PEtOx-DOPE nanoliposomes functionalized with peptide 563 in targeted BikDDA delivery to prostate cancer.

Background: Nanocarrier-based systems have cultivated significant improvements in prostate cancer therapy. However, the efforts are still limited in clinical applicability, and more research is required for the development of effective strategies. Here, we describe a novel nanoliposomal system for targeted apoptotic gene delivery to prostate cancer. Methods: Poly (2-ethyl-2-oxazoline) (PEtOx) dioleoyl phosphatidylethanolamine (DOPE) nanoliposomes were conjugated with the prostate-specific membrane antigen (PSMA)-targeting peptide GRFLTGGTGRLLRIS (P563) and loaded with BikDDA, a mutant form of the proapoptotic Bik. We selected 22Rv1 cells with moderate upregulation of PSMA to test the in vitro uptake, cell death, and in vivo anticancer activity of our formulation, P563-PEtOx-DOPE-BikDDA. Results: BikDDA was upregulated in 22Rv1 cells, inducing cell death, and CD-1 nude mice xenografts administered with the formulation showed significant tumor regression. Conclusion: We suggest that P563-PEtOx-DOPE-BikDDA nanoliposomes can serve as prominent gene carriers against prostate cancer.

Evaluation of Biodegradable Microparticles for Mucosal Vaccination Against Diphtheria Toxoid: Nasal Efficacy Studies in Guinea Pigs.

Objectives: In this study, poly-(ɛ-caprolactone) (PCL) and poly-(lactic-co-glycolic acid) (PLGA) microparticles encapsulating diphtheria toxoid (DT) were investigated for their potential as a mucosal vaccine delivery system. Materials and Methods: Antigen-containing microparticles were prepared using the double emulsion (w/o/w) solvent evaporation method. Results: The average geometric diameter of the particles was found to be between 7 and 24 µm, which is suitable for uptake by the antigen-presenting cells in the nasal mucosa. Although the differences were insignificant, the PLGA polymer-containing formulations exhibited the highest encapsulation efficiency. Microparticle formulations, prepared with both PLGA and PCL polymers, were successfully produced at high production yields. The in vitro release profile was presented as a biexponential process with an initial burst effect due to the release of the protein adsorbed on the microsphere surface, and the subsequent sustained release profile is the result of protein diffusion through the channels or pores formed in the polymer matrix. DT-loaded microparticles, DT solution in phosphate-buffered saline (PBS), and empty microparticles (control) were administered via nasal route and subcutaneously to guinea pigs. The antibody content of each serum sample was determined using an enzyme-linked immunosorbent assay (ELISA). Conclusion: Absorbance values of the ELISA test showed that PLGA- and PCL-bearing microparticles could stimulate an adequate systemic immune response with intranasal vaccination. In addition, PLGA and PCL microparticles resulted in significantly increased IgG titers with intranasal administration as a booster dose following subcutaneous administration. PCL polymer elicited a high immune response compared with PLGA polymer (p <0.05).

Development of Zoledronic Acid Containing Biomaterials for Enhanced Guided Bone Regeneration.

In recent years, biomaterial-based treatments, also called guided bone regeneration (GBR), which aim to establish a bone regeneration site and prevent the migration of gingival connective tissue and / or peripheral epithelium through the defective area during periodontal surgical procedures have come to the fore. In this report, we have developed a nanoparticle bearing thermosensitive in situ gel formulation of Pluronic F127 and poly(D,L-lactic acid) based membrane to reveal their utilization at GBR by in-vivo applications. In addition, the encouragement of the bone formation in defect area via inhibition of osteoclastic activity is intended by fabrication these biodegradable biomaterials at a lowered Zoledronic Acid (ZA) dose. Both of the developed materials remained stable under specified stability conditions (25 °C, 6 months) and provided the extended release profile of ZA. The in-vivo efficacy of nanoparticle bearing in situ gel formulation, membrane formulation and simultaneous application for guided bone regeneration was investigated in New Zealand female rabbits with a critical size defect of 0.5 × 0.5 cm in the tibia bone for eight weeks. Based on the histopathological findings, lamellar bone and primarily woven bone formations were observed after 8 weeks of post-implantation of both formulations, while fibrosis was detected only in the untreated group. Lamellar bone growth was remarkably achieved just four weeks after the simultaneous application of formulations. Consequently, the simultaneous application of ZA-membrane and ZA-nanoparticles loaded in-situ gel formulations offers enhanced and faster GBR therapy alternatives.

Guided bone regeneration by the development of alendronate sodium loaded in-situ gel and membrane formulations.

Biocompatible materials applied in guided bone regeneration are needed to prevent leakage caused by the invasion of peripheral epithelium. (2.1) The aim of this study is to develop a thermosensitive in situ gel system containing alendronate sodium loaded PLGA nanoparticles and alendronate sodium loaded membranes for guided bone regeneration. Thermosensitive Pluronic F127 gel system was preferred to prevent soft tissue migration to the defect site and prolong the residence time of the nanoparticles in this region. In situ gel system was combined with membrane formulation to enhance bone regenaration activity. Efficacy of combination system was investigated by implanting in 0.5 × 0.5 cm critical size defect in tibia of New Zealand female rabbits. According to the histopathological results, fibroblast formations were found at defect area after 6 weeks of post implantation. In contrast, treatment with the combination of in-situ gel containing nanoparticles with membrane provided woven bone formation with mature bone after 4 weeks of post implantation. As a results, the combination of in-situ gel formulation containing alendronate sodium-loaded nanoparticles with membrane formulation could be effectively applided for guided bone regeneration.

Formulation and investigation of 5-FU nanoparticles with factorial design-based studies.

This study describes an orthogonal experimental design to optimize the formulation of 5-fluorouracil (5-FU) loaded poly D,L (lactide-co-glycolide) (PLGA) nanoparticles (5FU-NP) by a nanoprecipitation-solvent displacement technique. The type of surfactant, amount of acetone and molecular weight of the polymer with three levels of each factor were selected and arranged in an L18(3(5)) orthogonal experimental table. From the statistical analysis of the data polynominal equations were generated. Optimized formulations have the particle size ranging from 160 to 250 nm. Smallest nanoparticles (161+/-1.22 nm) were obtained using Resomer PLGA 755 and pluronic F-68 with 10 ml acetone amount. Under these conditions the 5-FU entrapment percentage was maximum 78.30%, suggesting 5-FU might be entrapped and adsorbed on the nanoparticle surface. In vitro release of three formulations with maximum drug entrapment efficiency and minimum particle size, were also investigated by release kinetics. According to the determined coefficients, release data fit to Higuchi's diffusion kinetics. The in vitro release of 5FU-NP in phosphate buffered saline (PBS, pH 7.4) is suggested to be controlled by a combination of diffusion with slow and gradual erosion of the particles. Also, the antimicrobial activity was observed even on the end of seventh day with all formulations.

Chitosan-DNA nanoparticles: effect on DNA integrity, bacterial transformation and transfection efficiency.

While somatic gene therapy has the potential to treat many genetic disorders, recent clinical trials suggest that an efficient and safe delivery vehicle for successful gene therapy is lacking. The current study examines the influence of two different preparation (the solvent evaporation method and the complex coacervation method) methods on the encapsulation of a model plasmid with chitosan. The ability of different molecular weights of chitosan to form nanoparticles with a plasmid, and particulated polymers to stabilize a plasmid in a supercoiled form, were examined by agarose gel electrophoresis. Protection of encapsulated pDNA offered by these nanoparticles from nuclease attack was confirmed by assessing degradation in the presence of DNase I, and the transformation of the plasmids with incubated nanoparticles were examined by beta-galactosidase assay. Model pDNA existed as a mixture of both supercoiled (84.2%) and open circular (15.8%) forms. Our results demonstrated that supercoiled forms decreased while open circular forms and fragmented linear forms increased during the preparation of formulations. F1 formulation prepared by the complex coacervation method protected the supercoiled form of pDNA effectively. There weren't any significant changes in nanoparticle size and zeta potential values at pH 5.5 for a period of 3 months, but differences in particle sizes were observed after lyophilization with a cryoprotective agent. The efficiency of nanoparticles mediated transformation to Escherichia coli cells was significantly higher than naked DNA or poly-L-lysine (PLL)-DNA polycation complexes. The transfection studies were performed in COS-7 cells. A 3-fold increase in gene expression was produced by nanoparticles as compared to the same amount of naked plasmid DNA (pDNA). These observations suggest that formulations with high molecular weight (HMW) chitosan can be an effective non-viral method of gene vector in animal studies.

Chitosan nanoparticles for plasmid DNA delivery: effect of chitosan molecular structure on formulation and release characteristics.

Chitosan can be useful as a nonviral vector for gene delivery. Although there are several reports to form chitosan-pDNA particles, the optimization and effect on transfection remain insufficient. The chitosan-pDNA nanoparticles were formulated using complex coacervation and solvent evaporation techniques. The important parameters for the encapsulation efficiency were investigated, including molecular weight and deacetylation degree of chitosan. We found that encapsulation efficiency of pDNA is directly proportional with deacetylation degree, but there is an inverse proportion with molecular weight of chitosan. DNA-nanoparticles in the size range of 450-820 nm depend on the formulation process. The surface charge of the nanoparticles prepared with complex coacervation method was slightly positive with a zeta potential of +9 to +18 mV; nevertheless, nanoparticles prepared with solvent evaporation method had a zeta potential approximately +30 mV. The pDNA-chitosan nanoparticles prepared by using high deacetylation degree chitosan having 92.7%, 98.0%, and 90.4% encapsulation efficiency protect the encapsulated pDNA from nuclease degradation as shown by electrophoretic mobility analysis. The release of pDNA from the formulation prepared by complex coacervation was completed in 24 hr whereas the formulation prepared by evaporation technique released pDNA in 96 hr, but these release profiles are not statistically significant compared with formulations with similar structure (p > .05). According to the results, we suggest nanoparticles have the potential to be used as a transfer vector in further studies.

Preparation and in vitro/in vivo evaluation of mucosal adjuvant in situ forming gels with diphtheria toxoid.

Studies on preparation of in situ gel formulations containing diphtheria toxoid as the model active substance and their intranasal administration have been conducted in this study. The objective of mucosal vaccination is to stimulate both systemic and mucosal immune responses. In situ gel formulations were prepared by using, in different ratios, mixtures of Poloxamer 407 and Poloxamer 188 polymers, which gelate in a temperature-dependent manner, and mucoadhesive polymers carbopol 934, hydroxypropyl methyl cellulose, hydroxypropyl cellulose or chitosan. Following pre-formulation studies, F1, F2, F3, F4, F5, F6 and F7 formulations, which gelate at intervals and temperatures in accordance with nasal temperatures, were subjected to more comprehensive studies. For this purpose, organoleptic characteristics of the formulations were identified, their pH and mucoadhesive potencies were measured and rheological behaviors were characterized. Calculated amounts of diphtheria toxoid were added to formulations after optimization of formulations was achieved, and assay and in vitro release studies were carried out. Formulations coded F3 and F7 were considered to be superior to other formulations given the in vitro test results. Therefore, these formulations were tested in guinea pigs to determine immune responses, which they would produce following intranasal and subcutaneous administration. Absorbance values of ELISA tests and antibody neutralization test showed that formulations coded F3 and F7 were unable to stimulate adequate systemic immune response when either of the formulations was administered alone intranasally, whereas F7 resulted in significantly increased neutralizing antibody titers with intranasal administration as a booster dose following subcutaneous administration.

Formulation and in vitro characterization of PEGylated chitosan and polyethylene imine polymers with thrombospondin-I gene bearing pDNA.

An ideal gene carrier is required both in safety and efficiency for transfection. We examined the use of water soluble chitosan and polyethyleneimine as a carrier for anti-angiogenic protein, TSP-1 coded, in gene delivery. The aim of this study was to synthesize and characterize polyethylene glycol conjugated cationic polymers to increase anti-angiogenic gene transfection and reduce possible cytotoxicity. Gel electrophoresis study showed strong DNA binding ability of modified cationic polymers. Also structural properties of pegylated polymers were confirmed by (1)H-NMR. We investigated in vitro properties of PEG conjugated and coated particles which were observed between 145 and 250 nm with the positive zeta potential value. In addition, the chitosan-based DNA complexes did not induce remarkable cytotoxicity against MCF-7 cells. Due to low cytotoxicity, we observed high transfection efficiency at chitosan-based formulations compared with PEI ones. Although transfection studies carried on in vitro conditions, we measured slight increases at transfection with PEGylation. PEG-conjugated chitosan formulations can be a promising candidate due to its efficiency in condensing and transfection of pDNA, its low cytotoxicty and comparatively high encapsulation degree.