A randomised clinical trial study assessing the efficacy of 5% losartan potassium loaded in ethosomal gel to treat human keloids: a trial protocol.

BACKGROUND: Keloid is a skin disorder that results from excessive fibrous tissue growth in the area of the initial trauma. Treating keloids can be challenging since the success of various treatments varies from one study to another. Triamcinolone acetonide injection, a standard treatment, can cause undesirable side effects. Meanwhile, the effectiveness of existing topical therapies for keloids is not always reliable. The pro-inflammatory, pro-proliferative, and pro-fibrotic effects of angiotensin II in human skin contribute to keloid formation. Losartan potassium, an angiotensin II blocker, has the potential to act as an anti-keloid agent. Due to the thicker skin structure of a keloid and ease of application, ethosome gel is chosen as a safe and comfortable carrier for losartan potassium, making it a good choice for treating keloids. METHODS: In this randomised clinical trial, 46 adults with keloids were divided into two treatment groups. One group of 23 participants received 5% losartan potassium loaded in ethosomal gel, while the other group of 23 participants received intralesional injections of 10% triamcinolone acetonide. Over 12 weeks, changes in POSAS 3.0 scores, degree of erythema and pigmentation, surface area, thickness, and pliability of the keloids will be measured at four different times: baseline, 4 weeks, 8 weeks, and 12 weeks. Statistical analysis will be conducted using SPSS software version 24, with a significance level of p < 0.05. DISCUSSION: Losartan potassium is believed to be beneficial for keloid management because it inhibits the angiotensin II receptor, which plays a role in inflammation, proliferation, and fibrosis. This study examines the efficacy of 5% losartan potassium loaded in ethosomal gel for human keloids. TRIAL REGISTRATION: Clinicaltrial.gov identifier NCT05893108 . Registered on 7 June 2023.

Organically surface engineered mesoporous silica nanoparticles control the release of quercetin by pH stimuli.

Controlling the premature release of hydrophobic drugs like quercetin over physiological conditions remains a challenge motivating the development of smart and responsive drug carriers in recent years. This present work reported a surface modification of mesoporous silica nanoparticles (MSN) by a functional compound having both amines (as a positively charged group) and carboxylic (negatively charged group), namely 4-((2-aminoethyl)amino)-4-oxobut-2-enoic acid (AmEA) prepared via simple mechanochemistry approach. The impact of MSN surface modification on physical, textural, and morphological features was evaluated by TGA, N2 adsorption-desorption, PSA-zeta, SEM, and TEM. The BET surface area of AmEA-modified MSN (MSN-AmEA) was found to be 858.41 m2 g-1 with a pore size of 2.69 nm which could accommodate a high concentration of quercetin 118% higher than MSN. In addition, the colloidal stability of MSN-AmEA was greatly improved as indicated by high zeta potential especially at pH 4 compared to MSN. In contrast to MSN, MSN-AmEA has better in controlling quercetin release triggered by pH, thanks to the presence of the functional groups that have a pose-sensitive interaction hence it may fully control the quercetin release, as elaborated by the DFT study. Therefore, the controlled release of quercetin over MSN-AmEA verified its capability of acting as a smart drug delivery system.

Induction of protein specific antibody by carbonated hydroxy apatite as a candidate for mucosal vaccine adjuvant.

Buccal mucosae are considered as a site for vaccine delivery since they are relatively abundant with antigen-presenting dendritic cells, mainly Langerhans cells. In this study, we formulated carbonated hydroxy apatite (CHA) with ovalbumin (OVA) (denoted as CHA-OVA), incorporated it into bilayer buccal membrane to form hydrogel films containing CHA-OVA complex for vaccination via buccal mucosae. Ethylcellulose blend with polyethylene glycol 400 were used as impermeable backing layer. Physical properties of all tested buccal membranes were found suitable for mucosal application. In vitro and ex vivo release study showed there was no burst release of OVA found from all tested formula. From the in vivo examination, rabbit buccal mucosae vaccinated by mucoadhesive membranes containing CHA-OVA complex demonstrated mucosal specific antibody induction, represented the potential of CHA as a candidate of needle-free vaccine adjuvant. Future research is awaiting to investigate proper CHA crystallinity in complex with protein against targeted diseases.

Self-nanoemulsifying Delivery of Andrographolide: Ameliorating Islet Beta Cells and Inhibiting Adipocyte Differentiation.

Purpose: Insulin resistance is a characteristic of non-insulin-dependent diabetes mellitus associated with obesity and caused by the failure of pancreatic beta cells to secrete sufficient amount of insulin. Andrographolide (AND) improves beta-cell reconstruction and inhibits fat-cell formation. This research aimed to improve the delivery of water-insoluble AND in self-nanoemulsifying (ASNE) formulation, tested in streptozotocin (STZ)-induced diabetic rats and 3T3-L1 preadipocyte cells. Methods: A conventional formulation of AND in suspension was used as a control. The experimental rats were orally administered with self-nanoemulsifying (SNE) and suspension of AND for 8 days. Measurements were performed to evaluate blood glucose levels in preprandial and postprandial conditions. Immunohistochemistry was used to assess the process of islet beta cell reconstruction. In vitro study was performed using cell viability and adipocyte differentiation assay to determine the delivery of AND in the formulation. Results: ASNE lowered blood glucose levels (day 4) faster than AND suspension (day 6). The histological testing showed that ASNE could regenerate pancreatic beta cells. Therefore, ASNE ameliorated pancreatic beta cells. The in vitro evaluation indicated the inhibition of adipocyte differentiation by both AND and ASNE, which occurred in a time-dependent manner. ASNE formulation had better delivery than AND. Conclusion: ASNE could improve the antidiabetic activity by lowering blood glucose levels, enhancing pancreatic beta cells, and inhibiting lipid formation in adipocyte cells.

MicroRNA-155-5p Diminishes in Vitro Ovarian Cancer Cell Viability by Targeting HIF1α Expression.

Purpose: Ovarian cancer is the most lethal of gynecological malignancies. Recently, the development of microRNA (miRNA) -based therapeutics that could impact broad cellular programs, leading to inhibition of cancer cell viability, is gaining attention in the therapeutic landscape. The therapy is based on the presence of aberrant expressions of miRNA in cancer cells. Decreasing of tumor suppressor miRNA expression causes upregulation of oncoprotein, which worsens the prognosis of the ovarian cancer. Methods: miR-155-5p mimics were carried by chitosan nanoparticles using new nanotechnology methods. Cellular uptake of miRNA was assessed by fluorescence microscope while MTT and qPCR assay were used to determine miRNA profile and the effect of CS-NP/miRNA on SKOV3 cells. Results: Results of profiling validated using quantitative realtime-polymerase chain reaction (PCR) found one of the most altered tumor suppressor miRNAs, miR-155-5p was downregulated 892.15-fold. According to bioinformatic analysis we identified the miRNA could recognize and regulate HIF1α expression. Transfection of mimics for miR-155-5p showed significantly increased miR-155-5p endogen SKOV3 expression level compared to the control group. We found differences after transfection mimics for miR-155-5p 31.5 and 63 nanoMolar. Increasing of miR-155-5p endogen lead to diminished SKOV3 viability (by 30%; <0.05 at concentration 80 nanoMolar). These mimics may cause an increase in upregulated miR-155-5p endogen that can reduce HIF1α expression. Here we found 2-fold and 2.8-fold reduction of HIF1α expression level after transfection compared to the control group. Conclusion: According to these findings, the mimics miR-155-5p can inhibit ovarian cancer cell proliferation by regulating HIF1α expression.

Development of polymeric nanoparticle gel prepared with the combination of ionic pre-gelation and polyelectrolyte complexation as a novel drug delivery of timolol maleate.

OBJECTIVE: The purpose of this study was to overcome the undesired systemic absorption of skin topical administration of timolol maleate (TM) by developing the TM nanoparticle gel. METHODS: TM-loaded nanoparticle (TMNP) was prepared by ionic pre-gelation of pectin (PCN) and calcium ions (CI) followed with polyelectrolyte complex using chitosan (CHI). TMNP was characterized by measuring the particle size, polydispersity index, zeta potential, encapsulation efficiency (EE), and the interaction between formula constituents. TM-loaded nanoparticle gel (TMNG) was prepared by using hydroxypropyl methylcellulose (HPMC) and was characterized by measuring the spreadability, pH, viscosity, and drug content. The drug release kinetics were analyzed using DDSolver add-in program. RESULTS: TMNP possessed particle size of 175.2 ± 19.7 nm, polydispersity index of 0.528 ± 0.113, zeta potential of -10.86 ± 0.87 mV, and EE of 27.45 ± 2.34%. The electrostatic interactions between PCN, CI, and CHI that formed the nanoparticles were confirmed by the result of vibrational spectroscopy analysis. TMNG possessed spreadability of 60.80 ± 1.38 cm2, pH of 5.154 ± 0.004, viscosity of 269.07 ± 5.83 cP, and drug content of 107.38 ± 1.77%. TM showed a sustained release manner within 24 h by following Korsmeyer-Peppas kinetical model with non-Fickian release mechanism. CONCLUSION: The prepared nanoparticle gel can be an effective controlled release system of TM that administered topically on the skin surface.

The Effects of Combination of Mimic miR-155-5p and Antagonist miR-324-5p Encapsulated Chitosan in Ovarian Cancer SKOV3.

OBJECTIVE: Ovarian cancer is a malignant tumor that attacks reproductive organs of women. MicroRNA is known to have an involvement in the prognosis of ovarian cancer. One of them is miR-155-5p which is down regulated and miR-324-5p which is up regulated. Chitosan is used as microRNA delivery system. The aims of this study is to find out the effects of combination microRNA encapsulated chitosan in cell line SKOV3. METHODS: Cell line SKOV3 obtained from Stem Cell and Cancer Institute (Kalbe). Mimic miR-155-5p and Antagonist miR-324-5p formulated with chitosan. Total RNA was extracted from nine samples (three as control and six as treatment), and prepared for cDNA synthesis. Expression of RNA and mRNA target was measured using q-PCR Biorad CFX96 C.100 and Gen Ex 7 software. Statistics analysis was measured using SPSS 16.0. RESULTS: The administration of combination microRNA encapsulated with chitosan affect the expression of miR-155-5p and miR-324-5p endogen (p <0.05). The expression of mRNA target HIF1α and GLI1 was down regulated after treatment. The correlation between expression of microRNA and mRNA target was strongly (p <0.05). CONCLUSION: This study successfully presented effects of combination of mimic miR-155-5p and antagonist miR-324-5p encapsulated chitosan which be considered as a potential therapy targets for ovarium cancer.

pH-Triggered Drug Release Controlled by Poly(Styrene Sulfonate) Growth Hollow Mesoporous Silica Nanoparticles.

In the current report, hollow mesoporous silica (HMS) nanoparticles were successfully prepared by means of a hard-templating method and further modified with poly(styrene sulfonate) (PSS) via radical polymerization. Structural analysis, surface spectroscopy, and thermogravimetric characterization confirmed a successful surface modification of HMS nanoparticles. A hairy PSS was clearly visualized by high-resolution transmission electron microscopy measurement, and it is grown on the surface of HMS nanoparticles. The Brunauer-Emmett-Teller surface area and average pore size of HMS nanoparticles were reduced after surface modification because of the pore-blocking effect, which indicated that the PSS lies on the surface of nanoparticles. Nevertheless, the PSS acts as a "nano-gate" to control the release of curcumin which is triggered by pH. The drug-release profile of unmodified HMS nanoparticles showed a stormed release in both pH 7.4 and 5.0 of phosphate buffer saline buffer solution. However, a slow release (9.92% of cumulative release) of curcumin was observed at pH 7.4 when the surface of HMS nanoparticles was modified by PSS. The kinetic release study showed that the curcumin release mechanism from PSS@HMS nanoparticles followed the Ritger-Peppas kinetic model, which is the non-Fickian diffusion. Therefore, the PSS-decorated HMS nanoparticles demonstrate potential for pH-triggered drug release transport.

Development of Nanoemulsion-based Hydrogel Containing Andrographolide: Physical Properties and Stability Evaluation.

INTRODUCTION: Andrographolide is a compound that shows various pharmacological activities, which can be applied topically or orally. Nanoemulsion can improve drug solubility and stability, but has limitations for topical application. Incorporation of nanoemulsion into hydrogel can increase the viscosity of the system which can prolong the drug residence time. The aim of this study was to develop andrographolide nanoemulsion-based hydrogel for topical application. METHOD: Andrographolide nanoemulsion was prepared using Capryol 90 as the oil, Kolliphor RH 40 as the surfactant, and propylene glycol as the cosurfactant. Droplet size and polydispersity index of the nanoemulsions were evaluated using particle size analyzer. D-optimal mixture design was employed to generate the total number of runs (formulation), and obtain the optimum formulation. Fourteen formulations of nanoemulsion-based hydrogel were prepared by incorporating nanoemulsion into the hydrogel base (1:1). Carbopol was employed as the gelling agent, whereas other excipients including propylene glycol, oleic acid, triethanolamine, methylparaben, and propylparaben were also added to produce hydrogel base. Nanoemulsion-based hydrogel was evaluated for its pH, viscosity, and physical appearance (after 8 weeks of storage). RESULTS: The result revealed that nanoemulsion-based hydrogel containing 34.65% of carbopol, 1.35% of triethanolamine, and 9% of propylene glycol was selected as an optimum formulation which shows acceptable pH, viscosity, and physical appearance. This optimum nanoemulsion-based hydrogel has pH of 6.50 ± 0.02, and 2492.33 ± 36.91 cP of viscosity with milky white color, and smooth homogeneous texture. CONCLUSION: This study suggested that andrographolide can be successfully formulated into an acceptable nanoemulsion-based hydrogel.

Assessment of Fractional Factorial Design for the Selection and Screening of Appropriate Components of a Self-nanoemulsifying Drug Delivery System Formulation.

Purpose: Recently, a self-nanoemulsifying drug delivery system (SNEDDS) has shown great improvement in the enhancement of drug bioavailability. The selection of appropriate compositions in the SNEDDS formulation is the fundamental step towards developing a successful formulation. This study sought to evaluate the effectiveness of fractional factorial design (FFD) in the selection and screening of a SNEDDS composition. Furthermore, the most efficient FFD approach would be applied to the selection of SNEDDS components. Methods: The types of oil, surfactant, co-surfactant, and their concentrations were selected as factors. 26 full factorial design (FD) (64 runs), 26-1 FFD (32 runs), 26-2 FFD (16 runs), and 26-3 FFD (8 runs) were compared to the main effect contributions of each design. Ca-pitavastatin (Ca-PVT) was used as a drug model. Screening parameters, such as transmittance, emulsification time, and drug load, were selected as responses followed by particle size along with zeta potential for optimized formulation. Results: The results indicated that the patterns of 26 full FD and 26-1 for both main effects and interactions were similar. 26-3 FFD lacked adequate precision when used for screening owing to the limitation of design points. In addition, capryol, Tween 80, and transcutol P were selected to be developed in a SNEDDS formulation with a particle size of 69.7± 5.3 nm along with a zeta potential of 33.4± 2.1 mV. Conclusion: Herein, 26-2 FFD was chosen as the most efficient and adequate design for the selection and screening of SNEDDS composition. The optimized formulation fulfilled the requirement of a quality target profile of a nanoemulsion.

Understanding the effect of lipid formulation loading and ethanol as a diluent on solidification of pitavastatin super-saturable SNEDDS using factorial design approach.

Solidification of a preconcentrate lipid formulation namely self-nano emulsifying drug delivery system (SNEDDS) is required to achieve feasibility, flexibility, and a new concept of "dry nano-emulsion". The purpose of this study was to assess the effect of SNEDDS loading and ethanol as a diluent on the solidification of pitavastatin supersaturable SNEDDS (S-SNEDDS). A 22 full factorial design approach with a center point addition as a curvature was implemented to determine the effect of S-SNEDDS loading and ethanol on the physical characteristics, namely flowability, compactibility, and drug release behavior. Vibrational spectra, thermal behavior, and morphology of solid S-SNEDDS formulation were also evaluated. The results indicated that there was no interaction between S-SNEDDS and carrier, based on vibrational spectra. However, thermal behaviors (enthalpy and weight loss) were depending on SNEDDS loading. Thereafter, the ethanol as a diluent of preconcentrated formulation had no effect on the morphology of carrier structure. However, the S-SNEDDS loading altered the structure of carrier owing to either solubilization or abrasion processes. The statistical model suggested that ethanol as diluent reduced the flowability, compactibility, and drug releases. Meanwhile, the liquid SNEDDS loading affected the reducing of flowability and compactibility. Finally, solidification without diluent and 20% lipid formulation load was recommended. In addition, it was very useful because of ease on handling, flexibility for further formulation, and desired characteristics of final solid dosage form.

Evaluation of the efficacy and toxicity of massoia oil nanoemulsion.

In order to enhance essential oil's stability and water insolubility, Massoia aromatica oil nanoemulsion was formulated and tested on the planktonic growth and biofilm formation of Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans; macrophage phagocytosis and on Vero cells viability. Oil in water nanoemulsion formula was optimized by using several solvents and co-solvents composition. The stability test of the formula was conducted by using a six cycle's freeze-thaw technique. Particle size and morphology were analyzed using a particle size analyzer and transmission electron microscopy. Microbial growth, biofilm formation inhibition, and cytotoxicity assays were performed on the optimized formula by using micro dilution methods. Mice macrophage phagocytosis activities against latex and C. albicans in the presence of samples were evaluated. Massoia nanoemulsion was obtained as a transparent yellowish emulsion having 99.6-99.9% of transmittance; physically and chemically stable; showed stronger antibacterial and antibiofilm on P. aeruginosa and S. aureus, moderate to C. albicans; no significant different on phagocytic activities. The IC50 of massoia oil nanoemulsion and massoia oil towards Vero cells were 35.9µg/mL and 107.5µg/mL respectively. Massoia oil nanoemulsion can protect the stability and decreases the hydrophobicity of the oil, conserve the antimicrobial and immunomodulatory activities, but increases its cytotoxicity.

In Vitro Evaluation of Chitosan-DNA Plasmid Complex Encoding Jembrana Disease Virus Env-TM Protein as a Vaccine Candidate.

INTRODUCTION: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. MATERIAL AND METHODS: Cloning of the DNA vaccine was successfully performed on E. coli DH5α and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. RESULTS: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. CONCLUSION: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.

Formulation of nanoparticles ribosome inactivating proteins from Mirabilis jalapa L. (RIP MJ) conjugated AntiEpCAM antibody using low chain chitosan-pectin and cytotoxic activity against breast cancer cell line.

Ribosome Inactivating Proteins (RIPs) isolated from Mirabilis jalapa L. (MJ protein) leaves showed high cytotoxic effect on malignant. Chitosan nanoparticles have frequently been used in protein delivery applications. The aim of this study was to develop targeted drug delivery system of RIP MJ for breast cancer therapy with chitosan nanoparticles conjugated antiEpCAM antibody. RIP MJ nanoparticles were prepared using low viscous chitosan and pectin using polyelectrolit complex method, followed by conjugation process with antiEpCAM antibody. Characterization of this formula was then carried out for its entrapment efficiency, particles size, zeta potential, morphology using transmission electron microscope (TEM) and cytotoxic assay against T47D and Vero cell line. The optimal concentration of MJ protein; low viscous chitosan; pectin for preparing AntiEpCAM conjugated of RIP MJ nanoparticles was 0.1%; 0.01%;1% (m/v) respectively and showed satisfactory formula with the average particle size of 376.8±105.2nm, polydispersity index (PI) 0.401, zeta potential 43,71 mV, high entrapment efficiency 98,97±0,12%. Transmission electron microscope (TEM) imaging showed a spherical and homogenous structure for nanoparticles. The in vitro cytotoxicity analysis showed that RIP MJ nanoparticle had more cytotoxic effect compared to unformulated RIP against T47D cell-lines. AntiEpCAM conjugated RIP MJ nanoparticles however, increased cytotoxic effect of RIPs on Vero cell-lines not for T47D cell-lines. Chitosan-Pectin nanoparticles suitable for delivering protein to target cancer cells.

Formulation and Cytotoxicity of Ribosome-Inactivating Protein Mirabilis Jalapa L. Nanoparticles Using Alginate-Low Viscosity Chitosan Conjugated with Anti-Epcam Antibodies in the T47D Breast Cancer Cell Line.

Ribosome-inactivating protein (RIP) from Mirabilis jalapa L. leaves has cytotoxic effects on breast cancer cell lines but is less toxic towards normal cells. However, it can easily be degraded after administration so it needs to be formulated into nanoparticles to increase its resistance to enzymatic degradation. The objectives of this study were to develop a protein extract of M. jalapa L. leaves (RIP-MJ) incorporated into nanoparticles conjugated with Anti-EpCAM antibodies, and to determine its cytotoxicity and selectivity in the T47D breast cancer cell line. RIP-MJ was extracted from red-flowered M. jalapa L. leaves. Nanoparticles were formulated based on polyelectrolyte complexation using low viscosity chitosan and alginate, then chemically conjugated with anti-EpCAM antibody using EDAC based on carbodiimide reaction. RIP-MJ nanoparticles were characterised for the particle size, polydispersity index, zeta potential, particle morphology, and entrapment efficiency. The cytotoxicity of RIP-MJ nanoparticles against T47D and Vero cells was then determined with MTT assay. The optimal formula of RIP-MJ nanoparticles was obtained at the concentration of RIP-MJ, low viscosity chitosan and alginate respectively 0.05%, 1%, and 0.4% (m/v). RIP-MJ nanoparticles are hexagonal with high entrapment efficiency of 98.6%, average size of 130.7 nm, polydispersity index of 0.380 and zeta potential +26.33 mV. The IC50 values of both anti-EpCAM-conjugated and non-conjugated RIP-MJ nanoparticles for T47D cells (13.3 and 14.9 µg/mL) were lower than for Vero cells (27.8 and 33.6 µg/mL). The IC50 values of conjugated and non- conjugated RIP-MJ for both cells were much lower than IC50 values of non-formulated RIP-MJ (>500 µg/mL).

HEC-cysteamine particles: influence of particle size, zeta potential, morphology and sulfhydryl groups on permeation enhancing properties.

Within this study, the influence of particle size and zeta potential of hydroxyethyl cellulose-cysteamine particles on permeation enhancing properties was investigated. Particles were prepared by four different methods namely ionic gelation, spray drying, air jet milling and grinding. Particles prepared by grinding were additionally air jet milled. All particles were characterized in terms of particle size and zeta potential. The transport of fluorescein isothiocyanate-dextran 4 (FD4) across Caco-2 cell monolayers in the presence of these particles and the decrease in transepithelial electrical resistance (TEER) was evaluated. The cytotoxic effect of the particles was investigated using resazurin assay. Nanoparticles displaying a zeta potential of 3.3 ± 1.3 mV showed the highest enhancement of FD4 transport among all particles with a 5.83-fold improvement compared to buffer only. Due to the larger particle size, particles generated by grinding exhibited a lower capability in opening of tight junctions compared to smaller particles generated by air jet milling. In addition, the results of the transport studies were supported by the decrease in the TEER. All particle formulations tested were comparatively non-cytotoxic. Accordingly, the zeta potential and particle size showed a significant impact on the opening of tight junctions and hence could play an important role in the design of hydroxyethyl cellulose (HEC)-cysteamine-based nano- and micro-particles as drug delivery systems.

Thiolated hydroxyethyl cellulose: design and in vitro evaluation of mucoadhesive and permeation enhancing nanoparticles.

Within this study, HEC-cysteamine nanoparticles with free thiol groups in the range of 117-1548 µmol/g were designed and characterized. Nanoparticles were generated via ionic gelation of the cationic polymer with tripolyphosphate (TPP) followed by covalent crosslinking via disulfide bond formation using H2O2 as oxidant. The mean diameter of the particles was in the range of 270-360 nm, and zeta potential was determined to be +4 to +10 mV. Nanoparticles were evaluated in terms of mucoadhesive, permeation enhancing, and biocompatible properties as well as biodegradability. The particles remained attached to porcine intestinal mucosa up to 70% after 3h of incubation. The more nanoparticles were oxidized; however, the less were their mucoadhesive properties. Nanoparticles applied in a concentration of 0.5% (m/v) with the highest content of free thiol groups improved the transport of fluorescein isothiocyanate dextran 4 (FD4) across Caco-2 cell monolayer 3.94-fold in comparison with control (buffer). In addition, the transport of FD4 was even 1.84-fold enhanced in the presence of 0.5% (m/v) nanoparticles with the lowest free thiol group content. The higher the disulfide bond content within nanoparticles was, to a lower degree nanoparticles were hydrolyzed by cellulase. None of these nanoparticles showed pronounced cytotoxicity. Accordingly, HEC-cysteamine could be a promising excipient for nanoparticulate delivery systems for poorly absorbed drugs.

An oral oligonucleotide delivery system based on a thiolated polymer: Development and in vitro evaluation.

The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 µmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41% (DNase I) and 60% (intestinal fluid) were degraded in the presence of 0.02% (m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes.

Thiolated chitosan nanoparticles: transfection study in the Caco-2 differentiated cell culture.

The aim of this study was to monitor the expression of secreted protein in differentiated Caco-2 cells after transfection with nanoparticles, in order to improve gene delivery. Based on unmodified chitosan and thiolated chitosan conjugates, nanoparticles with the gene reporter pSEAP (recombinant Secreted Alkaline Phosphatase) were generated at pH 4.0. Transfection studies of thiolated chitosan in Caco-2 cells during the exponential growth phase and differentiation growth phase of the cells led to a 5.0-fold and 2.0-fold increase in protein expression when compared to unmodified chitosan nanoparticles. The mean particle size for both unmodified chitosan and cross-linked thiolated chitosan nanoparticles is 212.2 ± 86 and 113.6 ± 40 nm, respectively. The zeta potential of nanoparticles was determined to be 7.9 ± 0.38 mV for unmodified chitosan nanoparticles and 4.3 ± 0.74 mV for cross-linked thiolated chitosan nanoparticles. Red blood cell lysis evaluation was used to evaluate the membrane damaging properties of unmodified and thiolated chitosan nanoparticles and led to 4.61 ± 0.36% and 2.29 ± 0.25% lysis, respectively. Additionally, cross-linked thiolated chitosan nanoparticles were found to exhibit higher stability toward degradation in gastric juices. Furthermore the reversible effect of thiolated chitosan on barrier properties was monitored by measuring the transepithelial electrical resistance (TEER) and is supported by immunohistochemical staining for the tight junction protein claudin. According to these results cross-linked thiolated chitosan nanoparticles have the potential to be used as a non-viral vector system for gene therapy.

Chitosan-thioglycolic acid conjugate: an alternative carrier for oral nonviral gene delivery?

Regarding safety concerns, nonviral gene delivery vehicles that have the required efficiency and safety for use in human gene therapy are being widely investigated. The aim of this study was to synthesize and evaluate a thiolated chitosan to improve the efficacy of oral gene delivery systems. Thiolated chitosan was synthesized by introducing thioglycolic acid (TGA) to chitosan via amide bond formation mediated by a carbodiimide. Based on this conjugate, nanoparticles with pDNA were generated at pH 4.0 and 5.0. Cytotoxicity of the thiolated chitosan/pDNA nanoparticles on Caco-2 cells was evaluated. The diameter of thiolated chitosan/pDNA nanoparticles was in the range of 100-200 nm. The zeta potential was determined to be 5-6 mV. Due to stability toward nucleases, the transfection rate of thiolated chitosan/pDNA nanoparticles was fivefold higher than that of unmodified chitosan/pDNA nanoparticles. Lactate dehydrogenase tests for thiolated chitosan/pDNA (pH 4.0 and 5.0) showed that (3.79 +/- 0.23)% and (2.9 +/- 0.13)% cell damage. According to these results, thiolated chitosan represents promising excipients for preparation DNA nanoparticles in nonviral gene delivery system.