Synthesis and characterization of chitosan-PVA hydrogel containing PEGylated recombinant epidermal growth factor on cell culture for wound healing substitute

Abstract The aim of the current study was to assess the physicochemical characteristics and wound healing activity of chitosan-polyvinyl alcohol (PVA) crosslinked hydrogel containing recombinant human epidermal growth factor (rh-EGF) or recombinant mouse epidermal growth factor (rm-EGF). The hydrogels were prepared and analyses were made of the morphological properties, viscosity, water absorption capacity, mechanical and bio-adhesive properties. The viscosity of the formulations varied between 14.400 - 48.500 cPs, with the greatest viscosity values determined in K2 formulation. F2 formulation showed the highest water absorption capacity. According to the studies of the mechanical properties, H2 formulation (0.153±0.018 N.mm) showed the greatest adhesiveness and E2 (0.245±0.001 mj/cm2) formulation, the highest bio-adhesion values. Hydrogels were cytocompatible considering in vitro cell viability values of over 76% on human keratinocyte cells (HaCaT, CVCL-0038) and of over 84% on human fibroblast cells (NIH 3T3, CRL-1658) used as a model cell line. According to the BrdU cell proliferation results, B1 (197.82±2.48%) formulation showed the greatest NIH 3T3 and C1 (167.43±5.89%) formulation exhibited the highest HaCaT cell proliferation ability. In addition, the scratch closure assay was performed to assess the wound healing efficiency of formulation and the results obtained in the study showed that F2 formulation including PEGylated rh-EGF had a highly effective role.

Preparation and in vitro characterization of monoclonal antibody ranibizumab conjugated magnetic nanoparticles for ocular drug delivery

Gold coated magnetite nanoparticles were prepared and coated with ranibizumab as an ocular drug delivery system. The surface morphologies of the nanoparticles were determined by Scanning Electron Microscopy (SEM). The size and surface charge were determined by using the dynamic light scattering (DLS) technique. Crystallographic properties of the gold coated Fe3O4 nanoparticles were recorded on X-ray diffractometer (XRD) the XRD pattern of nanoparticlees were shown to have uniqe Fe3O4 and gold peaks. Conjugation of ranibizumab onto nanoparticles was achieved using the physical adsorption method. The amount of ranibizumab on the surface of the nanoparticles was determined by thermogravimetric analysis (TGA). In the in vitro release studies performed using UV spectroscopy; it was found that almost 60% of antibodies were released within the first 30 minutes. Antibody activity after release studies was also proved with ELISA. Non-toxicity of gold coated Fe3O4 particles were proved with MTT. Results of the studies, showed that the antibody conjugated magnetic nanoparticle system could be a potential treatment system for ocular diseases.

Targeting of temozolomide using magnetic nanobeads: an in vitro study

Temozolomide, a chemotherapeutic drug that is often administered for the treatment of brain cancer has severe side effects and a poor aqueous solubility. In order to decrease the detrimental effect of the drug over healthy cells, a novel drug delivery vehicle was developed where the therapeutic drug was encapsulated within the hydrophobic cavities of b-CD modified magnetite nanoparticles, which are embedded in chitosan nanobeads prepared by salt addition. In-vitro studies have shown that the magnetic properties of the novel delivery vehicle are adequate for targeted drug delivery applications under an external magnetic field. Additionally, an increase in the amount of chitosan was shown to exhibit a strong shielding effect over the magnetic properties of the delivery vehicle, which lead to deterioration of the amount of captured drug at the targeted area, suggesting a delicate balance between the amounts of constituents composing the drug delivery vehicle.

Complexation and enhancement of temozolomide solubility with cyclodextrins

ABSTRACT Temozolomide is a poorly soluble anti-cancer drug used in the treatment of some brain cancers. Following literature reports about the enhancement of solubility and stability for these kinds of drugs upon complexation with cyclodextrins, we aimed to form an inclusion complex between temozolomide and the different types of cyclodextrins (CDs) to enhance its solubility. In this study, three different cyclodextrins (ß -CD, hydroxyl-ß-CD and γ-CD) were used, and changes in solubility was measured by UV-Vis Spectroscopy and HPLC. Morphological changes upon complexation were shown by the Scanning Electron Microscope (SEM), and weight loss profiles with respect to temperatures which were unique to the compounds were shown by Thermogravimetric Analysis. Changes in heat release profiles were shown by Differential Scanning Calorimeter (DSC). Drug solubility was measured to be increased to around 25% for 1:1 molar ratio for all used CD complexations. Changes of morphology, heat release and weight loss profiles are consistent with the formation of an inclusion complex between CDs and temozolomide. In this study, success was shown in the enhancement of temozolomide solubility upon complexation with different types of CDs. It has been demonstrated that cyclodextrins can be used as complexing agents for poorly soluble anti-cancer drugs, increasing their solubility and hence drug availability

Future prospects for gene delivery systems.

INTRODUCTION: Gene therapy is the challenging area of biotechnology. Despite its promise for critical diseases, it has serious safety and efficiency issues, particularly with regards to gene transfer systems. Areas covered: We examined the current situation with gene transfer systems and addressed problems this technology. We then searched patent applications about in the area from the Patentscope online system, the international patent database. We analyzed the data obtained to get a general idea about gene delivery systems designed for future use and assessed approaches for more efficient, safer and valid delivery systems. Expert opinion: When quality assurance terms are fulfilled, some of these issues (genetic changes, mutations) could be minimized during the production process. Modification of vectors for improving their efficiency and safety or development of alternative transfer systems could be the solutions for these problems. Gene transfer technologies are important for gene therapy and should demonstrate effective, target-specific and acceptable safety profiles. For this reason, searching for alternatives to current systems is a necessity.

Development and characterization of vancomycin-loaded levan-based microparticular system for drug delivery.

Encapsulation of vancomycin (VANCO) into biodegradable levan microparticles was achieved using a simple preparation technique. Microparticles were prepared by using levan polysaccharide produced by a halophilic bacterium Halomonas smyrnensis AAD6T. To optimize efficiency of encapsulation process by precipitation method, three parameters were studied: drug and polymer concentrations and preparation rotating speed. The particles were characterized in vitro. The size of levan microparticles was changed between 0.404 µm and 1.276 µm. The surface charge was detected between +4.1 mV and +6.5 mV. The highest drug encapsulation capacity of the system was 74.7% and was depending on the polymer concentration. In dissolution studies, initial burst effect around 10-20% from all the formulations was observed and then the release was slowed down and continued at a constant level. In vitro antibiotic release from the microparticles was controlled with the drug carrier system and release fit to Higuchi kinetic model. All the released samples collected at different time intervals during dissolution studies have exhibited intrinsic bactericidal activity against Bacillus subtilis ATCC 6633. WST-1 cell proliferation and viability studies showed that VANCO-loaded levan microparticles at concentrations between 100 µg/mL and 1000 µg/mL were nontoxic to L929 cells. As conclusion, levan microparticulate system could be a potential carrier of antibiotic drugs such as VANCO.

Topical drug delivery using chitosan nano- and microparticles.

INTRODUCTION: Topical drug delivery offers important benefits for improving the therapeutic effect and reducing systemic side effects of the administered compounds. In addition, utilization of biopolymeric material-based systems can play a key role in developing new topical dosage forms and their applications. This review describes the advances that have been made, new strategies and as well as possible challenges of particular systems of chitosan used in topical drug delivery, including challenging innovations in topical usage of these systems that can make significant impact on clinical practice. AREAS COVERED: The main area covered is hypothesis that particulate carriers based on chitosan and its derivatives can penetrate the topical barriers from the body. For this reason, the novel studies described emphasize the fact that chitosan-based particular systems are popular that can be tailor-made according to in vitro and in vivo characterization. Such parameters, which are known to influence their in vivo performance, can be modulated by adjusting the formulation conditions of the chitosan-based particular systems for topical application. EXPERT OPINION: The topical application of drugs with particulate systems comprising a natural polymer, chitosan, is one of the most popular drug delivery routes. The aim of topical use of chitosan particles is to improve the drug bioavailability by prolonging the residence time of drugs applied topically or by enhancing the passing of drugs through the epithelial cells by opening the tight junctions between epithelial cells and also to reduce the side effects of the drugs.

The design of biodegradable ofloxacin-based core-shell microspheres: influence of the formulation parameters on in vitro characterization.

Ofloxacin (OFL), second-generation fluoroquinolone, is a broad-spectrum antibiotic which is active against both Gram-positive and Gram-negative bacteria. However, OFL has a short biological half life (8-9 h) and poor stability in serum and needs frequently repeated doses during the treatment. The objective of this study was to fabricate the fucospheres and chitosan microspheres containing a poorly soluble drug, OFL, and to compare the formulation parameters influencing the in vitro properties of microparticles such as size, zeta potential, encapsulation efficiency and drug release characteristics. Particle size of fucospheres and chitosan microspheres has been found to be 0.61-1.48 µm and 1.05-2.08 µm, respectively. The zeta potentials have changed between 5.6 mV and 28.0 mV for fucospheres; 22.3 mV and 42.4 mV for chitosan microspheres. The fucospheres have had higher drug encapsulation efficiency than those of chitosan microspheres. The particle size, surface charge, encapsulation efficiency and in vitro drug release from both fucospheres and chitosan microspheres have been affected by type and concentration of the polymers used. The release mechanism from most of the microsphere formulations has been fitted to Higuchi kinetic model. It can be concluded that OFL-encapsulated fucospheres can be a potential delivery system for antibiotics.

Topical application of antisense oligonucleotide-loaded chitosan nanoparticles to rats.

Skin delivery of antisense oligonucleotides (AsODNs) has exciting potential in the treatment of skin diseases. However, the therapeutic applications of oligonucleotide-based therapies are limited by the instability of these molecules toward nucleases, short half-life in vivo, and insufficient cellular uptake. The purpose of this study was to investigate in vivo antisense effect of AsODN-loaded chitosan nanoparticles after topical application. AsODN-loaded chitosan nanoparticles were topically applied to Sprague Dawley rats (adult and baby). At 1, 3, 6, 9, and 12 days posttransfection, animals' skin samples were taken for measurement of beta-galactosidase (beta-Gal) expression and histological control. After topical application of AsODN-loaded chitosan nanoparticles in different doses, beta-Gal expression reduced significantly. Highest inhibition was observed after 6 days of transfection of nanoparticles. Free AsODNs exhibited 35% of beta-Gal inhibition on the first day. beta-Gal expression was inhibited in approximately 82-85% with transfection of nanoparticles containing 30 microg AsODNs at 6 days. The antisense effect of AsODN-loaded chitosan nanoparticle in baby skin was evaluated at 6 days: 77-86% of beta-Gal suppression was measured and differences between the doses were not significant. Thus, chitosan nanoparticles are useful carrier for delivery of AsODNs into skin cells of rats and may be used for topical application on human skin.

Comparison on in vitro characterization of fucospheres and chitosan microspheres encapsulated plasmid DNA (pGM-CSF): formulation design and release characteristics.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine used in the treatment of serious conditions resulting from chemotherapy and bone marrow transplantation such as neutropenia and aplastic anemia. Despite these effects, GM-CSF has a very short biological half-life, and it requires frequent injection during the treatment. Therefore, the cytokine production is possible in the body with plasmid-encoded GM-CSF (pGM-CSF) coding for cytokine administered to the body. However, the selection of the proper delivery system for the plasmid is important. In this study, two different delivery systems, encapsulated plasmid such as fucoidan-chitosan (fucosphere) and chitosan microspheres, were prepared and the particle physicochemical properties evaluated. Fucospheres and chitosan microspheres size ranges are 151-401 and 376-681 nm. The zeta potential values of the microspheres were changed between 8.3-17.1 mV (fucosphere) and +21.9-28.9 mV (chitosan microspheres). The encapsulation capacity of fucospheres changed between 84.2% and 94.7% depending on the chitosan molecular weight used in the formulation. In vitro plasmid DNA release from both delivery systems exhibited slower profiles of approximately 90-140 days. Integrity of released samples was checked by agarose gel electrophoresis, and any additional band was not seen. All formulations were analyzed kinetically. The calculated regression coefficients showed a higher r2 value with zero-order kinetics. In conclusion, the characterizations of the microspheres can be modulated by changing the formulation variables, and it can be concluded that fucospheres might be a potential carrier system for the controlled delivery of GM-CSF encoding plasmid DNA.

Preparation of fucoidan-chitosan hydrogel and its application as burn healing accelerator on rabbits.

Treatment of dermal wounds with macromolecular agents such as natural polymers is one of the research areas of the biomaterial science. Fucoidan is a sulphated polysaccharide which is commonly obtained from seaweeds. The great number of studies on the different pharmacological properties of fucoidan is present, but there is limited information about using of fucoidan in the treatment of dermal burns. The aim of this study was to prepare fucoidan-chitosan hydrogels and to investigate their treatment efficiency on dermal burns. Hydrogels were prepared by swelling the polymers in acidic solution and their swelling, mechanical (hardness, cohesiveness and adhesiveness) and bioadhesive properties were investigated. The viscosity and water absorption capacity of formulations increased with increase in the polymer concentration. In contrast to the cohesiveness results, the adhesiveness of hydrogels increased with the polymer concentration. The bioadhesion was changed between 0.012-0.142 mJ x cm(-2) and enhanced with addition of fucoidan into gel formulations. It was formed dermal burns on seven adult male New Zealand white rabbits and the optimum gel formulation applied on the wounds. Control and treatment group biopsy samples were taken on days 7, 14 and 21 and each burn wound site was evaluated histopathologically. No edema was seen in tested groups except control after 3 d treatment. After 7 d treatment, fibroplasia and scar were fixed on wounds treated with fucoidan-chitosan gel and fucoidan solution. The best regeneration on dermal papillary formation and the fastest closure of the wounds were observed in fucoidan-chitosan hydrogels after 14 d treatment.

Chitosan film containing fucoidan as a wound dressing for dermal burn healing: preparation and in vitro/in vivo evaluation.

The aim of this study was to develop chitosan film containing fucoidan and to investigate its suitability for the treatment of dermal burns on rabbits. Porous films, thickness between 29.7 and 269.0 mum, were obtained by the solvent dropping method. Water vapor permeability (3.3-16.6/0.1 g), the swelling (0.67-1.77 g/g), tensile strength (7.1-45.8 N), and bioadhesion (0.076-1.771 mJ/cm(2)) of the films were determined. The thinnest films were obtained with the lowest chitosan concentration (P < .05). The water absorption capacity of the films sharply increased with the freeze-drying technique. The film having the thickness of 29.7 mum showed the highest amount of moisture permeability (16.6 g/0.1 g). Higher chitosan concentration significantly increased tensile strength of the films (P < .05). Using higher concentration of lactic acid made films more elastic and applicable, and these films were selected for in vivo studies. Seven adult male New Zealand white rabbits were used for the evaluation of the films on superficial dermal burns. Biopsy samples were taken at 7, 14, and 21 days after wounding, and each wound site was examined macroscopically and histopathologically. After 7 days treatment, fibroplasia and scar were observed on wounds treated with fucoidan-chitosan film. The best regenerated dermal papillary formation, best re-epithelization, and the fastest closure of wounds were found in the fucoidan-chitosan film treatment group after 14 days compared with other treatment and control groups. It can be concluded that fucoidan-chitosan films might be a potential treatment system for dermal burns and that changing formulation variables can modulate the characterizations of the films.

In vitro evaluation of enrofloxacin-loaded MLV liposomes.

Fluoroquinolones are broad-spectrum antimicrobial agents that seem to reach their intracellular target site (DNA gyrase) in Escherichia coli by means of an uptake process through the outer and inner membranes. Delivery of quinolones with liposomes has many advantages than the free form of the drug. Liposomes may represent an excellent device for improving the selective transport of antibiotics in these respects. In this study, enrofloxacin-loaded multilamellar vesicles (MLVs) were prepared and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared by using the dry lipid film method. A number of variables, such as phospholipid (DL-alpha -phosphatidylcholine dipalmitoyl), cholesterol, enrofloxacin (ENF), stearylamine, and dicetyl phosphate molar ratios and alpha -tocopherol amounts, were studied. The liposome size, encapsulation capacity, drug release, stability, and electrophoretic mobility of ENF-loaded liposomes were determined. Using this method, spherical MLVs with high drug content could be produced. Particle size of liposomes changed between 1.63 and 3.31 micro m and liposome size was affected by all formulation variables (p < 0.05) except molar ratio of ENF. MLVs can be used as a carrier system for the controlled release of ENF. The highest encapsulation of ENF amount can be obtained using positively charged SA in the formulation and changing the formulation parameters can vary drug release patterns.

Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV.

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 microm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.