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

Cellular crosstalk mechanism of Toll-like receptors in gingival overgrowth (review)

Gingival overgrowth is an undesirable outcome of systemic medication and is evidenced by the accretion of collagenous components in gingival connective tissues along with diverse degrees of inflammation. Phenytoin therapy has been found to induce the most fibrotic lesions in gingiva, cyclosporine caused the least fibrotic lesions, and nifedipine induced intermediate fibrosis in drug induced gingival overgrowth. In drug induced gingival overgrowth, efficient oral hygiene is compromised and has negative consequences for the systemic health of the patients. Toll like receptors (TLRs) are involved in the effective recognition of microbial agents and play a vital role in innate immunity and inflammatory signaling responses. TLRs stimulate fibrosis and tissue repairs in several settings, although with evident differences between organs. In particular, TLRs exert a distinct effect on fibrosis in organs with greater exposure to TLR ligands, such as the gingiva. Cumulative evidence from diverse sources suggested that TLRs can affect gingival overgrowth in several ways. Numerous studies have demonstrated the expression of TLRs in gingival tissues and suggested its potential role in gingival inflammation, cell proliferation and synthesis of the extracellular matrix which is crucial to the development of gingival overgrowth. In the present review, we assessed the role of TLRs on individual cell populations in gingival tissues that contribute to the progression of gingival inflammation, and the involvement of TLRs in the development of gingival overgrowth. These observations suggest that TLRs provide new insight into the connection among infection, inflammation, drugs and gingival fibrosis, and are therefore efficient therapeutic target molecules. We hypothesize that TLRs are critical for the development and progression of gingival overgrowth, and thus blocking TLR expression may serve as a novel target for antifibrotic therapy

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)

The Role of Natural Dietary Products in Nanomedicine

It has long been established that a diet rich in fresh fruits, vegetables, seeds, grains and legumes and antioxidants, and other beneficial compounds may help prevent various human diseases. However, diet is not a cure for treatment of severe diseases, but it may help prevent some ailments, and it can help the body overcome the effects of conventional treatments. Natural compounds not only serve as a drug or template for drugs but also, in many instances, had been a source of discovery of novel biology that provided better understanding of target and pathway involved in the disease processes. In addition, drugs derived from natural compounds work better for patients than do drugs manufactured synthetically. Approximately, 40% of drugs in the pipeline and 70% of synthetic therapeutic molecules are plagued with poor solubility, oral bioavailability, and delivery. Drugs with poor solubility encounter limited transport during oral administration because of low concentration gradient between the gut and the blood vessels. To increase body fluid saturation solubility of poorly soluble drug, new delivery methods need to be developed using natural dietary plant metabolites

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

Molecular typing of Aeromonas species using RAPD and ERIC-PCR fingerprinting

Two molecular typing methods were used in this study to examine the intra/inter-specific genetic relatedness among the A. hydrophila, A. caviae and A. veronii biovar sobria strains. In the analysis by RAPDPCR and ERIC-PCR, the size for RAPD and ERIC fragments ranged from 0.25 to 10.0 kb with an average number of sixteen and eight bands, respectively. For the inter-species similarity, RAPD could generate 16 major clusters and 36 single isolates at the similarity of 30%. 10 clusters are significant since it grouped the strains according to their own species, however, ERIC-PCR could discriminate the strains into 4 significant clusters and 64 single isolates at the similarity of 50%. Moreover, there was no genetic similarity between food and environmental strains of Aeromonas sp. isolated from different geographical areas as well as from the same geographical area. Eighty five genotypes among the 85 A. hydrophila, A. caviae and A. veronii biovar sobria isolates were generated using RAPD and ERIC-PCR which indicated that the strains were very diverse