Ruhi V. Ubale

Implementation of mixture design for formulation of albumin containing enteric-coated spray-dried microparticles

Context: Oral delivery of proteins has been a challenging as well as rapidly developing field. Objective: To implement mixture design of experiment to develop enteric-coated microparticles containing bovine serum albumin. Materials and methods: Microparticles were prepared using Buchi Spray Dryer 191. Simplex lattice mixture design computed using JMP software was implemented to compare the gastric protection rendered by Eudragit FS30D, Eudragit L100-55, and Eudragit S100 in microparticulate form. Further, an extreme vertices mixture design was used to incorporate hydroxypropyl methylcellulose (HPMC) Chitosan in the formulation to delay the release. Microparticle recovery yield and protein content in microparticles were evaluated. Results and discussions: The design was statistically significant with Eudragit S100 resulting in protein release of < 5% in acidic buffer. The selected optimal formulation had 70% of Eudragit S, 25% HPMC, and 5% Chitosan. The release profiles of protein from Eudragit S alone and along with HPMC were compared. About 25% decrease in the amount of protein release was observed 6 h post exposure of microparticle to buffer of pH 6.8. The microparticle recovery yield reduced from 77.99% to 71.56% which is due to addition of HPMC into the formulation matrix. Conclusion: Although all three Eudragit polymers can be used for enteric coating, in the microparticulate form Eudragit S resulted in higher gastric protection. Also use of HPMC along with Eudragit S resulted in further sustained release.

Formulation and Characterization of Atropine Sulfate in Albumin–Chitosan Microparticles for In Vivo Ocular Drug Delivery

The overall study goal was to produce a microparticle formulation containing atropine sulfate for ocular administration with improved efficacy and lower side effects, compared with that of the standard marketed atropine solution. The objective was to prepare an atropine sulfate-loaded bovine serum albumin-chitosan microparticle that would have longer contact time on the eyes as well as better mydriatic and cycloplegic effect using a rabbit model. The microparticle formulation was prepared by method of spray-drying technique. The percent drug loading and encapsulation efficiency were assessed using a USP (I) dissolution apparatus. The particle sizes and zeta potential were determined using laser scattering technique and the surface morphology of the microparticles was determined using a scanning electron microscope. The product yield was calculated from relative amount of material used. In vitro cytotoxicity and uptake by human corneal epithelial cells were examined using AlamarBlue and confocal microscopy. The effects of the microparticle formulation on mydriasis in comparison with the marketed atropine sulfate solution were evaluated in rabbit eyes. The prepared microparticle formulation had ideal physicochemical characteristics for delivery into the eyes. The in vivo studies showed that the microparticles had superior effects on mydriasis in rabbits than the marketed solutions

Formulation of meningococcal capsular polysaccharide vaccine-loaded microparticles with robust innate immune recognition

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis associated with a high mortality rate. Capsular polysaccharides (CPSs) are a major virulence factor and form the basis for serogroup designation and protective vaccines. The current polysaccharide meningococcal vaccines are available but are very expensive and require chemical conjugation. Here, we report a novel meningococcal vaccine formulation consisting of meningococcal CPS polymers encapsulated in albumin-based biodegradable microparticles that slowly release antigen and induce robust innate immune responses. Vaccines that elicit innate immunity are reported to have enhanced and protective adaptive immune responses. In this study, the meningococcal CPS-loaded microparticles, but not the empty microparticles, induced the release of IL-8, TNF-α and IL-1β, enhanced phagocytic capacity and induced robust autophagy in macrophages. The novel meningococcal vaccine microparticles are robustly taken up by macrophages and elicit strong innate immune responses that enhance antigen presentation which is a prerequisite for inducing adaptive immunity.

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.

Development and Validation of a UPLC Method for Rapid and Simultaneous Analysis of Proton Pump Inhibitors

Proton pump inhibitors (PPIs) are used extensively for the relief of gastroesophageal reflux, peptic ulcers, and other hypersecretory conditions. Some of the commonly used PPIs—omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole—were used in this study with the aim of developing a rapid ultra performance liquid chromatography (UPLC) method for detecting each and allowing separation and quantification of a mixture of PPIs. An analysis of samples was performed on a UPLC system equipped with a quaternary solvent delivery system, a refrigerated sample manager, a column heater, a photo diode array detector scanning from 210 to 400 nm, and a C18 analytical column (50 mm × 3.0 mm, 1.7-μm particle size). The chromatographic analysis of the PPI samples and standards was performed using gradient elution with acetonitrile and water. The calibration curve range varied for each of the PPIs ranging from a lower limit of 0.75–1.78 μg/mL to a maximum concentration of 200 μg/mL with a regression coefficient (r2) of ≥0.98. The accuracy and precision were calculated, and the %RSD was determined to be ≤0.21% (intraday) and ≤5% (interday). The LOD was 0.23–0.59 μg/mL and the LOQ was 0.71–1.78 μg/mL for each of the drugs analyzed. The method was capable of detecting and quantifying each drug in a mixture with good resolution and a total run time of less than 5 min. Herein, we report an efficient and rapid analytical method for the simultaneous detection of multiple PPIs in a mixture.

The effect of antisense to NF-κB in an albumin microsphere formulation on the progression of left-ventricular remodeling associated with chronic volume overload in rats

Abstract Background: Increased NF-κB levels play a crucial role in the pathophysiology of heart failure and are known to cause ventricular remodeling. Antisense therapy can be used for blocking the expression of NF-κB and subsequently avoiding heart failure. However, as with most biotechnology products, molecular instability and overall cost are often the major issues and concerns limiting the advancement of most antisense drugs to the market. Therefore, a cost-efficient biodegradable sustained release particle drug delivery system to transport and target NF-kB antisense to its intended site of action would be ideal. Purpose: To evaluate the in vivo performance of a sustained release spray-dried albumin microsphere formulation for effective delivery and treatment of left ventricular remodeling with antisense to NF-κB. Methods: Albumin-based microspheres encapsulating antisense to NF-kB were prepared by spray drying and studied in a rat model to treat congestive heart failure. Results: The NF-κB activation and TNF-α release seen in treated animals were significantly lower than control animals. Ventricular remodeling was controlled in animals with antisense-treated AV fistulas as ΔV0–25 and ΔV0 were significantly lower compared to animals with untreated AV fistulas. Conclusion: This treatment was successful in curbing ventricular remodeling by suppressing NF-κB activation.

Background of Ocular Drug Delivery

Delivery of drugs to the eye is one of the challenges facing clinicians due to the unique anatomy, physiology, and biochemistry of the eye. Current treatment protocols for administration of drugs in eye diseases are primarily solution, gels or ointments. However, these modes of delivery have several drawbacks such as short residence time, short duration of action, the need for repeated administrations and non-specific toxicity. This chapter will introduce a brief discussion on various topics and issues relevant to ocular therapeutics and drug delivery.

Liposome-Based Nanomedicine Therapeutics for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a very painful severe autoimmune disease with complex pathology characterized by progressive chronic inflammation, and devastation of the synovium, cartilage, and other joint-associated structures. Significant advances in research in the area of pathophysiology, diagnosis, drug development, and targeted delivery have led to improved RA therapy and better patient compliance. Targeted drug delivery using liposomal nanomedicines significantly alleviate the challenges with conventional anti-RA medications such as off-target effects, short biological half-life, poor bioavailability, high dose-related toxicity, etc. Liposomal nanomedicines in RA drug targeting offer the opportunity for passive targeting [based on size and polyethylene glycol (PEG)-ylation-mediated enhanced permeability and retention] and active targeting (ligation with antibody or peptides, etc.) and encapsulation of lipophilic, hydrophilic drugs, and/or combinational drugs. However, it has been found recently that such injectable nanomedicines raise the concern of an adverse immune phenomenon called complement activationrelated pseudo allergy (CARPA) and failure of therapy on multiple doses due to accelerated body clearance caused many by anti-PEG immunoglobulin M. To ensure safety and efficacy of RA therapy, these need to be considered along with the common formulation quality parameters. Here, we discuss nanotherapeutic targeting in RA therapy using liposomes. Liposomal nanoparticles are investigated for individual anti-RA drug categories. CARPA issues and pathophysiology with such nanomedicines are also discussed in detail.

In Vitro and in Vivo Evaluation of Ocular Drugs and Delivery Systems

Animal models like the rabbit have traditionally been used to study the safety and efficacy of ophthalmic products. However, these models are now steadily being replaced by in vitro and ex vivo models that are more convenient and less expensive. The chapter provides an overview of the physicochemical tests used for ocular products as well as the established and recently constructed ocular models and the advantages and limitations associated with these models.

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.