Novel peptide inhibitor of human tumor necrosis factor-α has antiarthritic activity.

The inhibition of tumor necrosis factor (TNF)-α trimer formation renders it inactive for binding to its receptors, thus mitigating the vicious cycle of inflammation. We designed a peptide (PIYLGGVFQ) that simulates a sequence strand of human TNFα monomer using a series of in silico methods, such as active site finding (Acsite), protein-protein interaction (PPI), docking studies (GOLD and Flex-X) followed by molecular dynamics (MD) simulation studies. The MD studies confirmed the intermolecular interaction of the peptide with the TNFα. Fluorescence-activated cell sorting and fluorescence microscopy revealed that the peptide effectively inhibited the binding of TNF to the cell surface receptors. The cell culture assays showed that the peptide significantly inhibited the TNFα-mediated cell death. In addition, the nuclear translocation of the nuclear factor kappa B (NFκB) was significantly suppressed in the peptide-treated A549 cells, as observed in immunofluorescence and gel mobility-shift assays. Furthermore, the peptide protected against joint damage in the collagen-induced arthritis (CIA) mouse model, as revealed in the micro focal-CT scans. In conclusion, this TNFα antagonist would be helpful for the prevention and repair of inflammatory bone destruction and subsequent loss in the mouse model of CIA as well as human rheumatoid arthritis (RA) patients. This calls upon further clinical investigation to utilize its potential effect as an antiarthritic drug.

Stimuli-responsive magnetic silica-poly-lactic-co-glycolic acid hybrid nanoparticles for targeted cancer chemo-immunotherapy.

Chemotherapy and immunotherapy are two important modalities in cancer management. However, due to multiple reasons, a monotherapy is only partially effective. Hence, if used concurrently in targeted and stimuli-responsive manner, it could have been superior therapeutically. To facilitate co-delivery of chemotherapeutic and immunotherapeutic agent to the target cancer cells, engineered nanoparticles, i.e., a pH-responsive polymer PLGA-coated magnetic silica nanoparticles (Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs) encapsulating paclitaxel (PTX) and siRNA against programmed cell death ligand-1 (PD-L1) are synthesized and characterized. Developed nanoparticles demonstrated pH-sensitive sustained drug release up to 10 days. In vitro 4T1 cell line studies showed efficient cellular uptake, PD-L1 gene downregulation, and apoptosis. Further, in vivo efficacy studies carried out in the mice model demonstrated a significant reduction of tumor growth following treatment with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs as compared with monotherapy with Fe3O4-SiO2-PLGA-PDA-PTX NPs. The high therapeutic efficacy observed with dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs was mainly due to the cytotoxic effect of PTX combined with targeted silencing of the gene of interest, i.e., PD-L1, which in turn improve CD8+ T cell-mediated cancer cell death as evident with increased proliferation of CD8+ T cells in co-culture experiments. Thereby, dual-Fe3O4-SiO2-PLGA-PDA-PTX-siRNA NPs may have a promising anti-cancer treatment potential against breast cancer; however, the beneficial effects of dual loading of PTX + PD-L1 siRNA may be corroborated against other cancer models such as lung and colorectal cancer models as well as in clinical trials.

Beyond the polysaccharide and glycoconjugate vaccines for Streptococcus pneumoniae: Does protein/peptide nanovaccines hold promises?

Streptococcus pneumoniae having almost 98 serotypes and being common cause of acute otitis media, pneumonia, bacteremia, meningitis etc., which results in high mortality and morbidity globally. Although vaccines like PCV-13 and PPV-23 are available, some problems like serotype replacement and poor immunogenicity in children, old age and immunocompromised people has been observed. To overcome these drawbacks protein/peptide-based vaccine can be a good strategy as these provides wide serotype coverage. However, immunogenicity of protein subunit vaccines is lower, that issue can be solved by using adjuvants. Recently nanoparticles as an adjuvant for vaccine delivery being used, which has provided not only good immunogenicity but also improved delivery and efficiency of protein-based vaccines. In this review we have discussed the latest advancement of nanoparticles-based protein/peptide vaccine delivery for Streptococcus pneumoniae.

RBD decorated PLA nanoparticle admixture with aluminum hydroxide elicit robust and long lasting immune response against SARS-CoV-2.

Nanoparticles-based multivalent antigen display has the capability of mimicking natural virus infection characteristics, making it useful for eliciting potent long-lasting immune response. Several vaccines are developed against global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However these subunit vaccines use mammalian expression system, hence mass production with rapid pace is a bigger challenge. In contrast E. coli based subunit vaccine production circumvents these limitations. The objective of the present investigation was to develop nanoparticle vaccine with multivalent display of receptor binding domain (RBD) of SARS-CoV-2 expressed in E. coli. Results showed that RBD entrapped PLA (Poly lactic acid) nanoparticle in combination with aluminum hydroxide elicited 9-fold higher immune responses as compared to RBD adsorbed aluminum hydroxide, a common adjuvant used for human immunization. It was interesting to note that RBD entrapped PLA nanoparticle with aluminum hydroxide not only generated robust and long-lasting antibody response but also provided Th1 and Th2 balanced immune response. Moreover, challenge with 1 µg of RBD alone was able to generate secondary antibody response, suggesting that immunization with RBD-PLA nanoparticles has the ability to elicit memory antibody against RBD. Plaque assay revealed that the antibody generated using the polymeric formulation was able to neutralize SARS-CoV-2. The RBD entrapped PLA nanoparticles blended with aluminum hydroxide thus has potential to develop asa subunit vaccine against COVID-19.

Design of expert guided investigation of native L-asparaginase encapsulated long-acting cross-linker-free poly (lactic-co-glycolic) acid nanoformulation in an Ehrlich ascites tumor model.

Present study explores native L-asparaginase encapsulated long-acting cross-linker-free PLGA-nanoformulation in an Ehrlich ascites tumor model. L-asparaginase-PLGA nanoparticles for tumor were prepared using a double emulsion solvent evaporation technique, optimized and validated by Box-Behnken Design. L-ASN-PNs showed a particle size of 195 nm ± 0.2 nm and a PDI of 0.2. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques revealed its smooth morphology and elicited an in-vitro release of 80% of the drug, following the Higuchi drug release model. In-vivo studies of L-ASN-PNs on an Ehrlich ascites tumor (EAT) model were completed and compared with the standard medication of 5-fluorouracil (5-FU) treatment. L-ASN-PN treated mice showed a 51.15% decrease in tumor volume and 100% survival rate with no reduction in body weight, no haemotoxicity and no hepatotoxicity, as evident from the hematological parameters, and liver enzyme parameters that were well within the prescribed limits. Chemotherapy has severe side effects and restricted therapeutic success. Henceforth, the purported L-Asparaginase PLGA nanoparticles are a suitable entity for better tumor regression, intra-tumor accumulation and no hematological side-effects.

Modulation of immune response and enhanced clearance of Salmonella typhi by delivery of Vi polysaccharide conjugate using PLA nanoparticles.

Polysaccharide antigens do not promote antibody class switching and memory antibody response, thus require conjugation with a T cell dependent carrier protein to generate protective immune response. The intensity of immune responses varies with the carrier proteins for the same carbohydrate antigen and most of the carrier proteins do not generate strong immune responses. Vi polysaccharide and r-flagellin of Salmonella typhi were conjugated and formulated in PLA particles as nanoglycoconjugate which not only generated strong immune response but also promoted antibody class switching and elicited memory antibody response from single point immunization. Nanoglycoconjugate immunization also modulate anti-inflammatory property of Vi polysaccharide with an enhance secretion of pro-inflammatory cytokine TNF-α and IL-6. This was with concomitant decrease of IFN-γ production, antibody class switching from IgG3 to IgG2 with memory antibody generation against Vi polysaccharide. Antibody elicited by nanoglycoconjugate showed better opsonization and clearance of Salmonella typhi in THP-1 macrophages as compared to Vi-flagellin glycoconjugate and Vi TT (Typhbar®). Delivery of glycoconjugate through nanoparticles provides a platform technology for improving the immunogenicity of polysaccharide based vaccines.

Antioxidant activity and protective effect of suramin against oxidative stress in collagen induced arthritis.

It is imperative to interrupt the link between arthritis and regulation of oxidative stress with the administration of antioxidants. Suramin is known for its anti-inflammatory, antineoplastic and antiangiogenic activities implying its possible antioxidant property. In this study, the antioxidant activity of suramin in cell free system was found to be higher than l-ascorbic acid (l-AA) with respect to its scavenging effect on nitric oxide (NO), hypochlorous acid and hydrogen peroxide radicals. Besides, suramin was found to be nontoxic to cultured RAW cells even at high concentrations along with marked inhibition of NO production. Suramin was found to curb the inflammation associated with the collagen induced arthritis (CIA) model. Administration of suramin significantly reduced the malondialdehyde and protein carbonyl content in joints, liver, kidney and spleen of rats as studied ex vivo. Furthermore, the increased antioxidant enzymes such as SOD, catalase, GST, GPx and GR activities in the tissues were restored significantly after suramin treatment. In silico experiments using Vlife MDS4.4-GRIP docking method showed strong affinity of suramin towards erythrocyte catalase followed by glutathione peroxidase thus corroborating with the findings of antioxidant enzyme assays. Our studies clearly indicate that suramin has remarkable antioxidant potential and can ameliorate arthritis via modulation of oxidative stress.

Intravaginal Delivery of Polyphenon 60 and Curcumin Nanoemulsion Gel.

Polyphenon 60 (P60) and curcumin (CUR) were loaded in a single nanoemulsion system and their combined antibacterial action was studied against uropathogenic Escherichia coli. To enhance availability at target organs and to inhibit enzymatic degradation in gastro intestinal tract, vaginal route of administration was explored. P60 + CUR nanoemulsion (NE) was formulated by ultra-sonication and optimized using Box-Behnken design. Optimized NE showed Z-average of 211.2 nm, polydispersity index of 0.343, and zeta potential of -32.7 mV. Optimized P60+ CUR NE was characterized by stability testing and transmission electron microscopy, and it was observed that NE was stable at 4°C for 30 days and monodisperse in nature with particle size of 195-205 nm. P60+ CUR NE was further formulated as gel and characterized by viscosity, growth curve analysis, and in vitro permeation studies. In vitro drug permeation studies in simulated vaginal media showed maximum permeation (84 ± 0.21%) of curcumin within 5 h and (91 ± 0.16%) of P60 within 8 h. Both the drugs maintained sustained permeation for 12 h. To investigate the transport via intravaginal route, gamma scintigraphy and biodistribution study of P60 + CUR NBG was performed on Sprague-Dawley rats using 99mtechnetium pertechnetate for radiolabeling to P60 molecule. Following intravaginal administration, P60 + CUR NBG dispersed in the kidney and urinary bladder with (3.07 ± 0.15) and (3.35 ± 0.45) percentage per gram after 3 h for P60 and CUR, respectively, and remained active for 12 h. Scintigraphy images suggested that the P60 + CUR NBG given by intravaginal route led to effective distribution of actives in urinary tract, and this observation was in agreement with the biodistribution results.

Chondroitin sulfate-capped super-paramagnetic iron oxide nanoparticles as potential carriers of doxorubicin hydrochloride.

Chondroitin-4-sulfate (CS), a glycosaminoglycan, was used to prepare CS-capped super-paramagnetic iron oxide nanoparticles, which were further employed for loading a water-soluble chemotherapeutic agent (doxorubicin hydrochloride, DOX). CS-capped SPIONs have potential biomedical application in cancer targeting. The optimized formulation had a hydrodynamic size of 91.2±0.8nm (PDI; 0.228±0.004) and zeta potential of -49.1±1.66mV. DOX was loaded onto the formulation up to 2% (w/w) by physical interaction with CS. TEM showed nano-sized particles having a core-shell structure. XRD confirmed crystal phase of iron oxide. FT-IR conceived the interaction of iron oxide with CS as bidentate chelation and also confirmed DOX loading. Vibration sample magnetometry confirmed super-paramagnetic nature of nanoparticles, with saturation magnetization of 0.238emug(-1). In vitro release profile at pH 7.4 showed that 96.67% of DOX was released within 24h (first order kinetics). MTT assay in MCF7 cells showed significantly higher (p<0.0001) cytotoxicity for DOX in SPIONs than DOX solution (IC50 values 6.294±0.4169 and 11.316±0.1102µgmL(-1), respectively).

Novel flavonoid-based biodegradable nanoparticles for effective oral delivery of etoposide by P-glycoprotein modulation: an in vitro, ex vivo and in vivo investigations.

A receptor level interaction of etoposide with P-glycoprotein (P-gp) and subsequent intestinal efflux has an adverse effect on its oral absorption. The present work is aimed to enhance the bioavailability of etoposide by co-administering it with quercetin (a P-gp inhibitor) in dual-loaded polymeric nanoparticle formulation. Poly-lactic-co-glycolic acid (PLGA) nanoparticles were optimized for various parameters like o/w phase volume ratio, poly-vinyl alcohol concentration, PLGA concentration and sonication time. The cytotoxicity studies (MTT assay) revealed a 9- and 11-fold decrease in the IC 50 values for etoposide-loaded nanoparticles (ENP) and etoposide + quercetin dual-loaded nanoparticles (EQNP) when compared to that of free etoposide, respectively, and the results were further supported by florescent-activated cell sorter studies. The confocal imaging of the intestinal sections treated with ENP and EQNP containing fluorescent probe (rhodamine) showed the superiority of the EQNP to permeate deeper. Furthermore, pharmacokinetic studies on rats revealed that EQNP exhibited a 2.4-fold increase in bioavailability of etoposide than ENP with no quercetin. The developed loaded nanoparticles have the high potential to enhance the bioavailability of the etoposide and sensitize the resistant cells.

Delivery of polysaccharides using polymer particles: implications on size-dependent immunogenicity, opsonophagocytosis, and protective immunity.

Bacterial capsular polysaccharides are components of many modern vaccines, but they are weakly immunogenic. Herein, we describe the delivery of pneumococcal capsular polysaccharide serotype-1 (PCP-1) in polylactide polymeric particles to enhance its immunogenicity. Immunization with PCP-1-entrapped particles elicited long-term memory antibody responses from a single intramuscular injection. PCP-1-entrapped nanoparticles (NPs) elicited significantly higher anti-PCP-1 IgG responses than that observed with soluble and microparticles (MPs) formulations. Delivering PCP-1 and pneumococcal proteins in same particles did not improve the IgG response. The sera of animals immunized with PCP-1-entrapped particles promoted efficient opsonophagocytosis of pneumococci by macrophages. Single-dose immunization with PCP-1-entrapped particles conferred a long-term serotype-specific protection against lethal pneumococcal challenge. The higher immunogenicity of PCP-1 nanoparticles showed correlation with enhanced uptake by antigen-presenting cells. The results highlight the potential of polymeric nanoparticles as an efficient means of presenting polysaccharide antigens to the immune system.

Solubilization and refolding of bacterial inclusion body proteins.

Inclusion bodies produced in Escherichia coli are composed of densely packed denatured protein molecules in the form of particles. Refolding of inclusion body proteins into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost in production of recombinant proteins from E. coli. With new information available on the structure and function of protein aggregates in bacterial inclusion bodies, it has been possible to develop improved solubilization and refolding procedures for higher recovery of bioactive protein. Inclusion bodies are formed from partially folded protein intermediates and are composed of aggregates of mostly single types of polypeptide. This helps to isolate and purify the protein aggregates to homogeneity before solubilization and refolding. Proteins inside inclusion body aggregates have native-like secondary structures. It is assumed that restoration of this native-like secondary structure using mild solubilization conditions will help in improved recovery of bioactive protein in comparison to solubilization using a high concentration of chaotropic agent. Analysis of the dominant forces causing aggregation during inclusion body formation provides information to develop suitable mild solubilization procedures for inclusion body proteins. Refolding from such solubilized protein will be very high due to restoration of native-like secondary structure. Human growth hormone inclusion bodies were purified to homogeneity from E. coli cells before solubilization and refolding. Pure inclusion bodies were solubilized at alkaline pH in the presence of 2 M urea solution. The solubilized proteins were refolded using a pulsatile renaturation process and subsequently purified using chromatographic procedures. More than 40% of the inclusion body proteins could be refolded back to the bioactive native conformation. Mild solubilization is thus the key for high recovery of bioactive protein from inclusion bodies.