Intracellular trafficking and drug release from fluorescently-labeled chitosan nanoparticle systems for development of innovative drug delivery systems

The increased bioavailability of essential biomolecules such as drugs, DNA and peptides is pre-requisite for efficient intracellular efficacy on drug delivery systems. Nanotechnological-based approaches for drug delivery applications potentially promotes a better distribution of energy in vivo, increasing the intracellular uptake of biomolecules for enhanced therapeutic uptake. Realising the ubiquitous utilization of nanoparticles in an increasing myriad of research fields, investigations into nanoparticle uptake, cargo release, as well as nanoparticle carrier persistence are pertinent towards their consequent optimization and development. We describe in this work, the elucidation of nanoparticle uptake and sustained release of its encapsulated cargo in colon cancer cells to model a nanoparticle-mediated drug delivery system. Chitosan nanoparticles were synthesized through ionic gelation routes and characterized by means of light scattering, electron microscopy, and infrared spectroscopic analysis. The nanoparticles were encapsulated with a fluorescently-modified amino acid for in vitro tracking, and its intracellular release was quantitated in a time-dependent study using flow cytometry and fluorescent microscopy. Cytotoxic analysis was subsequently performed to evaluate any inherent efficacy of the nanoparticle for use as a candidate delivery system. Findings arising from our analyses showed that intracellular uptake of nanoparticles occurred within 30 mins of cell treatment; and continually took place up to 48 hours post treatment. Interestingly, release of cargo only occurred 6 hours post treatment and a controlled release system was exhibited up to 48 hours without extracellular leakage. MTT assay showed very low toxicity of the 60-180nm size particles; demonstrating a potential of the chitosan nanoparticle system for use as a systemic, slow release system for drug delivery. Conclusions derived from this study is hoped to provide sufficient data towards more critical developments of nanoparticle delivery systems for targeted and enhanced drug delivery parameters, most clinically relevant in the pharmaceutical and medical fields

Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography

Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP)

Microbial degradation of chitin materials by Trichoderma virens UKM1

The current increase in amount of seafood wastes produced by the shrimp industry has lead to the finding of new methods for shrimp waste disposal or waste reused. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma viren UKM1 was used in this study. From preliminary study, commercialized Trichoderma Minimal Medium (TMM) was selected for the degradation study. The substrates used were colloidal chitin as control substrate, sun dried ground and unground shrimp shells. Scanning Electron Microscopy (SEM) studies showed penetration of fungus mycelium into the colloidal chitin as compare to sun dried ground and unground. This observation suggested that colloidal chitin was the best carbon source for modeling the degradation of chitin materials. Stereo microscope studies suggested that the fungus removed (degraded) the chitinous materials layer by layer as indicated by the significant reduction in shell thickness. Shrimp shells were further evaluated for end products in the crude medium using High Performance Liquid Chromatography (HPLC). A simple, rapid, selective and specific HPLC method was developed to quantify glucosamine indirectly using the value of total N-acetyl-glucosamine (NAG) produced which the production of chitooligomer was used as marker. Results showed that the Trichoderma virens UKM1 secretes a significant amount of exochitinase compared to endochitinase by the identification of monomeric N-acetyl-glucosamine (NAG) from the chitinous substrate. The highest specific enzyme activity obtained using colloidal chitin was 14.59 U mg-1. Percentage of residual chitooligomer in impure chitinases samples was 86%

A simple add-and display method for immobilisation of cancer drug on his-tagged virus-like nanoparticles for controlled drug delivery

pH-responsive virus-like nanoparticles (VLNPs) hold promising potential as drug delivery systems for cancer therapy. In the present study, hepatitis B virus (HBV) VLNPs harbouring His-tags were used to display doxorubicin (DOX) via nitrilotriacetic acid (NTA) conjugation. The His-tags served as pH-responsive nanojoints which released DOX from VLNPs in a controlled manner. The His-tagged VLNPs conjugated non-covalently with NTA-DOX, and cross-linked with folic acid (FA) were able to specifically target and deliver the DOX into ovarian cancer cells via folate receptor (FR)-mediated endocytosis. The cytotoxicity and cellular uptake results revealed that the His-tagged VLNPs significantly increased the accumulation of DOX in the ovarian cancer cells and enhanced the uptake of DOX, which improved anti-tumour effects. This study demonstrated that NTA-DOX can be easily displayed on His-tagged VLNPs by a simple Add-and-Display step with high coupling efficiency and the drug was only released at low pH in a controlled manner. This approach facilitates specific attachment of any drug molecule on His-tagged VLNPs at the very mild conditions without changing the biological structure and native conformation of the VLNPs

Cytotoxicity, regulation of apoptotic and anti-apoptotic gene expression by IL-27 in MCF-7 and MDA-MB-231 breast cancer cell lines

Breast cancer is one of the commonest cancers among women. Conventional therapies cause adverse side effects in patients. Cytokine immunotherapy such as interleukin-27 (IL-27) has been sought as an alternative cancer treatment in recent years. IL-27 has been shown to improve anticancer immunity and anti-angiogenesis in cancers, however, its effect on apoptotic and anti-apoptotic gene expression especially in breast cancers is yet to be explored. Cytotoxicity of IL-27 in non-cancerous (184b5) and cancerous (MCF-7 and MDA-MB-231) breast cell lines was first determined for 24-72 h in this study. The results indicated that IL-27 treatment did not retard 184b5 cell growth, however, did inhibit MCF-7 (48 h) and MDA-MB-231 (72 h) cell growth with IC50 at 442 and 457 ng/ml, respectively. Apoptotic (TRAIL, FADD, FAS, caspase-3 and caspase-8) and anti-apoptotic (BCL-2, AKT, and COX-2) genes were then amplified from untreated (control) and treated breast cancer cells and studied. TRAIL, caspase-3, caspase-8 gene expression was significantly (p < 0.05) upregulated in treated MCF-7 (442 ng/ml) and MDA-MB-231 (457 ng/ml) cells. Expression of FADD and FAS genes was not detected in both control and treated MCF-7 and MDA-MB-231 cells. COX-2 gene was also not expressed by MCF-7 cells, but reduced significantly (p < 0.05) in treated MDA-MB-231 cells. In MDA-MB-231 cells, IL-27 treatment seemed to slightly enhance the expression of AKT and BCL-2 genes which, on the other hand, was downregulated in treated MCF-7 cells. Conclusively, IL-27 is able to inhibit breast cancer cell growth and regulate apoptotic and anti-apoptotic gene expression in breast cancer cells

Tissue engineering approach to repair abdominal wall defects using cell-seeded bovine tunica vaginalis in a rabbit model

The aim of this study was to engineer skeletal muscle tissue for repair abdominal wall defects. Myoblast were seeded onto the scaffolds and cultivated in vitro for 5 days. Full thickness abdominal wall defects (3 × 4 cm) were created in 18 male New Zealand white rabbits and randomly divided into two equal groups. The defects of the first group were repaired with myoblast-seeded-bovine tunica vaginalis whereas the second group repaired with non-seeded-bovine tunica vaginalis and function as a control. Three animals were sacrificed at 7th, 14th, and 30th days of post-implantation from each group and the explanted specimens were subjected to macroscopic and microscopic analysis. In every case, seeded scaffolds have better deposition of newly formed collagen with neo-vascularisation than control group. Interestingly, multinucleated myotubes and myofibers were only detected in cell-seeded group. This study demonstrated that myoblast-seeded-bovine tunica vaginalis can be used as an effective scaffold to repair severe and large abdominal wall defects with regeneration of skeletal muscle tissue

Current techniques in reprogramming cell potency

The first successful attempt to reprogram somatic cell into embryonic-like stem cell was achieved on 2006. Since then, it had sparked a race against time to bring this wonderful invention from bench to bedside but it is not easily achieved due to severe problems in term of epigenetic and genomic. With each problem arise, new technique and protocol will be constructed to try to overcome it. This review addresses the various techniques made available to create iPSC with problems hogging down the technique

Antibiotic resistance and plasmid profiling of Vibrio parahaemolyticus isolated from cockles in Padang, Indonesia

Vibrio parahaemolyticus is one of the most widely recognized pathogenic Vibrio species due to numerous outbreaks and its’ wide occurrence in marine environment. In this study, 32 isolates of V. parahaemolyticus isolated from cockles were tested for sensitivity to 16 antibiotics and the presence of plasmids. All the isolates were multi-resistance, defined as resistant to at least three different antibiotics with multiple antibiotic resistance indexes ranging from 0.31 to 0.69, indicating the isolates originate from high risk sources of contamination where antibiotics are often used. In the plasmid profiling test, only 15 isolates (47%) harbored plasmid DNA, which ranged in size from 2.7 to 56.2 kb, separating the isolates into 14 plasmid profiles. Hence, food contaminated with antibiotic resistant V. parahaemolyticus could be a major threat to public health due to the distinct possibility that they can be a significant reservoir of genes encoding antibiotic resistance determinants that can be transferred intra or interspecies. As in many developing countries, raw food hygiene and antimicrobial resistance epidemiology is still in the infancy stage in the locality of the study and thus our data provide a current baseline profile of antimicrobial resistance and plasmid of V. parahaemolyticus from cockles in Padang, Indonesia

Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes

Vibrio parahaemolyticus is a gram negative bacterium and causes gastrointestinal illness in humans. In this study, twenty five out of fifty cockle samples from Padang, Indonesia produced purple colonies when they were grown on selective medium, CHROMagarTM Vibrio. Specific–PCR for toxR gene detection gave positive results in which a band with 368 base pairs size appeared on the gel for all the isolates that confirmed the presence of V. parahaemolyticus. In the virulence properties test, all the isolates showed negative results for tdh and trh genes detection. The results indicate that the isolates under this study do not contain virulence properties that correlate to the ability of infection and diseases, which means that they are nonpathogenic

Comparative study of antioxidant level and activity from leaf extracts of Annona muricata Linn obtained from different locations

Annona muricata Linn possesses an anti-tumorigenic effect towards cancer. Several of its bioactive components have already been assessed in previous findings. However, none of the previous studies actually addressed the important consideration of the association between cultivation area of this medicinal plant and its bioactive compounds/antioxidants. In this study, the antioxidant level and antioxidant activity of 19 Annona muricata collected from different locations were evaluated by phenolic and flavonoid assays together with Oxygen Radical Absorbance Capacity (ORAC), Ferric Reducing Ability of Plasma (FRAP) and 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assays. M1 was found to have an attractive antioxidant profile as it had the highest content of phenolics (73.2 µg/mL GAE) and flavonoids (191.4 µg/mL CE) and also the highest antioxidant capacity in ORAC assay (254.7 µM). Additionally, it had a favourably high ferric ion reducing capacity (15.55 µM Fe2+/µg) and the best free DPPH-radical scavenging activity (IC50=143.5 µg/mL). On the contrary, R1 showed the lowest level of phenolics with a GAE value of 21.92 µg/mL, ranked second lowest in flavonoid content (65.42 µg/mL CE), and it had the least antioxidant capacity in ORAC (94.66 µM), FRAP (4.17 µM Fe2+/µg) and DPPH assays (1597 µg/mL), making it the least desirable antioxidant source. Based on this finding, it was concluded that Annona muricata Linn had varied antioxidant levels and activity regarding its cultivation area; hence, it would be a guide in the selection of potential candidates for natural antioxidants in phytopharmacy