Hassan Borteh

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.

Acetalated dextran encapsulated AR-12 as a host-directed therapy to control Salmonella infection.

AR-12 has been evaluated in clinical trials as an anti-cancer agent but also has demonstrated host-directed, broad-spectrum clearance of bacteria. We have previously shown that AR-12 has activity in vitro against Salmonella enterica serovar Typhimurium and Francisella species by inducing autophagy and other host immune pathways. AR-12 treatment of S. Typhimurium-infected mice resulted in a 10-fold reduction in bacterial load in the liver and spleen and an increased survival time. However, AR-12 treatment did not protect mice from death, likely due poor formulation. In the current study, AR-12 was encapsulated in a microparticulate carrier formulated from the novel degradable biopolymer acetalated dextran (Ace-DEX) and subsequently evaluated for its activity in human monocyte-derived macrophages (hMDMs). Our results show that hMDMs efficiently internalized Ace-DEX microparticles (MPs), and that encapsulation significantly reduced host cell cytotoxicity compared to unencapsulated AR-12. Efficient macrophage internalization of AR-12 loaded MPs (AR-12/MPs) was further demonstrated by autophagosome formation that was comparable to free AR-12 and resulted in enhanced clearance of intracellular Salmonella. Taken together, these studies provide support that Ace-DEX encapsulated AR-12 may be a promising new therapeutic agent to control intracellular bacterial pathogens of macrophages by targeting delivery and reducing drug toxicity.

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.

Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

ABSTRACT Francisella tularensis causes tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activity in vitro against Salmonella enterica serovar Typhimurium and F. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to control S. Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type A F. tularensis SchuS4 infection were examined in vitro and in vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden of F. tularensis in infected human macrophages, in a manner similar to that of free AR-12. However, in vivo, AR-12/MPs significantly enhanced the survival of F. tularensis SchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival of F. tularensis SchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

Host-mediated Leishmania donovani treatment using AR-12 encapsulated in acetalated dextran microparticles.

Leishmaniasis is a disease caused by parasites of Leishmania sp., which effects nearly 12 million people worldwide and is associated with treatment complications due to widespread parasite resistance toward pathogen-directed therapeutics. The current treatments for visceral leishmaniasis (VL), the systemic form of the disease, involve pathogen-mediated drugs and have long treatment regimens, increasing the risk of forming resistant strains. One way to limit emergence of resistant pathogens is through the use of host-mediated therapeutics. The host-mediated therapeutic AR-12, which is FDA IND-approved for cancer treatment, has shown activity against a broad spectrum of intracellular pathogens; however, due to hydrophobicity and toxicity, it is difficult to reach therapeutic doses. We have formulated AR-12 into microparticles (AR-12/MPs) using the novel biodegradable polymer acetalated dextran (Ace-DEX) and used this formulation for the systemic treatment of VL. Treatment with AR-12/MPs significantly reduced liver, spleen, and bone marrow parasite loads in infected mice, while combinatorial therapies with amphotericin B had an even more significant effect. Overall, AR-12/MPs offer a unique, host-mediated therapy that could significantly reduce the emergence of drug resistance in the treatment of VL.

Fabrication of porous microtent structures toward an in vitro endothelium model

This paper reports the fabrication of compliant and permeable thin films with controlled curvature preferable to serve as engineering scaffolds for the production of in vivo like vascular endothelial constructs. A simple fabrication process was developed to fabricate three-dimensional ‘tent’ like microstructures by combining electrospinning and microfabrication. In particular, the ‘microtents’ were created by electrospinning mechanically flexible poly(etherurethane)urea(PEUU) polymer on a microstructured collecting substrate. The shape of the ‘microtents’ can be tuned by adjusting the geometries of microstructures on the collecting substrate and the operational parameters of electrospinning. Mechanical characterization showed the nonlinear mechanical behavior of porous polymeric thin films is similar to those of soft tissues, indicating that these thin films may serve as scaffolds for mimicking local mechanical environment of vascular tissues. Human endothelial cells were successfully cultured on the concave side of the porous thin film, constituting an endothelium model in vitro. This work addresses the need for engineered tissue scaffolds that can mimic both morphological and mechanical environments of natural vascular endothelium. The coupled effects of mechanical, structural and biochemical factors on vascular endothelium can thus be investigated.

Microparticles formulated from a family of novel silylated polysaccharides demonstrate inherent immunostimulatory properties and tunable hydrolytic degradability

Acid-degradable polymers are well-suited for use as drug delivery vehicles because numerous physiological sites (e.g., intracellular endocytic pathway) are acidic. Here we report the synthesis of acid-sensitive silylated polysaccharides derived from either dextran or inulin with various alkyl substitutions on the silicon center: trimethylsilyl dextran (TMS-DEX), ethyldimethylsilyl dextran (EDMS-DEX), triethylsilyl dextran (TES-DEX), and trimethylsilyl inulin (TMS-IN). The silylated dextran (Silyl-DEX) and silylated inulin (Silyl-IN) polymers were fabricated into microparticles (MPs) via emulsification followed by solvent evaporation. These MPs were relatively stable at extracellular pH 7.4 and displayed a wide range of pH 2.0 and 5.0 degradation half-lives (fifteen minutes to greater than nine days) that were dependent on the extent of silylation (40 to 98%) and steric crowding on the silicon center (trimethyl to ethyldimethyl to triethyl). Silyl-DEX and Silyl-IN MPs exhibited cytocompatibility when cultured in vitro with RAW 264.7 macrophages. TES-DEX and TMS-IN MPs, composed of highly hydrophobic moieties and the parent immunostimulatory inulin, respectively, elicited substantial in vitro production of tumor necrosis factor alpha, a cytokine associated with an innate immune response. In vivo immunization with a model ovalbumin antigen encapsulated in silylated polysaccharide MPs, without a separate adjuvant, resulted in a dual humoral and cellular response that was superior to an alum-adjuvanted formulation. Overall, we present Silyl-DEX and Silyl-IN as members of the acid-degradable polymer family for potential use in subunit vaccines and other drug delivery applications.

Porous microfluidics: A unique platform for transvascular study

This work reports the development of a porous microfluidic system which can serve as an in vitro model of natural vessels in circulatory and respiratory systems. The model possesses both structural and mechanical characteristics of the natural counterparts. To mimic the semi-permeable wall of natural vessels, microfluidic channels with porous walls are fabricated by spinning polymeric nanofibers on the collecting substrate with three-dimensional microelectrodes. The mechanical properties of the porous substrate are evaluated using conventional tensile testing. The permeability of the fibrous membrane is characterized by perfusion experiments. The results collectively show the utility of the membrane in transvascular study. Human umbilical vein endothelial cells (HUVECs) are cultured on the fibrous membrane and form an endothelial monolayer. The resulting structure is elastic and distensible, similar as natural blood vessels. The curved endothelium surface also helps to mimic the complex mechanical loading states of natural vascular endothelium under pulsatile flow. This work is expected to add a new dimension to the widely used microfluidic systems by allowing both in-vessel transport and trans-vessel transport. The immediate impacts of the porous microfluidic systems can be found in in vitro study of circulatory and respiratory systems.

Programmable micropatterning of nanofibers for functional tissue engineering

This paper reports programmable micropatterning of electrospun nanofibrous materials using micropatterned collecting chips that consist of independently programmable microelectrodes. By regulating the local electrical field using activated and floating electrodes, the collecting chip allows creating the pattern of electrospun fibrous microstructures with controllable local orientation. This work provides a simple yet highly reliable way to produce micro/nanostructures of polymer nanofibers in a cost effective manner. It has a great potential in creating functional tissue engineered scaffolds with morphological and functional similarities with natural tissues, and can also be used as filtering substrates in microdevices with designed performance for on-chip biochemical analysis.