Effect of Nutrient Formulations on Permeation of Proteins and Lipids through Porcine Intestine In vitro

Purpose: To investigate the effect of nutrient formulations on the permeation of proteins and lipids through porcine intestine in vitro.Method: In vitro permeation studies of proteins and lipids of two peptide-based formulations, composed of various compounds and sources of hydrolyzed protein was carried out, and compared with a conservative polymeric formulation as control, The test was undertaken using Franz diffusion cell apparatus incorporating porcine intestine.Results: The peptide-based formulation demonstrated higher protein absorption than the conservative polymeric one. However, there were some differences in protein absorption rates between the peptide based formulations obtained from various sources. Formulation A with 1.0 and 1.5 kcal/mL exhibited significantly (p < 0.05) higher cumulative protein permeation (11.97 ± 0.23 and 12.54 ± 0.94 μg/cm2) than formulations B (9.41 ± 0.36 and 9.67 ± 0.35 μg/cm2) and C (8.34 ± 0.56 and 8.61 ± 0.71 μg/cm2), respectively. Lipid permeation from formulations A and B (13.91 ± 0.26 and 12.94 ± 0.59 μg/cm2 respectively for 1.0 kcal/mL formulation, and 13.31 ± 0.21 and 12.86 ± 0.16 for 1.5 kcal/mL formulation) which consist mainly of medium chain triglycerides (MCTs), were significantly (p <0.05) higher than those from formulation C (11.49 ± 0.43 and 12.62 ± 0.38 μg/cm2 for 1.0 and 1.5 kcal/mL formulation, respectively) which mostly contained long chain triglycerides (LCTs).Conclusion: The results reveal that oligomeric formulations have higher absorption rate than polymeric formulations. However, the outcomes when administered to clinically ill patients need to be investigated.Keywords: Nutrient Formulations, Permeation, Proteins, Lipids, Porcine Intestine, Medium Chain Triglycerides, Long Chain Triglyceride

Development of Alginate/Chitosan Microparticles for Dust Mite Allerge

Purpose: To develop chitosan/alginate microparticles for the mucosal delivery of allergen from dust mite (Dermatophagoides pteronyssinus).Methods: Chitosan/alginate microparticles were prepared by ionotropic gelation. The effects of polymer content, crosslinking agent, and preparation method on the physicochemical characteristics of the microparticles as well as their in vitro cytotoxicity were investigated.Results: The microparticles were small (1 - 17 μm) and spherical in shape. The highest allergen content (0.30 ± 0.07 mg/g) was obtained with 2.5 % initial allergen loading in chitosan- triphosphate (CS-TPP) microparticles. Sustained allergen release (approx. 50 % over 24 h) was observed from alginate-coated chitosan microparticles. Allergen incorporation method and initial drug-loading could be varied to obtain optimum particle size with high allergen-loading and sustained release. The cytotoxicity of various microparticle formulations did not differ significantly (p > 0.05 ), as cell viability values were close to 100 %.Conclusion: This study indicates that alginate and alginate-coated chitosan microparticles are safe and can be further developed for mucosal allergen delivery.Keywords: Alginate, Chitosan, Microparticle, Allergen delivery, Dust mite, Dermatophagoides pteronyssinu

Fabrication of Cationic Exchange Polystyrene Nanofibers for Drug Delivery

Purpose: To prepare polystyrene nanofiber ion exchangers (PSNIE) with surface cation exchange functionality using a new method based on electrospinning and also to optimize crosslinking and sulfonation reactions to obtain PSNIE with maximum ion exchange capacity (IEC).Method: The nanofibers were prepared from 15% w/v polystyrene solution in dimethylacetamide (DMAc) containing 0.025 %w/v tetrabutylammonium bromide (TBAB) using electrospinning technique, followed by crosslinking with sulfuric acid/formaldehyde in a ratio ranging from 100/0 to 50/50 v/v and sulfonation in sulfuric acid. Degree of crosslinking was determined as the amount of fibers that remained in dichloromethane. The morphology and diameter of the fibers were evaluated by scanning electron microscopy (SEM) while IEC of PSNIE was performed by salt splitting titration.Results: PSNIE crosslinked with a sulfuric acid/formaldehyde ratio of 90/10 with 0.1 %w/v silver sulfate for 10 min at 70°C and sulfonated in 98 % sulfuric acid with 0.2 %w/v silver sulfate as the catalyst at 100°C for 30 min showed a maximum IEC of 3.21 meq/g-dry-PSNIE. Increase in  sulfonation temperature caused the IEC of PSNIE to increase due to faster sulfonation. It was observed that the higher the temperature the faster the rate of sulfonation reaction. The diameter of the fibers after sulfonation was 404 ± 42 nm.Conclusion: These results indicate that PSNIE can be successfully prepared by electrospinning. Furthermore, cationic drug can be loaded onto the novel PSNIE for controlled release delivery.Keywords: Polystyrene, Ion exchange capacity, Nanofibers, Ion exchangers, Crosslinking, Sulfonation

In Vitro Antioxidant Activity of Chitosan Aqueous Solution: Effect of Salt Form

Purpose: To investigate the effect of salt form on the antioxidant activities of chitosan aqueous solution.Methods: The antioxidant activities of chitosan acetate (CS-acetate), chitosan hydroxybenzotriazole (CS-HOBt), chitosan thiamine pyrophosphate (CS-TPP) and chitosan ethylenediaminetetraacetic acid (CS-EDTA) solution were determined employing various established in vitro system such as superoxide and hydroxyl radicals scavenging, metal ion chelating and reducing power. Their chemical structures were characterized by nuclear magnetic resonance (NMR) and Fourier transform infraredspectrophotometry (FT-IR).Results: NMR and FT-IR show confirmed formation of chitosan salts. The 50 % inhibition concentration (IC50) of superoxide and hydroxyl radicals was 0.349 – 1.34 and 0.34 – 1.54 mg/mL, respectively. Among the salt forms, CS-acetate (IC50 = 0.349 mg/mL) showed the highest superoxide radical scavenging effect while CS-HOBt (IC50 = 0.34 mg/mL) showed the highest hydroxyl radical scavenging effect. With regard to metal ion chelating activity, CS-EDTA showed the highest chelating activity (approx 100 % at 1 mg/mL) while the others showed 20 % activity at a concentration of 1 mg/mL. The results for reducing power indicate that CS-TPP had the highest reducing power.Conclusion: The results indicate that antioxidant activity varied with the salt form. Thus, CS salts may be used as a source of antioxidants for pharmaceutical applications.Keywords: Chitosan, Antioxidant, Hydroxybenzotriazole, Thiamine pyrophosphate, Ethylenediaminetetraacetic aci

Development and Evaluation of Ketoprofen Acrylic Transdermal Patches

Purpose: To fabricate ketoprofen transdermal patches (KTPs) using an acrylic pressure-sensitive adhesive (PSA) polymer.Methods: KTPs were prepared using solvent casting method. The influence of the amount of PSA, drug content, and terpenes as penetration enhancers on the characteristics of the patch, namely, thickness, W/A ratio, and adhesiveness and in vitro skin permeation, were investigated. Scanning electron microscope (SEM) and differential scanning calorimetry (DSC) studies were also performed on the patches. The physical and chemical stability of KTPs after storage at 40 oC, and 75 %RH for 1 month was also evaluated.Results: DSC thermograms demonstrate that the drug was dispersed molecularly in the polymer in all the formulations. Increase in PSA content increased the W/A ratio and adhesiveness of KTPs. Ketoprofen release from the transdermal patches followed the Higuchi diffusion model. Ketoprofen flux increased with increase in the ketoprofen content of the adhesive matrix. Inclusion of terpenes in the patch formulations significantly increased the permeation of ketoprofen through the skin, with enhancement ratio (ER) ranging from 1.4 to 2.6.Conclusion: KTPs formulated with acrylic pressure-sensitive adhesive and incorporating terpenes as permeation enhancers demonstrated suitable characteristics for transdermal delivery of ketoprofen.Keywords: Ketoprofen, Transdermal patch, Skin permeation, Acrylic matrix, Terpenes, Pressuresensitive adhesive

Characterization of 9-Nitrocamptothecin Liposomes: Anticancer Properties and Mechanisms on Hepatocellular Carcinoma In Vitro and In Vivo

BACKGROUND: Hepatocellular carcinoma (HCC) is the third most common cause of cancer related mortality worldwide. 9-Nitrocamptothecin (9NC) is a potent topoisomerase-I inhibitor with strong anticancer effect. To increase the solubility and stability, we synthesized a novel 9NC loaded liposomes (9NC-LP) via incorporating 9NC into liposomes. In the present study, we determined the effects of 9NC and 9NC-LP on in vitro and in vivo, and the underlying mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We first analyzed the characteristics of 9NC-LP. Then we compared the effects of 9NC and 9NC-LP on the proliferation and apoptosis of HepG2, Bel-7402, Hep3B and L02 cells in vitro. We also investigated their anticancer properties in nude mice bearing HCC xenograft in vivo. 9NC-LP has a uniform size (around 190 nm) and zeta potential (∼-11 mV), and exhibited a steady sustained-release pattern profile in vitro. Both 9NC and 9NC-LP could cause cell cycle arrest and apoptosis in a dose-dependent and p53-dependent manner. However, this effect was not ubiquitous in all cell lines. Exposure to 9NC-LP led to increased expression of p53, p21, p27, Bax, caspase-3, caspase-8, caspase-9 and apoptosis-inducing factor, mitochondrion-associated 1 and decreased expression of Bcl-2, cyclin E, cyclin A, Cdk2 and cyclin D1. Furthermore, 9NC-LP exhibited a more potent antiproliferative effect and less side effects in vivo. Western blot analysis of the xenograft tumors in nude mice showed similar changes in protein expression in vivo. CONCLUSIONS/SIGNIFICANCE: In conclusion, 9NC and 9NC-LP can inhibit HCC growth via cell cycle arrest and induction of apoptosis. 9NC-LP has a more potent anti-tumor effect and fewer side effects in vivo, which means it is a promising reagent for cancer therapy via intravenous administration

In Vivo Methods to Study Uptake of Nanoparticles into the Brain

Several in vivo techniques have been developed to study and measure the uptake of CNS compounds into the brain. With these techniques, various parameters can be determined after drug administration, including the blood-to-brain influx constant (Kin), the permeability-surface area (PS) product, and the brain uptake index (BUI). These techniques have been mostly used for drugs that are expected to enter the brain via transmembrane diffusion or by carrier-mediated transcytosis. Drugs that have limitations in entering the brain via such pathways have been encapsulated in nanoparticles (based on lipids or synthetic polymers) to enhance brain uptake. Nanoparticles are different from CNS compounds in size, composition and uptake mechanisms. This has led to different methods and approaches to study brain uptake in vivo. Here we discuss the techniques generally used to measure nanoparticle uptake in addition to the techniques used for CNS compounds. Techniques include visualization methods, behavioral tests, and quantitative methods

RNAi for Treating Hepatitis B Viral Infection

Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection

VLPs and particle strategies for cancer vaccines

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