Development of a polylactic acid-coated nanocellulose/chitosan-based film indicator for real-time monitoring of beef spoilage.

Food safety is one of the biggest challenges in global markets. There is a critical need to develop a simple, affordable, and environmentally friendly color indicator that can quickly and conveniently monitor and indicate the quality of packaged food products in the home, supermarkets, shops, etc. This study aimed to develop a nanocellulose/chitosan-based film coated with polylactic acid (PLA) to monitor beef spoilage in real-time. This film named PLA/NCM was fabricated by casting a suspension of a nanocellulose/chitosan mixture doped with methyl red, followed by a coating of PLA on the film surface. The film displayed a visible color change in response to different pH buffer solutions (2-10). The PLA/NCM film was applied to monitor the spoilage of beef under a refrigeration condition of 4 °C and showed an apparent color change after 5 days as a threshold for beef spoilage. The color modulation of the PLA/NCM films was processed each time via a colorimetric device and revealed substantial color difference values (ΔE) after 5 days of beef spoilage. The total viable microbial counts (TVC) and pH of the beef sample were determined, and the findings showed that the TVC and pH increased simultaneously during the beef spoilage. Although further research is necessary, the PLA/NCM film has the potential to be a color indicator for application in both smart food packaging and real-time monitoring of spoilage of beef and other meat products.

Toll-like Receptor-4 (TLR4) Agonist-Based Intranasal Nanovaccine Delivery System for Inducing Systemic and Mucosal Immunity.

Eliciting a robust immune response at mucosal sites is critical in preventing the entry of mucosal pathogens such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This task is challenging to achieve without the inclusion of a strong and safe mucosal adjuvant. Previously, inulin acetate (InAc), a plant-based polymer, is shown to activate toll-like receptor-4 (TLR4) and elicit a robust systemic immune response as a vaccine adjuvant. This study investigates the potential of nanoparticles prepared with InAc (InAc-NPs) as an intranasal vaccine delivery system to generate both mucosal and systemic immune responses. InAc-NPs (∼250 nm in diameter) activated wild-type (WT) macrophages but failed to activate macrophages from TLR4 knockout mice or WT macrophages when pretreated with a TLR4 antagonist (lipopolysaccharide-RS (LPS-RS)), which indicates the selective nature of a InAc-based nanodelivery system as a TLR4 agonist. Intranasal immunization using antigen-loaded InAc-NPs generated ∼65-fold and 19-fold higher serum IgG1 and IgG2a titers against the antigen, respectively, as compared to PLGA-NPs as a delivery system. InAc-NPs have also stimulated the secretion of sIgA at various mucosal sites, including nasal-associated lymphoid tissues (NALTs), lungs, and intestine, and produced a strong memory response indicative of both humoral and cellular immune activation. Overall, by stimulating both systemic and mucosal immunity, InAc-NPs laid a basis for a potential intranasal delivery system for mucosal vaccination.

Curcumin use in ulcerative colitis: is it ready for prime time? A systematic review and meta-analysis of clinical trials.

BACKGROUND: Curcumin, an active ingredient of the Indian herb turmeric (Curcuma longa), has shown promising anti-inflammatory properties. Studies of its potential benefits in treating patients with ulcerative colitis (UC) are limited. We performed a systematic review and meta-analysis of human randomized placebo controlled trials to evaluate the efficacy of adjunctive therapy with curcumin in treating patients with UC. METHODS: We conducted a search of several databases (from January 2000 to September 2018). A random-effects model was used for analysis. We assessed heterogeneity between study-specific estimates using the Cochran Q statistical test, 95% prediction interval (PI) and I2 statistics. The outcomes assessed were the pooled odds of clinical response and remission as well as the endoscopic response. RESULTS: A total of 7 studies with 380 patients (curcumin n=188; placebo n=190) were included in the final analysis. The pooled odds ratio for clinical remission with curcumin use was 2.9 (95%CI 1.5-5.5, I2=45, P=0.002), clinical response was 2.6 (95%CI 1.5-4.5, I2=74%, P=0.001), and endoscopic response/remission was 2.3 (95%CI 1.2-4.6, I2=35.5%, P=0.01). CONCLUSIONS: Based on our study, combined mesalamine and curcumin therapy was associated with roughly threefold better odds of a clinical response compared to placebo, with minimal side effects. This response was statistically significant, albeit with heterogeneity, probably due to the different severity scoring indices, curcumin dosages and routes of drug delivery used.

The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention.

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

Multifunctional approaches utilizing polymeric micelles to circumvent multidrug resistant tumors.

The concerns impeding the success of chemotherapy in cancer is descending efficacy of drugs due to the development of multiple drug resistance (MDR). The current efforts employed to overcome MDR have failed or are limited to only preliminary in-vitro investigations. Nanotechnology is at the forefront of the drug delivery research, playing pivotal role in chemotherapy and diagnosis, thereby providing innovative approaches for the management of the disease with minimal side effects. Recently, polymeric micelles (PMs) have witnessed significant developments in cancer therapy. PMs are self-assembled colloidal particles, with a hydrophilic head and a long hydrophobic tail, which enhance the solubility, permeability and bioavailability of drugs, due to the unique features of reaching higher concentration in the biological system, above critical micellar concentration. One of the effective approaches to improve the efficacy of chemotherapy and overcome drug resistance would be to employ multifunctional approach (combination of stimuli-responsive, utilization of drug resistance modulators and combination therapy) using PMs as drug delivery systems. Actively targeted, stimuli-sensitive and multifunctional approaches involve using single and/or combination of approaches (pH-responsive, temperature regulated, reduction-sensitive, ultrasound etc.) to combat drug resistant. The review will describe PMs, types of copolymers used in PMs, preparation and characterization of PMs. A comprehensive list of PMs tested in clinical trials is discussed. Lastly, this review covers stimuli-sensitive and multifunctional approaches to overcome MDR in cancer utilizing PMs.

Overcoming multiple drug resistance in cancer using polymeric micelles.

INTRODUCTION: A major concern that limits the success of cancer chemotherapy is multidrug resistance (MDR). The drug resistance mechanisms are either host related or tumor related. The host tumor interacting factors also contribute to MDR. Multifunctional polymeric micelles offer several advantages in circumventing MDR due to their design, selectivity, and stability in cancer microenvironment. AREAS COVERED: The review is broadly divided into two parts: the first part covers MDR and its mechanisms; the second part covers multifunctional polymeric micelles in combating MDR through its state-of-the-art design. This part covers various strategies like use of P-gp transporter inhibitors, TPGS, pH & thermo-sensitive, and siRNA for selectivity of PMs against multidrug-resistant tumors. EXPERT OPINION: Numerous approaches have been tested using polymeric micelles to overcome MDR tumors. However, these are either limited to only in-vitro investigations and/or preliminary preclinical models and do not investigate the underlying biological mechanism. Hence, there exists an unmet need to perform fundamental research that focuses on studying the underlying mechanism and preclinical/clinical testing of the micellar formulations.

Site-directed non-covalent polymer-drug complexes for inflammatory bowel disease (IBD): Formulation development, characterization and pharmacological evaluation.

Inflammatory Bowel Diseases (IBD) is a debilitating condition that affects ~70,000 new people every year and has been described as "an expensive disease with no known cure". In addition, IBD increases the risk of developing colon cancer. The current therapeutics for IBD focus on the established disease where the immune dysfunction and bowel damage have already occurred but do not prevent or delay the progression. The current work describes a polymer-based anti-inflammatory technology (Ora-Curcumin-S) specifically targeted to the luminal side of the colon for preventing and/or treating IBD. Ora-Curcumin-S was prepared by molecular complexation of curcumin with a hydrophilic polymer Eudragit® S100 using co-precipitation method. Curcumin interacted with the polymer non-covalently and existed in an amorphous state as demonstrated by various physicochemical techniques. Ora-Curcumin-S is a polymer-drug complex, which is different than solid dispersions in that the interactions are retained even after dissolving in aqueous buffers. Ora-Curcumin-S was >1000 times water soluble than curcumin and importantly, the enhanced solubility was pH-dependent, which was observed only at pHs above 6.8. In addition, around 90% of Ora-Curcumin-S was stable in phosphate buffer, pH 7.4 and simulated intestinal fluid after 24 h, in contrast to 10-20% unformulated curcumin. Ora-Curcumin-S inhibited Monophosphoryl Lipid-A and E. coli induced inflammatory responses in dendritic cells and cells over expressing Toll-Like Receptor-4 (TLR-4) suggesting that Ora-Curcumin-S is a novel polymer-based TLR-4 antagonist. Preliminary pharmacokinetics in mice showed targeted delivery of soluble curcumin to the colon lumen without exposing to the systemic circulation. Furthermore, Ora-Curcumin-S significantly prevented colitis and associated injury in a mouse model of ulcerative colitis estimated using multiple preclinical parameters: colonoscopy pictures, body weight, colon length, colon edema, spleen weight, pro-inflammatory signaling and independent pathological scoring. Overall, the outcome of this innovative proof-of-concept study provides an exciting and locally-targeted pathway for a dietary therapeutic option for IBD patients to help limit colonic inflammation and thus susceptibility to colitis-associated colorectal cancer.

Dendritic Cell-Targeted Nanovaccine Delivery System Prepared with an Immune-Active Polymer.

Targeting dendritic cells (DCs), either ex vivo (Ex. Sipuleucel-T) or in vivo, for stimulating cellular immunity has been a leading approach for cancer vaccines. We have rationally engineered a nanoparticle (NP)-based delivery system for vaccines (InAc-NPs) using inulin acetate (InAc) as the polymer to target DCs. The material and the antigen-encapsulated InAc-NPs (∼190 nm in diameter) were characterized for their physicochemical properties. As a potent vaccine adjuvant, InAc-NPs activated TLR4 on multiple immune cells, including DCs and primary swine and human cells, to secrete various cytokines as detected by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. In addition, InAc-NPs promoted the maturation of DCs as observed by a decreased phagocytic ability and enhanced capability to activate various maturation markers (MHC-I, MHC-II, CD40, and CD80) quantified using flow cytometry. In mice, the InAc-NPs produced strong serum antibody titers (total IgG, IgG1, and IgG2a) against the encapsulated antigen (ovalbumin) similar to complete Freund's adjuvant. Additionally, as a dose-sparing delivery system, antigen delivered through InAc-NPs generated higher antibody titers (IgG1, 1.57 times; IgG-total, 1.66 times; and IgG2a, 29.8 times) even at 100 times less antigen dose. High amounts of cytokines representing both humoral (IL4 and IL10) and cell-mediated (IL2 and IFN-γ) immunities were secreted from splenocytes of mice immunized with InAc-NPs. Importantly, InAc-NPs provided complete protection in 100% of the vaccinated mice from metastasis of intravenously injected melanoma cells (B16-F10) to lungs. In addition, the InAc-NPs were cleared from the injection site within 30 h of injection (in vivo imaging) and displayed no toxicity at the injection site (histology). The current study demonstrates that the multifunctional InAc-based nanovaccine delivery system has potential applications in cancer immunotherapy and delivering vaccines against various infectious diseases.

Evaluating Student Performance and Perception of a Workshop Integrating Pharmacy Practice and a Pharmaceutics Lab.

OBJECTIVE: Evaluating Student Performance and Perception of a Workshop Integrating Pharmacy Practice and a Pharmaceutics Lab. INNOVATION: Common methods for curricular integration are often time and faculty-intensive. An innovative approach to integration was developed and utilized in an introductory compounding workshop. Faculty members collaborated with a compounding pharmacist to design and facilitate a pharmaceutics workshop for first-year pharmacy students. The workshop was composed of four major sections, an introduction to pharmaceutical compounding and the regulations surrounding manufacturing and sterility, a case discussion involving a pediatric patient and the need to develop an appropriate drug delivery system, a short review of pharmaceutical calculations and labeling requirements, and then an introduction to logistics and active learning in a lab setting. CRITICAL ANALYSIS: After taking part in the workshop, students indicated a significantly higher comfort level going into the pharmaceutics lab (3.48±0.83 to 4.04±0.70) and in the compounding process (3.06±0.83 to 3.71±0.80). Their views of the clinical application of the lab and the need to use knowledge gained from other courses in the lab were also significantly improved (4.36±0.68 to 4.61±0.49 and 3.71±0.77 to 4.26±0.74, respectively). In addition, their perceptions of how they will utilize the skills developed as a practicing pharmacist, and their feelings towards the safety procedures involved in compounding, were also positively affected (3.96±0.87 to 4.45±0.59 and 3.28±0.92 to 3.91±0.72, respectively). Finally, students' average quiz score in Spring 2016, when the workshop was instituted, significantly increased from Spring 2015 (90.154±4.98 versus 85.89±10.87, respectively).

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer.

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

Pathogen-mimicking vaccine delivery system designed with a bioactive polymer (inulin acetate) for robust humoral and cellular immune responses.

New and improved vaccines are needed against challenging diseases such as malaria, tuberculosis, Ebola, influenza, AIDS, and cancer. The majority of existing vaccine adjuvants lack the ability to significantly stimulate the cellular immune response, which is required to prevent the aforementioned diseases. This study designed a novel particulate based pathogen-mimicking vaccine delivery system (PMVDS) to target antigen-presenting-cells (APCs) such as dendritic cells. The uniqueness of PMVDS is that the polymer used to prepare the delivery system, Inulin Acetate (InAc), activates the innate immune system. InAc was synthesized from the plant polysaccharide, inulin. PMVDS provided improved and persistent antigen delivery to APCs as an efficient vaccine delivery system, and simultaneously, activated Toll-Like Receptor-4 (TLR-4) on APCs to release chemokine's/cytokines as an immune-adjuvant. Through this dual mechanism, PMVDS robustly stimulated both the humoral (>32 times of IgG1 levels vs alum) and the cell-mediated immune responses against the encapsulated antigen (ovalbumin) in mice. More importantly, PMVDS stimulated both cytotoxic T cells and natural killer cells of cell-mediated immunity to provide tumor (B16-ova-Melanoma) protection in around 40% of vaccinated mice and significantly delayed tumor progression in rest of the mice. PMVDS is a unique bio-active vaccine delivery technology with broader applications for vaccines against cancer and several intracellular pathogens, where both humoral and cellular immune responses are desired.

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

Cancer metastasis is the major cause of cancer mortality. Despite extensive research efforts, effective treatment for cancer metastasis is still lacking. Cancer metastasis involves 4 essential steps: cell detachment, migration, invasion, and adhesion. Detachment is the first and required step for metastasis. Glutathione disulfide (GSSG) is derived from the oxidation of glutathione (GSH), which is present in biological systems in millimolar concentration. Although GSSG is commercially available, the impact of GSSG on cell functions/dysfunctions has not been fully explored due to the fact that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed GSSG liposomes that effectively deliver GSSG to cells. Unexpectedly, cells treated with GSSG liposomes were resistant to detachment by trypsinization. This observation led to the investigation of the antimetastatic effect of GSSG liposomes. Our data demonstrate that GSSG liposomes at 1 mg/mL completely blocked cell detachment and migration, and significantly inhibited cancer cell invasion. Aqueous GSSG showed no such effect, confirming that the effects on cell detachment, migration, and invasion were caused by the intracellular delivery of GSSG. An in vivo experiment with a murine melanoma experimental metastasis model showed that GSSG liposomes prevented melanoma lung metastasis. The unique antimetastatic mechanism through the effects on detachment and migration, and effective in vitro and in vivo metastasis inhibition, warrants further investigation of the GSSG liposomes as a potential treatment for cancer metastasis.

Hydrophobically modified inulin as an amphiphilic carbohydrate polymer for micellar delivery of paclitaxel for intravenous route.

Micellization offers several advantages for the delivery of water insoluble drugs including a nanoparticulate 'core-shell' delivery system for drug targeting. Recently, hydrophobically modified polysaccharides (HMPs) are gaining recognition as micelle forming polymers to encapsulate hydrophobic drugs. In this manuscript, for the first time, we have evaluated the self-assembling properties of a lauryl carbamate derivative of the poly-fructose natural polymer inulin (Inutec SP1(®) (INT)) to form paclitaxel (PTX) loaded micelles. INT self-assembled into well-defined micellar structures in aqueous environment with a low critical micellar concentration of 27.8 µg/ml. INT micelles exhibited excellent hemocompatibility and low toxicity to cultured cells. PTX loaded INT micelles exhibited a mean size of 256.37 ± 10.45 nm with excellent drug encapsulation efficiency (95.66 ± 2.25%) and loading (8.69 ± 0.22%). PTX loaded micelles also displayed sustained release of PTX and enhanced anti-cancer efficacy in-vitro in mouse melanoma cells (B16F10) compared to Taxol formulation with Cremophor EL as solvent. In addition, PTX loaded INT micelles exhibited comparable in-vivo antitumor activity in B16F10 allograft mouse model at half the dose of Taxol. In conclusion, INT offers safe, inexpensive and natural alternative to widely used PEG-modified polymers for the formulation of micellar delivery systems for paclitaxel.

Molecular complexation of curcumin with pH sensitive cationic copolymer enhances the aqueous solubility, stability and bioavailability of curcumin.

Curcumin is a natural dietary compound with demonstrated potential in preventing/treating several chronic diseases in animal models. However, this success is yet to be translated to humans mainly because of its poor oral bioavailability caused by extremely low water solubility. This manuscript demonstrates that water insoluble curcumin (~1µg/ml) forms highly aqueous soluble complexes (>2mg/ml) with a safe pH sensitive polymer, poly(butyl-methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl-methacrylate) when precipitated together in water. The complexation process was optimized to enhance curcumin loading by varying several formulation factors. Acetone as a solvent and polyvinyl alcohol as a stabilizer with 1:2 ratio of drug to polymer yielded complexes with relatively high loading (~280µg/ml) and enhanced solubility (>2mg/ml). The complexes were amorphous in solid and were soluble only in buffers with pHs less than 5.0. Hydrogen bond formation and hydrophobic interactions between curcumin and the polymer were recorded by infrared spectroscopy and nuclear magnetic resonance spectroscopy, respectively. Molecular complexes of curcumin were more stable at various pHs compared to unformulated curcumin. In mice, these complexes increased peak plasma concentration of curcumin by 6 times and oral bioavailability by ~20 times. This is a simple, economic and safer strategy of enhancing the oral bioavailability of curcumin.

Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention.

UNLABELLED: Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. IMPLICATIONS: Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2.

Discovery of inulin acetate as a novel immune-active polymer and vaccine adjuvant: synthesis, material characterization, and biological evaluation as a toll-like receptor-4 agonist.

Vaccine adjuvants are an essential part of modern vaccine design, especially against intracellular pathogens such as M. tuberculosis, malarial parasite, HIV, influenza virus and Ebola. The present work offers a unique approach to designing novel vaccine adjuvants by identifying polymers that mimic "pathogen associated molecular patterns" (PAMPS) and engineering an immune-active particulate vaccine delivery system that uses the polymer. By using this strategy, we report the discovery of the first plant polymer based toll-like receptor-4 (TLR-4) agonist, inulin acetate (InAc). InAc was synthesised from the plant polysaccharide inulin. Inulin acetate as a polymer and particles prepared using InAc were characterised using various physicochemical techniques. The TLR-4 agonistic activity of InAc was established in multiple immune, microglial, dendritic, peripheral blood mononuclear (human and swine) and genetically modified epithelial cells (HEK293) that exclusively express TLR-4 on their surface. InAc activated all the above-mentioned cells to release proliferative cytokines; however, InAc failed to activate when the were cells either pre-incubated with a TLR-4 specific antagonist or isolated from mice deficient in adapter proteins involved in TLR signalling (Mal/MyD88). Antigen encapsulated microparticles prepared with TLR-4 agonist InAc mimicked pathogens to offer improved antigen delivery to dendritic cells compared to soluble antigen (47 times) or antigen encapsulated poly(lactic-co-glycolic acid) (PLGA) particles (1.57 times). In conclusion, InAc represents a novel polymer-based modern vaccine adjuvant targeting specific signalling pathways of the innate immune system, which could be formulated into a platform vaccine delivery system against cancer and viral diseases.

Aspirin and salicylic acid decrease c-Myc expression in cancer cells: a potential role in chemoprevention.

Epidemiological studies have demonstrated a significant correlation between regular aspirin use and reduced colon cancer incidence and mortality; however, the pathways by which it exerts its anti-cancer effects are still not fully explored. We hypothesized that aspirin's anti-cancer effect may occur through downregulation of c-Myc gene expression. Here, we demonstrate that aspirin and its primary metabolite, salicylic acid, decrease the c-Myc protein levels in human HCT-116 colon and in few other cancer cell lines. In total cell lysates, both drugs decreased the levels of c-Myc in a concentration-dependent fashion. Greater inhibition was observed in the nucleus than the cytoplasm, and immunofluorescence studies confirmed these observations. Pretreatment of cells with lactacystin, a proteasome inhibitor, partially prevented the downregulatory effect of both aspirin and salicylic acid, suggesting that 26S proteasomal pathway is involved. Both drugs failed to decrease exogenously expressed DDK-tagged c-Myc protein levels; however, under the same conditions, the endogenous c-Myc protein levels were downregulated. Northern blot analysis showed that both drugs caused a decrease in c-Myc mRNA levels in a concentration-dependent fashion. High-performance liquid chromatography (HPLC) analysis showed that aspirin taken up by cells was rapidly metabolized to salicylic acid, suggesting that aspirin's inhibitory effect on c-Myc may occur through formation of salicylic acid. Our result suggests that salicylic acid regulates c-Myc level at both transcriptional and post-transcription levels. Inhibition of c-Myc may represent an important pathway by which aspirin exerts its anti-cancer effect and decrease the occurrence of cancer in epithelial tissues.

2-Deoxyglucose induces cell cycle arrest and apoptosisin colorectal cancer cells independent of its glycolysis inhibition.

2-Deoxyglucose (2DG) is an anticancer drug with excellent safety profile. Because of its higher dose requirements, its potential is yet to translate into a monotherapy. However, recently, 2DG has been tested as an adjunct in established chemotherapeutic regimens. 2DG enhanced the potency of several chemotherapeutic agents but not all. The rationale selection of known chemotherapeutic agents to use with 2DG is hampered because of the lack of complete understanding of mechanism behind 2DG anticancer effects. Although, 2DG is a well-known glycolytic inhibitor, which inhibits the key glycolytic enzyme hexokinase, its anticancer effects cannot be fully explained by this simplistic mechanism alone. In this article, we have shown for the first time that 2DG induced a transient expression of p21 and a continuous expression of p53 in colorectal cancer cells (SW620). The treatment also caused cell cycle arrest at G0/G1 phase and induced apoptosis through the mitochondrial pathway. The effects of 2DG on p21 and p53 protein levels were totally independent of its inhibitory effect on either hexokinase or ATP levels. Results from this study provides key insights into novel molecular mechanisms of 2DG and directs rational selection of other anticancer drugs to combine with 2DG in colorectal cancer treatment.

Development of soluble inulin microparticles as a potent and safe vaccine adjuvant and delivery system.

The goal of the present study is to develop a potent and safe vaccine adjuvant that can also stabilize vaccine formulations during lyophilization and storage. Inulin is a safe plant polysaccharide, and in its water soluble isoform, it is known to stabilize protein formulations during storage. However, soluble inulins have never been shown to stimulate the immune system. In this study, for the first time, we showed that water soluble inulins could be developed into vaccine adjuvants by formulating as antigen encapsulated microparticles. A method was developed to prepare soluble inulin microparticles (sIMs) with high encapsulation efficiency (∼75%) and loading (∼75 µg/mg) of the antigen. When immunized in mice, sIMs have generated robust Th2-type antibody titers (IgG1: 500,000) compared to unadjuvanted antigens (IgG1: 17,500) or alum adjuvanted antigens (IgG1: 80,000). In vitro assays showed that a higher proportion of antigen presenting cells (APC's) have taken up the antigen when presented in sIMs versus in solution (99 % vs 22 %). In addition, the amount of antigen taken up per cell has also been enhanced by more than 25 times when antigen was presented in sIMs. Efficient uptake of the antigen by APCs through sIMS was attributed to the observed enhancement in the immune response by antigen loaded sIMs. The sIMs neither caused any granuloma/tissue damage at the injection site in mice nor were they toxic to the APC's in cell culture. In conclusion, the current study has developed a safe, soluble inulin based vaccine adjuvant and delivery system.

Identification of a novel skin penetration enhancement peptide by phage display peptide library screening.

Skin is an important site for local or systemic application of drugs. However, a majority of drugs have poor permeability through the skin's topmost layer, stratum corneum (SC). The aim of this study was to identify safe and smaller peptides that could enhance the skin penetration of drug molecules. By screening phage display peptide library, we have identified a T2 peptide (LVGVFH), which enhanced the penetration of bacteriophages (~800 nm long bacterial viruses) across porcine and mouse skin. Pretreating the skin with synthetic T2 peptide at pH 4.5 resulted in significant penetration enhancement of hydrophilic drug 5-fluorouracil (5-FU) across skin. FTIR spectroscopy showed that the T2 peptide interacted with skin lipids to enhance the skin penetration. Pretreating the skin with T2 peptide enhanced the partitioning of small molecules with different lipophilicities (5-FU, fluorescein isothiocyanate, and rhodamine 123 hydrochloride) into skin. Fluorescence studies showed that T2 peptide enhanced the diffusion of these molecules into intercellular lipids of SC and thus enhanced the penetration into the skin. Histidine at the c-terminus of T2 peptide was identified to be critical for the skin penetration enhancement. T2 peptide interacted with skin lipids to cause skin penetration enhancement. The study identified a novel, safe, and noninvasive peptide to improve the skin penetration of drugs without chemical conjugation.