Development, Evaluation, and Molecular Dynamics Study of Ampicillin-Loaded Chitosan-Hyaluronic Acid Films as a Drug Delivery System.

Periodontitis is an inflammatory periodontal disease defined by the progressive loss of tissues surrounding the tooth. Ampicillin is an antibiotic for managing and treating specific bacterial infections, including periodontitis. Periodontal pockets occur due to periodontal disease progression and act as a natural reservoir that is easily reachable for the insertion of a delivery system, and the amount of drug to be released has a major role in the efficiency of treatment of the disease. Polyelectrolyte complexes (PECs), particularly those based on chitosan and hyaluronic acid combinations, offer a promising avenue to overcome the challenges associated with drug delivery. These complexes are both biodegradable and biocompatible, making them an optimal choice for enabling targeted drug delivery. This study centers on developing and assessing the structure and dynamic attributes of a drug-PEC system encompassing ampicillin and chitosan-hyaluronic acid components, which represents a targeted drug delivery system to better alleviate the periodontitis. To achieve this goal, we conducted experiments including weight and drug content uniformity, swelling index, drug release %, FT-IR and SEM analyses, and atomistic molecular dynamics simulations on the drug PECs loaded with ampicillin with varying amounts of hyaluronic acid. All simulations and the experimental analysis suggested that increased HA amount resulted in an increase in drug release % and swelling index. The simulation outcomes provide insights into the nature of the drug and PEC interactions alongside transport properties such as drug diffusion coefficients. These coefficients offer valuable insights into the molecular behavior of ampicillin-PEC drug delivery systems, particularly in the context of their application in periodontitis treatment.

Evaluation of the Effect of Honey-Containing Chitosan/Hyaluronic Acid Hydrogels on Wound Healing.

The 3D polymeric network structure of hydrogels imitates the extracellular matrix, thereby facilitating cell growth and differentiation. In the current study, chitosan/hyaluronic acid/honey coacervate hydrogels were produced without any chemicals or crosslinking agents and investigated for their wound-healing abilities. Chitosan/hyaluronic acid/honey hydrogels were characterized by FTIR, SEM, and rheology analysis. Moreover, their water content, water uptake capacities, and porosity were investigated. In FT-IR spectra, it was discovered that the characteristic band placement of chitosan with hyaluronic acid changed upon interacting with honey. The porosity of the honey-containing hydrogels (12%) decreased compared to those without honey (17%). Additionally, the water-uptake capacity of honey-containing hydrogels slightly decreased. Also, it was observed that hydrogels' viscosity increased with the increased hyaluronic acid amount and decreased with the amount of honey. The adhesion and proliferation of fibroblast cells on the surface of hydrogel formulations were highest in honey-containing hydrogels (144%). In in vivo studies, wound healing was accelerated by honey addition. It has been demonstrated for the first time that honey-loaded chitosan-hyaluronic acid hydrogels, prepared without the use of toxic covalent crosslinkers, have potential for use in wound healing applications.

Preparation and in vitro characterization of curcumin loaded Chitosan-Hyaluronic acid polyelectrolyte complex based hydrogels.

OBJECTIVE: The manuscript aims to prepare and comprehensively characterize curcumin-loaded chitosan-hyaluronic acid polyelectrolyte complex (PEC) hydrogels through in vitro assessments. By elucidating the formulation process, physicochemical attributes, and drug release kinetics, the study contributes to the producing of curcumin loaded new drug delivery system. SIGNIFICANCE: This approach shows the unique synergy of the chosen polymers with curcumin. The meticulous in vitro analysis of the hydrogels cements their novel attributes, underlining their potential as efficacious and biocompatible curcumin carriers. METHODS: To configure the optimum formulation variables, viscosity, swelling ratio, porosity, in vitro release, cell viability, and migration rate were determined. In addition, FTIR and SEM analyses were also carried out to define the characteristic of formulations. RESULTS: Release kinetic determination is essential in estimating the release behavior of formulation in the body. All formulations showed Higuchi release kinetics, indicating that drug release from the semi-solid matrix was diffusion controlled. CONCLUSION: As a result, in this study, a new formulation was produced based on a simple concept with acceptable quality parameter results promising to be conducted in the industry.

Combination therapy with chitosan/siRNA nanoplexes targeting PDGF-D and PDGFR-ß reveals anticancer effect in breast cancer.

BACKGROUND: Platelet derived growth factors (PDGF)-D and the expression of its receptor increase in neoplastic progression of cancer. Co-silencing of growth factor and receptor can be suggested as an important strategy for effective cancer therapy. In the present study, we hypothesized that suppression of PDGF-D signaling pathway with small interfering RNAs (siRNAs) targeting both PDGF-D and PDGF receptor (PDGFR)-ß is a promising strategy for anticancer therapy. METHODS: Chitosan nanoplexes containing dual and single siRNA were prepared at different weight ratios and controlled by gel retardation assay. Characterization, cellular uptake, gene silencing and invasion studies were performed. The effect of nanoplexes on breast tumor growth, PDGF expression and apoptosis was investigated. RESULTS: We have shown that downregulation of PDGF-D and PDGFR-ß with chitosan/siRNA nanoplex formulations reduced proliferation and invasion in breast cancer cells. In the in vivo breast tumor model, it was determined that the intratumoral administration of chitosan/siPDGF-D/siPDGFR-ß nanoplexes markedly decreased the tumor volume and PDGF-D and PDGFR-ß mRNA and protein expression levels and increased apoptosis. CONCLUSIONS: According to the results obtained, we evaluated the effect of PDGF-D and PDGFR-ß on breast tumor development and showed that RNAi-mediated inhibition of this pathway formulated with chitosan nanoplexes can be considered as a new breast cancer therapy strategy.

EGFR blocker lapatinib inhibits the synthesis of matrix metalloproteinases from synovial fibroblasts.

BACKGROUND: Epidermal growth factor receptor (EGFR) family members and their associated ligands may be related to bone and joint destruction in rheumatoid arthritis. Matrix metalloproteinases are responsible for joint and bone tissue degradation. This study is intended to investigate the effect of epidermal growth factor receptor inhibition by lapatinib on the synthesis of matrix metalloproteinases in in vitro. METHODS: Synovial fibroblast cell culture was obtained from a patient with rheumatoid arthritis who underwent knee arthroplasty. Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were added to the cell culture to stimulate synovial fibroblast cells and create an inflammatory character. Understimulated and nonstimulated conditions, lapatinib was applied to the culture in four different concentrations of 25, 50, 100, and 200 µmol. Then, matrix metalloproteinase -1, -3, and, -13 levels were assessed. RESULTS: When stimulated with IL-1ß and TNF-α, the synthesis of matrix metalloproteinases from synovial fibroblast was increased significantly. When lapatinib is added to the stimulated synovial fibroblasts, matrix metalloproteinases synthesis is significantly suppressed. DISCUSSION: Inhibition of the EGFR pathway with lapatinib suppresses matrix metalloproteinases synthesis. Our results suggest EGFR pathway inhibition may be a promising option to prevent joint destruction in the treatment of rheumatoid arthritis.

Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes

Abstract Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to promote the growth, proliferation, and migration of endothelial and keratinocyte cells. Chitosan has been widely used as a biopolymer in wound-healing studies. The aim of this study was to investigate the in vitro proliferative effects of chitosan/pGM-CSF complexes as well as the therapeutic role of the complexes in an in vivo rat wound model. The effect of complexes on cell proliferation and migration was examined. Wounds were made in Wistar-albino rats, and examined histopathologically. The cell proliferation and migration were increased weight ratio- and time-dependently in HaCaT and NIH-3T3 cell lines. Wound healing was significantly accelerated in rats treated with the complexes. These results showed that the delivery of pGM-CSF using chitosan complexes could play an accelerating role in the cell proliferation, migration, and wound-healing process.

Therapeutic Silencing of BCL-2 Using NK Cell-Derived Exosomes as a Novel Therapeutic Approach in Breast Cancer.

Overexpression of the anti-apoptotic protein BCL-2 is frequently observed in multiple malignancies, including about 85% of patients with estrogen receptor positive (ER+) breast cancer. Besides being studied as a prognostic marker, BCL-2 is investigated as a therapeutic target in ER+ breast cancer. Here, we introduce a new exosome-based strategy to target BCL-2 using genetically modified natural killer (NK) cells. The NK cell line NK92MI was lentivirally transduced to express and load BCL-2 siRNAs (siBCL-2) into exosomes (NKExos) and then evaluated for its potential to treat ER+ breast cancer. Transfected NK92MI cells produced substantial levels of BCL-2 siRNAs, without substantially affecting NK cell viability or effector function and led to loading of siBCL-2 in NKExos. Remarkably, targeting BCL-2 via siBCL-2 NKExos led to enhanced intrinsic apoptosis in breast cancer cells, without affecting non-malignant cells. Together, our prototypical results for BCL-2 in breast cancer provide proof of concept for a novel strategy to utilize NKExos as a natural delivery vector for siRNA targeting of oncogenes.

Evaluation of biological activities of Barbarea integrifolia and isolation of a new glucosinolate derivated compound.

The aim of the present study is to determine the potent biological activities and carry out isolation studies on Barbarea integrifolia. The antioxidant capacity of the species was evaluated by total phenolic content, FRAP, CUPRAC, and DPPH radical scavenging activity. Anticancer activity studies were performed by MTT assay in MDA-MB-231, MCF-7, Hep3B, PC-3, A549, HCT116, L-929 cell lines. It was observed that the remaining aqueous fraction has higher total phenolic content while higher activity in the CUPRAC and FRAP assays was displayed for the methanolic extract and chloroform fraction. The extracts showed anticancer activity as compared with vincristine. It was observed that chloroform fraction has the highest anticancer activity on MCF-7 cell line, while ethyl acetate fraction has the highest anticancer activity on Hep-3B and A549 cell lines. Methanolic extract has the highest anticancer activity on HCT116 and MDA-MB-23 cell lines. The isolation studies have been performed using several chromatographic methods. The chemical structures of compounds have been identified by means of 1H NMR, 13C NMR, 2D-NMR, and MS. Five major compounds, one steroid (ß-Sitosterol), one phenolic acid (Rosmarinic acid), one flavonol heteroside (kaempferol 7-O-α-l-rhamnoside-3-O-ß-d-(2-O-ß- d -glucosyl)-ß-d-glucoside), and two glucosinolates (Gluconasturtiin, Gluconasturtiin choline salt) have been isolated.

Relationship between guanosine triphosphate pathway and tetrahydrobiopterin in gestational diabetes mellitus.

PURPOSE: The present study assesses the change in tetrahydrobiopterin (BH4), one of the most important products in the guanosine triphosphate (GTP) pathway and in other parameters that might affect nitric oxide (NO) production, in gestational diabetes mellitus (GDM). METHODS: The study included 100 healthy pregnant women and 100 women diagnosed with GDM. Serum levels of neopterin, BH4 and NO were measured. The levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and guanosine triphosphate cyclohydrolase I (GCHI/GTPCH) gene expression were determined. RESULTS: It was found that diabetes led to an increase in neopterin and NO levels, and a decrease in BH4 levels. A stimulation was observed in eNOS gene expression in the GDM group when compared to the control group, while GCHI levels were found to decrease when compared to the control group. iNOS gene expression was detected in neither the healthy controls nor the patient group. CONCLUSIONS: Decreased NO bioavailability plays an important role in the progression of such macrovascular diseases as diabetes. BH4 levels decrease in diabetes patients, while the increased gene expression of GCHI reverses the diabetes-related BH4 deficiency and allows the endothelial cells to regain their ability to produce NO. Since GCHI is the rate-limiting enzyme in the biosynthesis of BH4, changes in GCHI levels directly affect the BH4 levels and the NO metabolism, leading to an increased risk of macrovascular complications. The significant increase in neopterin levels suggest that this is a potential biomarker for the early diagnosis of GDM.

Modulation of the dual-faced effects of miR-141 with chitosan/miR-141 nanoplexes in breast cancer cells.

BACKGROUND: miR-141, known as a tumor suppressive microRNA, is downregulated in breast cancer. However, recent contrasting studies report that it also acts as oncogene when it is upregulated. The present study aimed to investigate whether miR-141 is a tumor suppressor or oncogenic when it reaches normal levels in chitosan/miR-141 nanoplexes. METHODS: Chitosan nanoplexes were prepared using simple complexation method. Nanoplexes were characterized by a gel retardation assay and zeta potential and particle size measurements. To determine the expression level of miR-141, a quantitative real-time polymerase chain reaction was performed. The effects of miR-141 mimics were investigated with respect to angiogenesis by vascular endothelial growth factor (VEGF), epithelial-mesenchymal transition (EMT) by E-cadherin, metastasis by Igfbp-4 and Tinagl1 enzyme-linked immunosorbent assays, invasion by an invasion chamber, and apoptosis by Annexin V. RESULTS: The miR-141 expression levels of MDA-MB-231 and MDA-MB-435 cells by administration of chitosan/mimic miR-141 nanoplexes reached endogenous miR-141 levels of a non-tumorigenic epithelial breast cell line, MCF-10A. According to our results, metastasis, VEGF, EMT and invasion in breast cancer cells were diminished, whereas apoptosis increased by 1.5- and 2.4-fold in breast cancer cell lines as a result of the miR-141 mimics. CONCLUSIONS: In conclusion, we have demonstrated that administration of miR-141 mimics at the determined doses to breast cancer cells revealed a tumor suppressor effect, and not the oncogenic face. The delivery of miR-141 by chitosan nanoplexes presents a promising approach for the suppression of breast cancer.

The effectiveness of chitosan-mediated silencing of PDGF-B and PDGFR-ß in the mesangial proliferative glomerulonephritis therapy.

Platelet-derived growth factor-B (PDGF-B) is a growth factor that plays an important role in the progression of mesangial proliferative glomerulonephritis (MsPGN). PDGF-B may contribute to mesangioproliferative changes and is overexpressed in MsPGN. Recently, small interfering RNAs (siRNAs) have been widely used for gene silencing effects in experimental models of renal diseases. Nanoparticle-based therapeutics are preferred for reasons such as increasing therapeutic efficacy and reducing toxic effects caused by high doses. The distribution of nanoparticles to the kidney is a significant advantage in siRNA delivery. The aim of this study was to investigate the efficacy of chitosan/siRNA nanoplexes in silencing of PDGF-B and PDGFR-ß genes in kidney and to decrease mesangial cell proliferation and matrix accumulation in MsPGN model induced by anti-Thy-1.1 antibody. The therapeutic effects of chitosan/siPDGF-B + siPDGFR-ß nanoplexes in glomerulonephritic rats were studied by molecular, biochemical, and histopathologic evaluations. Chitosan/siPDGF-B + siPDGFR-ß nanoplexes markedly reduced PDGF-B and PDGFR-ß mRNA and protein expressions in experimental MsPGN model. Histopathologic examination results showed that the silencing of PDGF-B and its receptor PDGFR-ß led to reduction in mesangial cell proliferation and matrix accumulation. The use of chitosan/siPDGF-B + siPDGFR-ß nanoplexes for silencing the PDGF-B pathway in MsPGN can be considered as a new effective therapeutic strategy.

Syntheses of 1,2,3-triazole-bridged pyranose sugars with purine and pyrimidine nucleobases and evaluation of their anticancer potential.

With the aim to create a library of compounds with potential bioactivities by combining special characteristics of two important groups such as nucleobases and carbohydrates, twenty 1,4-disubstituted-triazole nucleosides were synthesized in good yields (80-94%) using the copper catalyzed 'Click' reaction between azido-modified pento- or hexopyranoses and alkyne-bearing pyrimidine or purine nucleobases. Structural elucidation was made with the assistance of spectroscopic techniques such as FTIR, 1D-, 2D-NMR, and ESI-TOFMS. All the synthesized triazole nucleosides were evaluated for their cytotoxic activity against three human cancer cell lines (MDA-MB-231, Hep3B, PC-3) by using the MTT assay. Particularly, compounds 3a and 1b were identified as potential hits against Hep3B cell.

In Vitro Dose Studies on Chitosan Nanoplexes for microRNA Delivery in Breast Cancer Cells.

Changes in microRNA (miRNA) expression levels that play important roles in regulation lead to many pathological events such as cancer. The miR-200 family is an important target in cancer therapy. The aim of this study is to equilibrate endogenous levels between cancer and noncancerous cells to prevent serious side effects of miR-200c- and miR-141-like metastatic colonization. For the first time, the characterization of miR-200c and miR-141 cluster containing chitosan nanoplexes was shown, and the optimization of miRNA expression levels by conducting dose studies in breast cancer cell lines was made. The mean diameter of chitosan/miR-141 and chitosan/miR-200c nanoplexes ranged from 296 to 355 nm and from 294 to 380 nm depending on the N/P ratio, respectively. The surface charge of nanoplexes was positive with zeta potential of +12 to +26 mV. While naked miRNA was degraded after 0 min in a 10% serum-containing medium, chitosan/miRNA nanoplexes were protected for 72 h. During the in vitro cellular uptake study, nanoplexes were observed to be accumulating in the cytoplasm or nucleus. After using different doses for miR-200c, the determined doses are 750, 100, and 750 ng in the MCF-7, MDA-MB-231, and MDA-MB-435 cell lines, respectively. Doses were determined as 100 ng for MDA-MB-231 and 150 ng for MDA-MB-435 to reach endogenous miR-141 levels of MCF-10A. Our results suggest that chitosan nanoplexes for miR-200c and miR-141 are an efficient delivery system in terms of formulation and transfection. As a conclusion, dose studies are important to provide effective treatment with miRNAs.

Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-ß-Containing Chitosan Nanoplexes.

Mesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-ß. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-ß, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-ß gene silencing efficiencies of PDGF-B and PDGFR-ß targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-ß-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease.

Tocilizumab's effect on cognitive deficits induced by intracerebroventricular administration of streptozotocin in Alzheimer's model.

Neuroinflammation plays pivotal roles in the pathogenesis of Alzheimer's disease (AD). IL-6 is pleiotropic cytokine which plays significant pathological role in inflammatory diseases and causes prolonged inflammation. Additionally, IL-6 activates microglia cells and enhances the accumulation of amyloid-ß peptides. Moreover, IL-6 signal transduction is mediated by membrane-bound and soluble IL-6 receptors. Tocilizumab which is a humanized anti-human IL-6 receptor (IL-6R) monoclonal antibody binds to both of these receptors and inhibits IL-6 signaling by this route. The objective was to investigate tocilizumab's potential effects in the treatment of AD. Male Sprague-Dawley rats were divided into three groups: sham (control), streptozotocin (STZ), and tocilizumab-STZ. We used a single dose of intracerebroventricular (ICV) tocilizumab, beginning 1 h prior to injection of STZ for 3 weeks. The rats in STZ and tocilizumab-STZ groups were given ICV-STZ (3 mg/kg). Behavioral parameters were evaluated on days 17-20 and the rats were sacrificed on day-21 to examine histopathological changes. STZ injection caused significant decrease in the mean escape latency in passive avoidance and also declined the performance improvement in Morris water maze tests. Tocilizumab-STZ group significantly improved learning and spatial memory functions by increasing RLT in the passive avoidance and by shortening escape latency in reaching the platform in the Morris water maze. Histopathological changes were examined using hematoxylin and eosin and immunohistochemical (IHC) stainings. IHC analysis revealed that while protein expressions of amyloid-ß (3.5 ± 0.2) and IL-6 (2.9 ± 0.4) showed intense immune-positivity in STZ group, amyloid-ß (1.3 ± 0.1) and IL-6 (1.5 ± 0.2) immunoreactivities were substantially decreased in tocilizumab treatment group. We conclude that tocilizumab treatment attenuated significantly STZ-induced cognitive impairment and histopathological changes. Further studies would be desirable to investigate clinically relevant protective effects of tocilizumab in AD.

Generation of stable cell line by using chitosan as gene delivery system.

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.

The enhancement of gene silencing efficiency with chitosan-coated liposome formulations of siRNAs targeting HIF-1α and VEGF.

RNA interference (RNAi) holds considerable promise as a novel therapeutic strategy in the silencing of disease-causing genes. The development of effective delivery systems is important for the use of small interfering RNA (siRNA) as therapy. In the present study, we investigated the effect on breast cancer cell lines and the co-delivery of liposomes containing siHIF1-α and siVEGF. In order to achieve the co-delivery of siHIF1-α and siVEGF and to obtain lower cytotoxicity, higher transfection and silencing efficiency, in this study, we used chitosan-coated liposomal formulation as the siRNA delivery system. The obtained particle size and zeta potential values show that the chitosan coating process is an effective parameter for particle size and the zeta potential of liposomes. The liposome formulations loaded with siHIF1-α and siVEGF showed good stability and protected siRNA from serum degradation after 24-h of incubation. The expression level of VEGF mRNA was markedly suppressed in MCF-7 and MDA-MB435 cells transfected with chitosan-coated liposomes containing HIF1-α and VEGF siRNA, respectively (95% and 94%). In vitro co-delivery of siVEGF and siHIF1-α using chitosan-coated liposome significantly inhibited VEGF (89%) and the HIF1-α (62%) protein expression when compared to other liposome formulations in the MDA-MB435 cell. The co-delivery of siVEGF and siHIF1-α was greatly enhanced in the vitro gene silencing efficiency. In addition, chitosan-coated liposomes showed 96% cell viability. Considering the role of VEGF and HIF1-α in breast cancer, siRNA-based therapies with chitosan coated liposomes may have some promises in cancer therapy.