Veronika Benson

Background/Objectives: Bio-produced gold nanoparticles (AuNPs) are effective carriers of short RNAs into specialized mammalian cells. Their potential application is still limited by scarce knowledge on their uptake and intracellular fate. Gold nanoparticles that are not biologically produced (NB-AuNPs) enter specialized cells primarily via clathrin-dependent endocytosis. Unlike the NB-AuNPs, the bio AuNPs possess natural surface coatings that significantly alter the AuNPs properties. Our research aimed to reveal the cellular uptake of the AuNPs with respect to delivering a functional RNA cargo. Methods: The AuNPs were conjugated with short inhibitory RNA specific to miR 135b. Mammary cancer cells 4T1 were pretreated with inhibitors of caveolin- and clathrin-mediated endocytosis and macropinocytosis. AuNPs’ uptake, fate, and miR 135b knock-down were assessed with TEM and qPCR. Results: The AuNPs-antimiR 135b conjugates entered 4T1 cells via all the tested pathways and could be seen inside the cells in early and late endosomes as well as cytoplasm. In contrast to the clathrin-dependent pathway, the caveolae-mediated endocytosis and the macropinocytosis of the AuNPs resulted in the effective targeting and reduction of the miR 135b. Conclusions: The bio-produced AuNPs can effectively enter mammalian cells simultaneously by different endocytic pathways but the delivery of functional cargo is not achieved via the clathrin-dependent endocytosis.

Background/Objectives: Extracellular vesicles (EVs) are involved in cell-to-cell communication and delivery of signaling molecules and represent an interesting approach in targeted therapy. This project focused on EV-mediated facilitation and cell-specific delivery of effector antimiR molecules carried by biologically produced gold nanoparticles (AuNPs). Methods: First, we loaded EVs derived from cancer cells 4T1 with AuNPs-antimiR. The AuNPs were also decorated with or without transferrin (Tf) molecules. We examined parental cell-specific delivery of the AuNPs-Tf-antimiR within monocultures as well as co-cultures in vitro. Subsequently, we used autologous EVs containing AuNPs-Tf-antimiR to target tumor cells in a xenograft tumor model in vivo. Efficacy of the antimir transfer was assessed by qPCR and apoptosis assessment. Results: In vitro, EVs loaded with AuNPs-antimiR were internalized only by the parental cells and the AuNPs-antimiR transfer was successful and effective only in EVs that were decorated with Tf. We achieved effective delivery of the antimiR molecule into cancer cells in vivo, which was proved by specific silencing of the target oncogenic miRNA as well as induction of cancer cells apoptosis. Conclusions: EVs represent an interesting and potent way for targeted cargo delivery and personalized medicine. On the other hand, there are various safety and efficacy challenges that remain to be addressed.

Appropriate in vitro systems are needed to evaluate effective strategies to prevent implant‐associated infections, which remain a significant complication in today's medicine. Conventional in vitro models involving monocultures cannot fully reflect the tissue environment in vivo. Coculturing eukaryotic and prokaryotic pathogenic and commensal cells in in vitro models configured according to the needs of each tissue could better mimic complex tissue structures and actual clinical conditions. This approach may also enable better investigations of cell–cell interactions and interactions with an implant surface than culturing each cohort separately. This review aims to provide an overview of current studies and techniques that demonstrate competitive colonization between mammalian and bacterial cell populations (pathogenic and commensal), highlight the gaps in current techniques, and speculate on the future of using these complex cell systems in the field of tissue engineering and regenerative medicine.

The biocompatible nanofiber wound dressing enables the controlled release of the antisense siRNA against matrix metalloproteinase 9. In a diabetic-like murine model, the treatment resulted in a significant shortening of the wound healing time.

This study recognized biologically produced gold nanoparticles (AuNPs) as multiple cargo carriers with a perspective of drug delivery into specialized tumor cells in vivo. Paclitaxel (PTX), transferrin, and antimiR-135b were conjugated with AuNPs and their uptake by mouse tumor cells in an induced breast cancer model was investigated. Each of the above-mentioned molecules was conjugated to the AuNPs separately as well as simultaneously, loading efficiency of each cargo was assessed, and performance of the final product (FP) was judged. After tumor induction in BALB/c mice, sub-IC₅₀ doses of FP as well as control AuNPs, PTX, and phosphate buffered saline were administered in vivo. Round AuNPs were prepared using Fusarium oxysporum and exhibited a size of 13 ± 1.3 nm and a zeta potential of -35.8 ± 1.3 mV. The cytotoxicity of individual conjugates and FP were tested by MTT assay in breast tumor cells 4T1 and nontumor fibroblasts NIH/3T3 cells. The conjugation of individual molecules with AuNPs was confirmed, and FP (size of 54 ± 14 nm and zeta potential of -31.9 ± 2.08 mV) showed higher 4T1-specific toxicity in vitro when compared to control conjugates. After in vivo application of the FP, transmission electron microscopy analyses proved the presence of AuNPs in the tumor cells. Hematoxylin and eosin staining of the tumor tissue revealed that the FP group exhibited the highest amounts of inflammatory, necrotic, and apoptotic cells in contrast to the control groups. Finally, qPCR results showed that FP could transfect and suppress miR-135b expression in vivo, confirming the tumor-targeting properties of FP. The capacity of biologically produced gold nanoparticles to conjugate with multiple decorative molecules while retaining their stability and effective intracellular uptake makes them a promising alternative strategy superior to current drug carriers.

The enormous therapeutic potential of selective ribonucleic acid (RNA) interference has recently been manifested by the approval of several small interfering RNA (siRNA)‐based drugs. However, the efficacy of siRNA delivery is still limited, and an extensive search for alternative and highly effective delivery approaches is ongoing. With this aim, three generations of non‐viral vectors based on modified nanodiamonds (NDs) have been gradually developed in the past decade. They show great promise due to the negligible toxicity of the ND core. Here, a robust methodological approach is presented to enable the evaluation of new vector nanomaterials. Using a new type of third‐generation ND vector coated with a copolymer with tunable charge density, variables such as colloidal stability, surface electrostatic properties, the molecular composition of the copolymer, and the mode of complexation with siRNA are optimized. Using an innovative data processing strategy, the results are related to biological potency, toxicity, and cell proliferation. Finally, the optimized composition of a coating copolymer consisting of a cationic component, 2‐dimethylaminoethyl methacrylate, and an electroneutral biocompatible component, N‐(2‐hydroxypropyl) methacrylamide, is evaluated. The optimized NDs vectors are colloidally and biologically stable siRNA delivery tools with broad potential for RNA interference‐based therapeutics.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x‐ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of –35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F‐actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell‐specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F‐actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

The extracellular vesicles (EVs) serve for cell-to-cell communication and delivery of signaling molecules and they represent interesting approach in targeted therapy. This study focuses on an EV-mediated transfer of functionalized nanoparticles into recipient tumor cells. We used biologically produced gold nanoparticles (AuNPs) with known physical-chemical properties and decorated them with an inhibitory RNA targeting oncogenic microRNA-135b (AuNPs-antimiR 135 b) and transferrin (AuNPs-antimiR 135 b-Tf). The AuNPs were added to the breast cancer 4T1 cell line at a non-toxic dose 0.03 µg/µL. The 4T1-autologous EVs (with or without the AuNPs) were extracted and analyzed. The amount of endocytosed and excreted AuNPs was determined by graphite furnace atomic absorption spectrometry. The protein content was analyzed using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and liquid chromatography with mass spectrometry (LC–MS). The fate of EVs and/or AuNPs was characterized using a Transmission Electron Microscopy and Zetasizer. The target-specific silencing effect of the antimir-135b was analyzed with a quantitative real-time PCR (qPCR). The AuNPs produced by Fusarium oxysporum possessed round shape, size of 5 nm and a zeta potential of -33 mV. We observed two types of EV populations: 60 nm and 250 nm. Out of the initial 58.7 µg of AuNPs added to the cells, 52.68 ± 0.65 µg were internalized and 38.64 ± 0.29 µg were found inside the EVs. The protein content in EV-AuNPs was higher compared to control EVs. LC-MS analysis showed that the AuNPs could alter the protein expression profile of the cancer cell. qPCR results revealed both EV-AuNPs-antimiR 135 b and EV-AuNPs-antimiR 135 b-Tf silenced target microRNA-135b, but higher efficacy was observed in the EV-AuNPs-antimiR 135 b-Tf group. Obtained data proved EV-mediated transportation of biological AuNPs bearing effector RNA among tumor cells. We also found the efficacy of loaded EVs is higher using AuNPs decorated with a tumor-specific ligand such as transferrin.

This study recognized biologically produced gold nanoparticles (AuNP) as multiple cargo carriers with a perspective of drugs delivery into specialized tumor cells in vivo. Paclitaxel (PTX), transferrin, and antimiR-135b were conjugated with AuNPs and their uptake by mouse tumor cells in an induced breast cancer model was investigated. Each of the above-mentioned molecules was conjugated to the AuNPs separately as well as simultaneously, loading efficiency of each cargo was assessed, and performance of the final product (FP) was assessed. After tumor induction in BALB/c mice, sub-IC50 doses of FP as well as control AuNPs, PTX, and PBS were administered in vivo. Round AuNPs were prepared using Fusarium oxysporum and exhibited size of 13 ± 1.3 nm and zeta potential of -35.8 ± 1.3 mV. The cytotoxicity of individual conjugates and FP were tested by MTT assay in breast tumor cells 4T1 and non-tumor fibroblasts NIH/3T3 cells. The conjugation of the individual molecules with AuNPs was confirmed and FP (size of 54 ± 14 nm and zeta potential of -31.9 ± 2.08 mV) showed higher 4T1-specific toxicity in vitro when compared to control conjugates. After in vivo application of the FP, TEM analyses proved presence of AuNPs in the tumor cells. Hematoxylin and eosin staining of the tumor tissue revealed that the FP group exhibited the highest amounts of inflammatory, necrotic, and apoptotic cells in contrast to control groups. Finally, qPCR results showed that FP could transfect and suppress miR-135b expression in vivo, confirming the tumor-targeting properties of FP. The capacity of biologically produced gold nanoparticles to conjugate with multiple decorative molecules while remaining their stability and effective intracellular uptake makes them promising alternative strategy superior to current drug carriers.

Non-healing wounds are a serious complication in diabetic patients. One of the detrimental factors contributing to limited wound healing is the accumulation of metalloproteinase-9 (MMP-9) in the wound. Selective inhibition of MMP-9 is one of the established therapeutic targets for diabetic wound healing and is therefore of great interest. Here we focused on development of gene silencing system for localized delivery of antisense siRNA against MMP-9 into the wound. We have developed a functional and biocompatible wound dressing allowing controlled release of a traceable vector loaded with the target siRNA. Specifically, the dressing consists of a degradable scaffold of polymer nanofibers embedded with the vector nanosystem, polymer-coated fluorescent nanodiamonds (FNDs). The biocompatible cationic polymer shell on FNDs was designed and optimized for binding of siRNA and formation of colloidally stable FND-siRNA complexes in physiological environment. A hybrid nanofiber scaffold consisting of poly(vinyl alcohol) and poly(caprolactone) ensured continuous release of FND-siRNA complexes from the dressing. The photostable luminescence of FNDs allowed us to monitor the vector system in the wound. Our dressing was tested on murine fibroblasts and also applied to wounds in a diabetic murine model to evaluate its suitability in terms of in vivo toxicity, biological efficacy and manipulation. The treatment resulted in significant local inhibition of MMP-9 and reduction of the wound healing time. The scar formation for treated diabetic-like animals became comparable with non-treated diabetes-free mice. Our results suggest that the application of our biocompatible dressing loaded with a non-toxic vector nanosystem is an effective and promising approach in gene therapy of non-healing wounds.