Sadhana Sharma

Nanoengineered device for drug delivery application

A high precision nanoengineered device was developed to yield long term zero-order release of drugs for therapeutic applications. The device contains nanochannels that were fabricated in between two directly bonded silicon wafers and therefore poses high mechanical strength. The fabrication is based upon selectively growing oxide and then removing it, and thus defining nanochannels by consuming a specified layer of silicon during oxide growth. Diffusion through the nanochannels is the rate limiting step for the release of drugs. A measurement of glucose released through such nanochannels validates the zero-order release profile. Device design, fabrication details and the glucose release profile through 60 nm channels are presented.

Electrospray encapsulation of toll-like receptor agonist resiquimod in polymer microparticles for the treatment of visceral leishmaniasis.

Leishmaniasis is a disease caused by the intracellular protozoan, Leishmania. A current treatment for cutaneous leishmaniasis involves the delivery of imidazoquinolines via a topical cream. However, there are no parenteral formulations of imidazoquinolines for the most deadly version of the disease, visceral leishmaniasis. This work investigates the use of electrospray to encapsulate the imidazoquinoline adjuvant resiquimod in acid sensitive microparticles composed of acetalated dextran (Ac-DEX) or Ac-DEX/Tween blends. The particles were characterized and tested both in vitro and in vivo. Solutions of Ac-DEX and resiquimod in ethanol were electrosprayed to generate approximately 2 μm Ac-DEX particles containing resiquimod with an encapsulation efficiency of 85%. To prevent particle aggregation, blends of Ac-DEX with Tween 20 and Tween 80 were investigated. Tween 80 was then blended with the Ac-DEX at ∼10% (w/w) of total polymer and particles containing resiquimod were formed via electrospray with encapsulation efficiencies between 40% and 60%. In vitro release profiles of resiquimod from Ac-DEX/Tween 80 particles exhibited the acid-sensitive nature of Ac-DEX, with 100% drug release after 8 h at pH 5 (phagosomal pH) and after 48 h at pH 7.4 (physiological pH). Treatment with Ac-DEX/Tween 80 particles elicited significantly greater immune response in RAW macrophages over free drug. When injected intravenously into mice inoculated with Leishmania, parasite load reduced significantly in the bone marrow compared to blank particles and phosphate-buffered saline controls. Overall, electrospray appears to offer an elegant, scalable way to encapsulate adjuvant into an acid sensitive delivery vehicle for use in treating visceral leishmaniasis.

Controlled-release microchips

Efficient drug delivery remains an important challenge in medicine: continuous release of therapeutic agents over extended time periods in accordance with a predetermined temporal profile; local delivery at a constant rate to the tumour microenvironment to overcome much of the systemic toxicity and to improve antitumour efficacy; improved ease of administration, and increasing patient compliance required are some of the unmet needs of the present drug delivery technology. Microfabrication technology has enabled the development of novel controlled-release microchips with capabilities not present in the current treatment modalities. In this review, the current status and future prospects of different types of controlled-release microchips are summarised and analysed with reference to microneedle-based microchips, as well as providing an in-depth focus on microreservoir-based and nanoporous microchips.

Enhanced stability of horseradish peroxidase encapsulated in acetalated dextran microparticles stored outside cold chain conditions

Micro- and nanoparticles have been shown to improve the efficacy of safer protein-based (subunit) vaccines. Here, we evaluate a method of improving the vaccine stability outside cold chain conditions by encapsulation of a model enzyme, horseradish peroxidase (HRP), in an acid-sensitive, tunable biodegradable polymer, acetalated dextran (Ac-DEX). Vaccines that are stable outside the cold chain would be desirable for use in developing nations. Ac-DEX particles encapsulating HRP were prepared using two different methods, probe sonication and homogenization. These particles were stored under different storage conditions (-20 °C, 4 °C, 25 °C or 45 °C) for a period of 3 months. On different days, the particles were characterized for various physical and chemical measurements. At all conditions, Ac-DEX particles remained spherical in nature, as compared to PLGA particles that fused together starting at day 3 at 45 °C. Furthermore, our results indicated that encapsulation of HRP in Ac-DEX reduces its storage temperature dependence and enhances its stability outside cold chain conditions. Homogenized particles performed better than probe sonicated particles and retained 70% of the enzymes initial activity as compared to free HRP that retained only 40% of the initial activity after 3 months of storage at 25 °C or 45 °C. Additionally, HRP activity was more stable when encapsulated in Ac-DEX, and the variance in enzyme activity between the different storage temperatures was not observed for either particle preparation. This suggests that storage at a constant temperature is not required with vaccines encapsulated in Ac-DEX particles. Overall, our results suggest that an Ac-DEX based micro-/nanoparticles system has wide applications as vaccines and drug delivery carriers, including those in developing nations.

Long-term biocompatibility of NanoGATE drug delivery implant

The fouling of components and the formation of a fibrotic tissue capsule around subcutaneously implanted medical devices are two major obstacles in developing viable, long-term implantable drug delivery systems. NanoGATE is a subcutaneous implant designed for constant-output passive diffusion of a drug of interest through a silicon nanopore membrane. To this end, we have investigated the long-term in vivo biocompatibility of the NanoGATE implant in terms of the fouling of the nanopore membrane and the formation of a fibrotic tissue capsule around the implant. We have also evaluated how these effects influence diffusion of a lysozyme surrogate from the device once implanted within the vascular compartment of a Sprague-Dawley rat model. Using several model biomolecules such as glucose, lysozyme, and albumin, our studies suggest that silicon nanopore membranes do not foul when implanted subcutaneously for 6 mo. This study also reveals the tissue capsule that naturally forms around the implant does not limit diffusion of molecules with molecular weights on the order of 14.4 kDa at therapeutic delivery rates of tens of micrograms per day. This indicates that our NanoGATE implant should be completely functional in vivo, providing constant release levels of a drug over an extended time period. Thus, by adjusting the release rate to fit the pharmacokinetic clearance profile of the Sprague-Dawley rat, long-term steady-state blood plasma concentrations can be achieved.

Rapid vaccination using an acetalated dextran microparticulate subunit vaccine confers protection against triplicate challenge by bacillus anthracis.

PurposeA rapid immune response is required to prevent death from Anthrax, caused by Bacillus anthracis.MethodWe formulated a vaccine carrier comprised of acetalated dextran microparticles encapsulating recombinant protective antigen (rPA) and resiquimod (a toll-like receptor 7/8 agonist).ResultsWe were able to protect against triplicate lethal challenge by vaccinating twice (Days 0, 7) and then aggressively challenging on Days 14, 21, 28. A significantly higher level of antibodies was generated by day 14 with the encapsulated group compared to the conventional rPA and alum group. Antibodies produced by the co-encapsulated group were only weakly-neutralizing in toxin neutralization; however, survival was not dependent on toxin neutralization, as all vaccine formulations survived all challenges except control groups. Post-mortem culture swabs taken from the hearts of vaccinated groups that did not produce significant neutralizing titers failed to grow B. anthracis.ConclusionsResults indicate that protective antibodies are not required for rapid protection; indeed, cytokine results indicate that T cell protection may play a role in protection from anthrax. We report the first instance of use of a particulate carrier to generate a rapid protective immunity against anthrax.

Evaluation of a biodegradable microparticulate polymer as a carrier for Burkholderia pseudomallei subunit vaccines in a mouse model of melioidosis.

Melioidosis, a potentially lethal disease of humans and animals, is caused by the soil-dwelling bacterium Burkholderia pseudomallei. Due to B. pseudomalleis classification as a Tier 1 Select Agent, there is substantial interest in the development of an effective vaccine. Yet, despite decades of research, no effective target, adjuvant or delivery vehicle capable of inducing protective immunity against B. pseudomallei infection has been identified. We propose a microparticulate delivery vehicle comprised of the novel polymer acetalated dextran (Ac-DEX). Ac-DEX is an acid-sensitive biodegradable carrier that can be fabricated into microparticles (MPs) that are relatively stable at pH 7.4, but rapidly degrade after phagocytosis by antigen presenting cells where the pH can drop to 5.0. As compared to other biomaterials, this acid sensitivity has been shown to enhance cross presentation of subunit antigens. To evaluate this platform as a delivery system for a melioidosis vaccine, BALB/c mice were vaccinated with Ac-DEX MPs separately encapsulating B. pseudomallei whole cell lysate and the toll-like receptor (TLR) 7/8 agonist resiquimod. This vaccine elicited a robust antibody response that included both Th1 and Th2 immunity. Following lethal intraperitoneal challenge with B. pseudomallei 1026b, vaccinated mice demonstrated a significant delay to time of death compared to untreated mice. The formulation, however, demonstrated incomplete protection indicating that lysate protein offers limited value as an antigen. Nevertheless, our Ac-DEX MPs may offer an effective delivery vehicle for a subunit B. psuedomallei vaccine.

Electrospun Acetalated Dextran Scaffolds for Temporal Release of Therapeutics

Electrospun acetalated dextran (Ac-DEX) scaffolds were fabricated to encapsulate resiquimod, an immunomodulatory toll-like-receptor (TLR) agonist. Ac-DEX has been used to fabricate scaffolds for sustained and temporal delivery of therapeutics because it has tunable degradation rates that are dependent on its synthesis reaction time or the molecular weight of dextran. Additionally, as opposed to commonly electrospun polyesters that shift the local pH upon degradation, the degradation products of Ac-DEX are pH-neutral: dextran, an alcohol, and the metabolic byproduct acetone. Formulations of Ac-DEX with two different degradation rates were used in this study. The effects of electrospinning conditions on the scaffold size and morphology were examined as well as fibroblast adhesion as imaged with fluorescence microcopy and scanning electron microscopy. Macrophage (MΦ) viability further indicates that the scaffolds are cytocompatible. Also, the controlled release profiles of resiquimod from loaded scaffolds and nitric oxide (NO) production by MΦ incubated with these scaffolds show the potential for Ac-DEX scaffolds to be used to temporally and efficiently deliver therapeutics. Overall, we present a novel scaffold that can have tunable and unique drug release rates for tissue engineering, drug delivery, immunomodulation, and wound healing applications.

Adsorption Equilibrium and Kinetics of Egg-White Proteins on Immobilized Metal Ion Affinity Gels for Designing Fractionation

Designing an Immobilized Metal ion Affinity (IMA) chromatographic process on large scale demands a thorough understanding to be developed regarding the adsorption behaviour of proteins on metal loaded IMA (IMA-M(II)) gels and the characteristic adsorption parameters to be evaluated. This research investigation illustrates the significance of these aspects for the proposed fractionation of chicken egg-white proteins on these gels. Consequently, a systematic investigation of the adsorption characteristics of three chicken egg-white proteins viz., ovalbumin, conalbumin and lysozyme on Cu(II) and Ni(II) loaded IMA gels, iminodiacetate (IDA) and tris(2-aminoethyl)amine (TREN), has been undertaken. These gels differ in their selectivity towards the proteins of interest under the identical sets of experimental conditions. While TREN-Ni(II) was selective only for lysozyme, IDA-Cu(II), IDA-Ni(II) and TREN-Cu(II) showed varying affinities for all the three proteins. The equilibrium and kinetic data were analysed using various theoretical models and adsorption parameters were quantified. On the basis of these investigations, various strategies have been proposed for the efficient large-scale fractionation of chicken egg-white proteins on these gels.

Gene delivery to cultured embryonic stem cells using nanofiber-based sandwich electroporation.

Multiple gene transfections are often required to control the differentiation of embryonic stem cells. This is typically done by removing the cells from the culture substrate and conducting gene transfection via bulk electroporation (in suspension), which is then followed by further culture. Such repetitive processes could affect the growth and behavior of delicate/scarce adherent cells. We have developed a novel nanofiber-based sandwich electroporation device capable of in situ and in culture gene transfection. Electrospinning was used to deposit poly(ε-caprolactone)/gelatin nanofibers on the Al(2)O(3) nanoporous support membrane, on top of which a polystyrene microspacer was thermally bonded to control embryonic stem cell colony formation. The applicability of this system was demonstrated by culturing and transfecting mouse embryonic stem cells. Measurements of secreted alkaline phosphatase protein and metabolic activity showed higher transfection efficacy and cell viability compared to the conventional bulk electroporation approach.