Rikhav P. Gala

A comprehensive production method of self-cryoprotected nano-liposome powders

This study provided a convenient approach for large scale production of hydrogenated soya phosphatidylcholine nano-liposome powders using beclometasone dipropionate as a model drug and sucrose as proliposome carrier. Fluid-bed coating was employed to manufacture proliposomes by coating sucrose with the phospholipid (5%, 10%, 15% and 20% weight gains), followed by hydration, size reduction using high pressure homogenization, and freeze-drying to yield stable nano-vesicles. High pressure homogenization was compared with probe-sonication in terms of liposome size, zeta potential and drug entrapment. Furthermore, the effect of freeze-drying on vesicle properties generated using both size reduction methods was evaluated. Results have shown that high-pressure homogenization followed by freeze-drying and rehydration tended to yield liposomes smaller than the corresponding vesicles downsized via probe-sonication, and all size measurements were in the range of 72.64-152.50 nm, indicating that freeze-drying was appropriate, regardless of the size reduction technique. The liposomes, regardless of size reduction technique and freeze drying had slightly negative zeta potential values or were almost neutral in surface charge. The entrapment efficiency of BDP in homogenized liposomes was found to increase following freeze-drying, hence the drug entrapment efficiency values in rehydrated liposomes were 64.9%, 57%, 69.5% and 64.5% for 5%, 10%, 15% and 20% weight gains respectively. In this study, we have reported a reliable production method of nano-liposomes based on widely applicable industrial technologies such as fluid-bed coating, high pressure homogenization and freeze-drying. Moreover, sucrose played a dual role as a carrier in the proliposome formulations and as a cryoprotectant during freeze-drying.

Induction of Death Receptor CD95 and Co-stimulatory Molecules CD80 and CD86 by Meningococcal Capsular Polysaccharide-Loaded Vaccine Nanoparticles

ABSTRACTNeisseria meningitidis is a leading cause of bacterial meningitis and sepsis, and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. We formulated a novel nanovaccine containing meningococcal CPS as an antigen encapsulated in albumin-based nanoparticles (NPs) that does not require chemical conjugation to a protein carrier. These nanoparticles are taken up by antigen-presenting cells and act as antigen depot by slowly releasing the antigen. In this study, we determined the ability of CPS-loaded vaccine nanoparticles to induce co-stimulatory molecules, namely CD80, CD86, and CD95 that impact effective antigen presentation. Co-stimulatory molecule gene induction and surface expression on macrophages and dendritic cells pulsed with meningococcal CPS-loaded nanoparticles were investigated using gene array and flow cytometry methods. Meningococcal CPS-loaded NP significantly induced the surface protein expression of CD80 and CD86, markers of dendritic cell maturation, in human THP-1 macrophages and in murine dendritic cells DC2.4 in a dose-dependent manner. The massive upregulation was also observed at the gene expression. However, high dose of CPS-loaded NP, but not empty NP, induced the expression of death receptor CD95 (Fas) leading to reduced TNF-α release and reduction in cell viability. The data suggest that high expression of CD95 may lead to death of antigen-presenting cells and consequently suboptimal immune responses to vaccine. The CPS-loaded NP induces the expression of co-stimulatory molecules and acts as antigen depot and can spare antigen dose, highly desirable criteria for vaccine formulations.

Evaluation of various adjuvant nanoparticulate formulations for meningococcal capsular polysaccharide-based vaccine

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and preventive vaccines. We have formulated a novel meningococcal nanoparticulate vaccine formulation that does not require chemical conjugation, but encapsulates meningococcal CPS polymers in a biodegradable material that slowly release antigens, thereby has antigen depot effect to enhance antigenicity. The novel vaccine formulation is inexpensive and can be stored as a dry powder with extended shelf life that does not require the cold-chain which facilitates storage and distribution. In order to enhance the antigenicity of meningococcal nanoparticulate vaccine, we screened various adjuvants formulated in nanoparticles, for their ability to potentiate antigen presentation by dendritic cells. Here, we report that MF59 and Alum are superior to TLR-based adjuvants in enhancing dendritic cell maturation and antigen presentation markers MHC I, MHC II, CD40, CD80 and CD86 in dendritic cells pulsed with meningococcal CPS nanoparticulate vaccine.

Novel Whole-Cell Inactivated Neisseria Gonorrhoeae Microparticles as Vaccine Formulation in Microneedle-Based Transdermal Immunization

Neisseria gonorrhoeae is a strict human pathogen responsible for more than 100 million new sexually transmitted infections worldwide each year. Due to the global emergence of antibiotic resistance, the Center for Disease control (CDC) recently listed N. gonorrhoeae as an urgent threat to public health. No vaccine is available in spite of the huge disease burden and the possibility of untreatable gonorrhea. The aim of this study is to investigate the immunogenicity of a novel whole-cell-based inactivated gonococcal microparticle vaccine formulation loaded in dissolvable microneedles for transdermal administration. The nanotechnology-based vaccine formulation consists of inactivated whole-cell gonococci strain CDC-F62, spray dried and encapsulated into biodegradable cross-linked albumin matrix with sustained slow antigen release. The dry vaccine nanoparticles were then loaded in a dissolvable microneedle skin patch for transdermal delivery. The efficacy of the whole-cell microparticles vaccine formulation loaded in microneedles was assessed in vitro using dendritic cells and macrophages as well as in vivo mouse model. Antibody titers were measured using an enzyme immunosorbent assay (ELISA) and antigen-specific T lymphocytes were assessed in spleens and lymph nodes. Here we report that whole-cell-based gonococcal microparticle vaccine loaded in dissolvable microneedles for transdermal administration induced significant increase in antigen-specific IgG antibody titers and antigen-specific CD4 and CD8 T lymphocytes in mice compared to gonococcal antigens in solution or empty microneedles. Significant increase in antigen-specific IgG antibody levels was observed at the end of week 2 in groups that received the vaccine compared to the group receiving empty nanoparticles. The advantages of using formalin-fixed whole-cell gonococci that all immunogenic epitopes are covered and preserved from degradation. The spherical shaped micro and nanoparticles are biological mimics of gonococci, therefore present to the immune system as invaders but without the ability to suppress adaptive immunity. In conclusion, the transdermal delivery of microparticles vaccine via a microneedle patch was shown to be an effective system for vaccine delivery. The novel gonorrhea nanovaccine is cheap to produce in a stable dry powder and can be delivered in microneedle skin patch obviating the need for needle use or the cold chain.

Formulation and testing of novel particulate vaccines for measles

Statement of the Problem: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and a major cause of cancer mortality in the world. Despite advances in CRC treatment, a greater proportion of patients, highlighting the need for new immunotherapy. Endothelial monocyte-activating polypeptide-II (EMAP-II) is a multifunctional cytokine with pro-inflammatory properties. Aminopeptidase N (CD13) is a zinc-binding protease that has a role in cancerogenesis. It is proposed that both EMAP II and CD13 are involved in tumor progression and metastasis. The purpose of this study is to develop a novel immunotherapy expressing anti-EMAP and anti-CD13 antibodies in CRC.P DNA (pDNA) vaccines have the potential to elicit an immune response against a wide range of diseases. However, the limitation of poor uptake of pDNA to antigen-presenting cells and rapid degradation of pDNA encapsulated in nanoparticles prompted us to fabricate an encapsulation free pDNA nanoparticulate vaccine. The negatively charge pDNA adsorbs on cationic PLGA (poly (d, l-lactide-co-glycolide)-chitosan nanoparticles and were used as means to deliver pDNA. Nanoparticles in the size range of 380-500 nm with a zeta potential of 50.0 mV were prepared using emulsification method. pDNA PLGA-chitosan nanoparticles were dispersed in poloxamer 407 that possess fluid property at 4oC and turns into gel at body temperature. Binding affinity of pDNA to cationic nanoparticles depends on pDNA to nanoparticles ratio (P/N) and complete immobilization of pDNA to cationic nanoparticles was achieved at a P/N ratio of 1/50. pDNA adsorption efficiency of 99.0 percent was achieved in pDNA PLGAchitosan nanoparticles prepared using chitosan glutamate concentration of 2 mg/mL. Complex of pDNA and cationic nanoparticles was well tolerated and maintained cell survival rate greater than 80.0 percent. Additionally, cellular uptake was found to be both time and concentration dependent and followed saturation kinetics with Vmax of 11.389 μg/mL.hr and Km value of 139.48 μg/mL. In-vitro release study of P/N, 1/50 showed that the nanoparticulate vaccine can sustain the release of pDNA up to 24 hours. In our study, we demonstrated that pDNA PLGA-chitosan nanoparticles were non-cytotoxic, showed enhance cellular uptake, and sustain the release of pDNA for extended period.neration of neutrophil extracellular traps (NETs), but also modulate the immune response according to the microenvironment and the cell-cell interactions. As it is known, NETs are formed by chromatin and granular proteins which after stimulation are released to the extracellular environment. NETs have not only been involved as defence mechanisms, but they have also been linked to tissue damage, thrombosis, cancer immune editing and autoimmunity. Poly morpho nuclear neutrophils (PMNs) activated in vitro may express molecules normally associated with antigen presenting cells (APCs).Objective: Some evidence suggests that irritable bowel syndrome (IBS) is affected by the immune system. This study focused effect of Azoximer bromide in concert with Probiotics in treatment of IBS.

Design and development of ovarian cancer vaccines

Statement of the Problem: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and a major cause of cancer mortality in the world. Despite advances in CRC treatment, a greater proportion of patients, highlighting the need for new immunotherapy. Endothelial monocyte-activating polypeptide-II (EMAP-II) is a multifunctional cytokine with pro-inflammatory properties. Aminopeptidase N (CD13) is a zinc-binding protease that has a role in cancerogenesis. It is proposed that both EMAP II and CD13 are involved in tumor progression and metastasis. The purpose of this study is to develop a novel immunotherapy expressing anti-EMAP and anti-CD13 antibodies in CRC.P DNA (pDNA) vaccines have the potential to elicit an immune response against a wide range of diseases. However, the limitation of poor uptake of pDNA to antigen-presenting cells and rapid degradation of pDNA encapsulated in nanoparticles prompted us to fabricate an encapsulation free pDNA nanoparticulate vaccine. The negatively charge pDNA adsorbs on cationic PLGA (poly (d, l-lactide-co-glycolide)-chitosan nanoparticles and were used as means to deliver pDNA. Nanoparticles in the size range of 380-500 nm with a zeta potential of 50.0 mV were prepared using emulsification method. pDNA PLGA-chitosan nanoparticles were dispersed in poloxamer 407 that possess fluid property at 4oC and turns into gel at body temperature. Binding affinity of pDNA to cationic nanoparticles depends on pDNA to nanoparticles ratio (P/N) and complete immobilization of pDNA to cationic nanoparticles was achieved at a P/N ratio of 1/50. pDNA adsorption efficiency of 99.0 percent was achieved in pDNA PLGAchitosan nanoparticles prepared using chitosan glutamate concentration of 2 mg/mL. Complex of pDNA and cationic nanoparticles was well tolerated and maintained cell survival rate greater than 80.0 percent. Additionally, cellular uptake was found to be both time and concentration dependent and followed saturation kinetics with Vmax of 11.389 μg/mL.hr and Km value of 139.48 μg/mL. In-vitro release study of P/N, 1/50 showed that the nanoparticulate vaccine can sustain the release of pDNA up to 24 hours. In our study, we demonstrated that pDNA PLGA-chitosan nanoparticles were non-cytotoxic, showed enhance cellular uptake, and sustain the release of pDNA for extended period.neration of neutrophil extracellular traps (NETs), but also modulate the immune response according to the microenvironment and the cell-cell interactions. As it is known, NETs are formed by chromatin and granular proteins which after stimulation are released to the extracellular environment. NETs have not only been involved as defence mechanisms, but they have also been linked to tissue damage, thrombosis, cancer immune editing and autoimmunity. Poly morpho nuclear neutrophils (PMNs) activated in vitro may express molecules normally associated with antigen presenting cells (APCs).Objective: Some evidence suggests that irritable bowel syndrome (IBS) is affected by the immune system. This study focused effect of Azoximer bromide in concert with Probiotics in treatment of IBS.

Trends in Nonparenteral Delivery of Biologics, Vaccines and Cancer Therapies

Abstract Recent years have witnessed the development and advancement of many nonparenteral biologics and vaccines for human use. This chapter discusses various nonparenteral routes of administration. The oral route of administration is the most preferred and patient compliant method of them all. Transdermal, buccal, and pulmonary routes are also discussed. We have developed novel technologies using nanoparticles and microparticles to deliver vaccines by the oral and transdermal route of administration. These new technologies enable the formulation of vaccine particles containing vaccine antigens, without loss of their biological activity during the formulation process. Also, multiple antigens, targeting ligands and adjuvants can all be encapsulated within the same particle. When administered orally, these particles are designed to withstand the acidic environment of the stomach and are targeted to the Peyer’s patches and the gut-associated mucosal immune system. Because these vaccines are particulate in nature, they are readily taken up by phagocytic antigen presenting cells (APCs), such as M cells, dendritic cells, and macrophages in the Peyer’s patches of the intestines, resulting in a strong immune response and antibody production. Of particular interest is the fact that the particles release the antigen in a slow and sustained manner over a prolonged time period, intracellularly into APCs, resulting in strong mucosal and systemic immunity after oral administration, without the need for added adjuvants that are typically present in current vaccine preparations. Because no needles are required for oral vaccines, this method of vaccine delivery is inexpensive and suitable for mass vaccination in the developing world as well as for the developed world. This chapter discusses studies conducted on a wide array of vaccines, including infectious disease vaccines and cancer vaccines. This method of vaccine delivery enables the delivery of a wide spectrum of vaccines for prophylactic and therapeutic use, including oral and transdermal vaccines for cancer such as human papillomavirus, melanoma, ovarian, breast, and prostate with encouraging results. With respect to cancer therapy, a comparison is made between the conventional cancer therapy and immunotherapy. With a wide range of nanocarriers available for delivery of biologics, vaccines, and cancer therapies, nanotechnology not only has gained the well-deserved limelight but has also attracted the attention of regulatory bodies, although it presents certain challenges that must be considered before marketing such nanocarriers.