mahdollisuuksia tulevassa kliinisessä sovelluksessa

Combined use of optical tweezers and scanning electron microscopy to reveal influence of nanoparticles on red blood cells interactions

Abstract As a promising drug delivery system, itself or coupled with red blood cells (RBC), nanoparticles (NP) should be studied in frames of their interaction at the cellular level. Experiments were performed on RBC in autologous blood plasma incubated with different NP — TiO₂, ZnO, nanodiamonds and polymeric nanocapsules. RBC aggregates formation in RBC suspension was observed with conventional microscopy, while quantitative interaction force measurements between individual RBC was assessed with optical tweezers. Scanning electron microscopy (SEM) imaging demonstrated NP localization and RBC membrane modifications upon binding with NP. Among tested NP, nanodiamonds caused increasing the size of aggregates in RBC suspensions, RBC interaction force increase and strong membrane surface modifications, comparing to other tested NP and control sample. Nanocapsules do not cause any adverse effects on RBC properties, confirming biocompatibility and applicability for drug delivery purposes. Optical tweezers combined with SEM imaging serves as fast informative assessment of NP effects on RBC

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

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 lipolysi