Co-Delivery of siRNA and Docetaxel to Cancer Cells by NLC for Therapy.

The present study aims to develop a delivery system that can carry small interference RNA (siRNA) with small-molecule chemotherapeutic drugs, which can be used in cancer treatment. The drug delivery system combines the advantages of a therapeutic agent with two different mechanisms to ensure that it is used efficiently for cancer therapy. In this study, a nanostructured lipid carrier system was prepared, Docetaxel was loaded to these systems, and the Eph siRNA was adsorbed to the outer surface. In addition, DOTAP was added to the lipophilic phase to load a positive charge on the lipidic structure for interaction with the cells. Moreover, characterization, cytotoxicity, and transfection procedures were performed on the whole system. This candidate system was also compared to Taxotere, which is the first approved Docetaxel-containing drug on the market. Given the results, it was determined that the particle size of NLC-DTX was 165.3 ± 3.5 nm, the ζ potential value was 38.2 ± 1.7 mV, and the PDI was 0.187 ± 0.024. Entrapment efficacy of nanoparticles was found to be 92.89 ± 0.21%. It was determined that the lipidic system prepared in vitro release analyses were able to provide sustained release and exhibit cytotoxicity, even at doses lower than the dose used for Taxotere. The formulations prepared had a higher level of effect on cells when compared with pure DTX and Taxotere, but they also exhibited time-dependent cytotoxicity. It was also observed that the use of Eph siRNA together with the chemotherapeutic agent via formulation also contributed to this cell death. The results of the present study indicate that there is a promising carrier system in order to deliver hydrophilic nucleic acids, such as siRNA, together with lipophilic drugs in cancer treatment.

Stem Cell-Based or Cell-Free Gene Therapy in Chondrocyte Regeneration: Synovial Fluid-Derived Mesenchymal Stem Cell Exosomes.

BACKGROUND: Cartilage injuries are currently the most prevalent joint disease. Previous studies have emphasized the use of stem cells as the effective treatment for regenerating cartilage damage. OBJECTIVE: In this study, considering the difficulties of the cellular therapy method, it was hypothesized that human synovial fluid-derived mesenchymal stem cell (hSFMSC) exosomes as a SC source could be used to treat these injuries as a safer and cell-free therapeutic alternative procedure due to its direct relevance to cartilage regeneration. Moreover, this study aimed to determine the miRNA and target genes required for the formation of SC treatment combined with gene therapy in order to reveal the mechanism of cartilage regeneration and increase its effectiveness. METHODS: MSCs were characterized by flow cytometry, and immunocytochemical and differentiation analyses were done. To characterize functionally isolated exosomes, in vitro uptake analysis was performed. RT-qPCR was used to examine in terms of the advantages of cellular and cell-free therapy, mature human chondroblasts derived by differentiation from hSF-MSCs and human chondrocyte profiles were compared in order to demonstrate the above profile of hSF-MSCs and exosomes isolated from them, and the effectiveness of SC therapy in repairing cartilage damage. RESULTS: According to our findings, the expression level of hsa-miR-155-5p was found to be considerably higher in chondrocytes differentiated from human synovial fluid MSCs than in mature human chondrocytes. These findings were also supported by the TGF-signalling pathway and chondrogenesis marker genes. CONCLUSION: It was concluded that hSF-MSCs and exosomes can be used in the treatment of cartilage damage, and hsa-miR-155-5p can be used as a target miRNA in a new gene therapy approach because it increases the therapeutic effect on cartilage damage.

HDL-Chitosan Nanoparticles for siRNA Delivery as an SR-B1 Receptor Targeted System.

AIMS: High-Density Lipoprotein (HDL) is a complex structure unique to the human body. ApoA-1 protein is a significant structural/functional protein of HDL and provides a natural interaction with the SR-B1 receptors on the cell membrane. The overexpression of the SR-B1 receptor in the membrane of malignant cells suggests that targeting cancer cells can be possible using HDL. The objective of this study was to prepare HDL-conjugated chitosan nanoparticles containing a genetic material that can be used for liver cancer. METHODS: HDL used in the preparation of the formulations have been obtained by isolating from blood samples taken from healthy volunteers. Bcl-2 siRNA inhibiting BCL-2 oncogene was selected as the genetic material. Chitosan nanoparticles were prepared using the ionic gelation method utilizing low molecular weight chitosan. Physicochemical properties of formulations, transfection efficacy, and cytotoxicity of them on 3T3 and HepG2 cell lines were examined. RESULTS: The average diameters of the selected formulations were below 250 nm with a positive zeta potential value between +36 ± 0.1 and +34 ± 0.5 mV. All formulations protected Bcl-2 siRNA from enzymatic degradation in the presence of serum. Cellular uptake ratios of particles by HepG2 cells were found to be between 76% and 98%. HDL/chitosan nanoparticles/Bcl-2 siRNA complex was found to be more toxic when compared to chitosan nanoparticles/Bcl-2 siRNA complex and naked Bcl-2 siRNA. CONCLUSION: According to attained results, the HDL-conjugated chitosan nanoparticles can bring advantages for targeted siRNA delivery to malignant cells that overexpress SR-B1 receptors, such as HepG2.

Formulation and characterization studies of inclusion complexes of voriconazole for possible ocular application.

In our study, Voriconazole (VOR) was selected as an active agent to be used for the treatment of ocular fungal infections. To overcome low aqueous solubility of VOR, inclusion complexes with α-cyclodextrin (α-CD), ß-cyclodextrin (ß-CD), γ-cyclodextrin (γ-CD), hydroxypropyl-cyclodextrin (HP-CD), hydroxypropyl-ß-cyclodextrin (HP-ß-CD) hydroxypropyl-γ-cyclodextrin (HP-γ-CD), methyl-ß-cyclodextrin (M-ß-CD) and sulfabutylether-ß-cyclodextrin (SBE-ß-CD) were formulated. Characterization studies revealed that inclusion complexes were formulated successfully with the lyophilization method. Aqueous solubility of VOR was enhanced up to 86 fold with the formation of the inclusion complexes. MTT analyses results revealed the safety of the complexes on 3T3 mouse fibroblast cell lines while Microbroth Dilution Method revealed the remarkable antifungal activities of the complexes. Analyses results revealed that inclusion complexes will overcome the poor ocular bioavailability of VOR resulting inefficient treatment of severe ocular fungal infections.

Preparation and Characterization of Palladium Derivate-Loaded Micelle Formulation in Vitro as an Innovative Therapy Option against Non-Small Cell Lung Cancer Cells.

Nanoparticles have been used in cancer treatments to target tumor and reduce side effects. In this study, we aimed to increase the effectiveness of palladium(II) complex [PdCl(terpy)](sac) ⋅ 2H2 O, which previously showed anticancer potential, by preparing the nanoparticle formulation. An inhalable micellar dispersion containing a palladium(II) complex (PdNP) was prepared and its physicochemical characteristics were evaluated using in vitro tests. Morphology, size and surface charges of particle and loading/encapsulation efficiency of PdNP were analyzed by scanning electron microscopy, zeta sizer and inductively coupled plasma mass spectrometry while aerosol properties of PdNP were measured by the next generation impactor. A549 and H1299 non-small lung cancer cell types were used for cytotoxicity using SRB and ATP assays. Fluorescent staining and M30 antigen assay were carried out for cell death evaluation. Apoptosis was confirmed by flow cytometry analyses. SEM, particle size, and zeta potential results showed the particles have inhalable properties. The amount of the palladium(II) complex loaded into the particles was quantified which indicated high encapsulation efficiencies (97 %). The micellar dispersion expected to reach the alveolar region and the brachial region was determined 35 % and 47 %, respectively. PdNP showed an anti-growth effect by increasing reactive oxygen species that is followed by the induction of mitochondria-dependent apoptosis that is evidenced by pyknotic nuclei and M30 antigen level increments and disruption of polarization of membrane in mitochondria (Δψm). The results show that PdNP might be a promising inhalable novel complex to be used in non-small cell lung cancer, which warrants animal studies in further.

Moxifloxacin Hydrochloride-Loaded Eudragit® RL 100 and Kollidon® SR Based Nanoparticles: Formulation, In vitro Characterization and Cytotoxicity.

BACKGROUND: Considering the low ocular bioavailability of conventional formulations used for ocular bacterial infection treatment, there is a need to design efficient novel drug delivery systems that may enhance precorneal retention time and corneal permeability. AIM AND OBJECTIVE: The current research focuses on developing nanosized and non-toxic Eudragit® RL 100 and Kollidon® SR nanoparticles loaded with moxifloxacin hydrochloride (MOX) for its prolonged release to be promising for effective ocular delivery. METHODS: In this study, MOX incorporation was carried out by spray drying method aiming ocular delivery. In vitro characteristics were evaluated in detail with different methods. RESULTS: MOX was successfully incorporated into Eudragit® RL 100 and Kollidon® SR polymeric nanoparticles by a spray-drying process. Particle size, zeta potential, entrapment efficiency, particle morphology, thermal, FTIR, NMR analyses and MOX quantification using HPLC method were carried out to evaluate the nanoparticles prepared. MOX loaded nanoparticles demonstrated nanosized and spherical shape while in vitro release studies demonstrated modified-release pattern, which followed the Korsmeyer-Peppas kinetic model. Following the successful incorporation of MOX into the nanoparticles, the formulation (MOX: Eudragit® RL 100, 1:5) (ERL-MOX 2) was selected for further studies because of its better characteristics like cationic zeta potential, smaller particle size, narrow size distribution and more uniform prolonged release pattern. Moreover, ERLMOX 2 formulation remained stable for 3 months and demonstrated higher cell viability values for MOX. CONCLUSION: In vitro characterization analyses showed that non-toxic, nano-sized and cationic ERL-MOX 2 formulation has the potential of enhancing ocular bioavailability.

Graphene quantum dots: Synthesis, characterization, cell viability, genotoxicity for biomedical applications.

We report the synthesis and applications of a novel N-doped graphene quantum dots (GQDs) using hydrothermal reaction between citric acid and p-aminophenol. The synthesized N-doped GQDs have been characterized physico-chemically and evaluated its antioxidant, antimicrobial, DNA binding and cleavage activities. siRNA loading studies were performed and their effects on cells were evaluated. Obtained results indicate that monodisperse solution of N-doped GQDs has been obtained with particles size ca. ∼10.9 ±â€¯1.3 nm. UV-Vis spectroscopy studies of the interactions between the N-doped GQDs and calf thymus DNA (CT-DNA) showed that the compound interact with CT-DNA via both intercalative and electrostatic binding. The DNA cleavage study showed that the N-doped GQDs cleaved DNA without any external agents. The antioxidant activity of N-doped GQDS was very active when compared to BHT. As the concentration of the compound increased, the antioxidant activity also increased. Cell viability assay demonstrated that the Ndoped GQDs showed cell viability (70%) when the concentration reached 200 µg/mL for A549 and also MDA-MB-231, 150 µg/mL for NIH-3T3 cell lines at 24 h incubation. N-doped GQDs were coated with Eudragit RS 100 and EphA2-siRNA was loaded. As a result of the studies on these formulations, it was concluded that there may be significant effects on A549 cells. The microscopy results revealed that N-doped GQDs was quickly internalized into the cell. Our novel N-doped-GQDs with siRNA are candidate for in situ tumor suppression via DNA and mRNA breakage.

Vaginal Suppositories with siRNA and Paclitaxel-Incorporated Solid Lipid Nanoparticles for Cervical Cancer: Preparation and In Vitro Evaluation.

The objective of this study is to prepare vaginal suppository containing chemotherapeutic agent and genetic material that can be applied locally for cervical cancer. Cervical cancer is one of the most life-threatening types of cancer among women and is generally resistant to chemotherapy. Paclitaxel has been selected as chemotherapeutic agent, and siRNA that inhibits the Bcl-2 oncogene has been selected as the genetic material for simultaneous vaginal delivery. For this purpose, three different solid lipid nanoparticles (SLNs) were prepared that include Bcl-2 siRNA and paclitaxel and paclitaxel/Bcl-2 siRNA combination separately, and these SLN formulations were dispersed in vaginal suppositories prepared with PEG 6000. First, the physicochemical properties of SLNs, their cytotoxicities on HeLa cell lines, and the transfection ability of siRNA-incorporated SLN on the cells have been examined. Afterward, the release of SLNs from the three different vaginal suppositories prepared has been determined via horizontal diffusion chamber system. The loaded amount to the SLNs and release amount from suppositories of paclitaxel have been determined via HPLC, whereas stability, loading, and release amount of siRNA has been determined via gel retardation system and UV spectrophotometer.

Preparation and in vitro evaluation of vaginal formulations including siRNA and paclitaxel-loaded SLNs for cervical cancer.

Cervical cancer is one of the most life threatening types of cancer among women and is generally resistant to chemotherapy. The objective of this study was to prepare a vaginal suppository containing a chemotherapeutic agent and a genetic material that can be applied locally for cervical cancer. Paclitaxel was selected as the chemotherapeutic agent and siRNA which inhibits BCL-2 oncogene was selected as the genetic material. Bcl-2 siRNA, paclitaxel and paclitaxel/Bcl-2 siRNA combination were incorporated into solid lipid nanoparticles (SLNs) and were dispersed separately in vaginal suppositories prepared with PEG 6000. Physicochemical properties of SLNs, their cytotoxicities on HeLa cell lines and also the effect of SLNs on the total protein amount of the cells were examined followed by the investigation of release rates of the active materials from the SLNs prepared. Average diameters of all SLNs prepared were below 180nm with a positive zeta potential value between +22.20 and +48.16mV at the pH range of 4.2 and 7.4. The release of Bcl-2 siRNA from SLNs incorporated Bcl-2 siRNA and the release of paclitaxel (PTX) from PTX incorporated SLNs were completed within 12h and 36h. SLNs incorporating Bcl-2 siRNA and paclitaxel/Bcl-2 siRNA were found to be more toxic when compared to paclitaxel incorporated SLN and placebo SLN. The disintegration of the vaginal suppositories as well as the release of the SLNs was completed within 2 h. This study indicates that vaginal suppository containing SLNs can bring the advantages of the simultaneous delivery of paclitaxel and siRNA via vaginal route with no help from professionals.

Chitosan nanoparticles for ocular delivery of cyclosporine A.

In the present study, cyclosporine A (CsA) was successfully incorporated into cationic chitosan nanoparticles by spray-drying method aiming ocular application. Physicochemical characterisation of particles was performed in detail. Among the particles prepared using three types of chitosan with different molecular weights, particles containing chitosan with medium molecular weight was selected for in vivo studies. Selection was dependent on higher incorporation and encapsulation efficiencies of CsA and also better release characteristic in simulated tear fluid. Sheep were used in in vivo studies. Biological samples were collected at predetermined time intervals and were analysed by enzyme immune assay. CsA could be detected in both aqueous and vitreous humour samples for the duration of 72 h. In vivo release profiles indicated prolonged release of active agent from positively charged chitosan formulations. This may be attributed to enhanced residence time at the corneal and conjunctival surfaces.