Kevin J. Peine

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.

Efficient Delivery of the Toll-like Receptor Agonists Polyinosinic:Polycytidylic Acid and CpG to Macrophages by Acetalated Dextran Microparticles

To enhance the immune activity of vaccine adjuvants polyinosinic:polycytidylic acid (poly I:C) and CpG acetalated dextran (Ac-DEX) microparticles can be used. Ac-DEX is a biodegradable and water-insoluble polymer that degrades significantly faster at pH 5.0 (phagosomal pH) than at pH 7.4 and has tunable degradation rates that can range from hours to months. This is an ideal characteristic for delivery of an antigen and adjuvant within the lysosomal compartment of a phagocytic cell. We evaluated poly I:C and CpG encapsulated in Ac-DEX microparticles using RAW macrophages as a model antigen-presenting cell. These cells were cultured with poly I:C or CpG in their free form, encapsulated in a fast degrading Ac-DEX, in slow degrading Ac-DEX, or in the Food and Drug Administration-approved polymer poly(lactic-co-glycolic acid) (PLGA). Ac-DEX had higher encapsulation efficiencies for both poly I:C and CpG than PLGA. Furthermore, poly I:C or CpG encapsulated in Ac-DEX also showed, in general, a significantly stronger immunostimulatory response than PLGA and unencapsulated CpG or poly I:C, which was indicated by a higher rate of nitric oxide release and increased levels of cytokines such as TNF-α, IL-6, IL-10, and IFN-γ. Overall, we have illustrated a method for enhancing the delivery of these vaccine adjuvants to further enhance the development of Ac-DEX vaccine formulations.

Treatment of experimental autoimmune encephalomyelitis by codelivery of disease associated Peptide and dexamethasone in acetalated dextran microparticles.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system that can cause loss of motor function and is thought to result, in part, from chronic inflammation due to an antigen-specific T cell immune response. Current treatments suppress the immune system without antigen specificity, increasing the risks of cancer, chronic infection, and other long-term side effects. In this study, we show treatment of experimental autoimmune encephalomyelitis (EAE), a model of MS, by coencapsulating the immunodominant peptide of myelin oligodendrocyte glycoprotein (MOG) with dexamethasone (DXM) into acetalated dextran (Ac-DEX) microparticles (DXM/MOG/MPs) and administering the microparticles subcutaneously. The clinical score of the mice was reduced from 3.4 to 1.6 after 3 injections 3 days apart with the coencapsulated microparticulate formulation (MOG 17.6 μg and DXM 8 μg). This change in clinical score was significantly greater than observed with phosphate-buffered saline (PBS), empty MPs, free DXM and MOG, DXM/MPs, and MOG/MPs. Additionally, treatment with DXM/MOG/MPs significantly inhibited disease-associated cytokine (e.g., IL-17, GM-CSF) expression in splenocytes isolated in treated mice. Here we show a promising approach for the therapeutic treatment of MS using a polymer-based microparticle delivery platform.

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.

Liposomal resiquimod for the treatment of Leishmania donovani infection

OBJECTIVES The imidazoquinoline family of drugs are Toll-like receptor 7/8 agonists that have previously been used in the treatment of cutaneous leishmaniasis. Because of the hydrophobic nature of imidazoquinolines, they are traditionally not administered systemically for the treatment of visceral leishmaniasis. We formulated liposomal resiquimod, an imidazoquinoline, for the systemic treatment of visceral leishmaniasis. METHODS By using lipid film hydration with extrusion, we encapsulated resiquimod in liposomes. These liposomes were then injected intravenously to treat BALB/c mice infected with Leishmania donovani. RESULTS Treatment with liposomal resiquimod significantly decreased the parasite load in the liver, spleen and bone marrow. In addition, resiquimod treatment increased interferon-γ and interleukin-10 production in an antigen recall assay. Resiquimod was shown to be non-toxic in histology and in vitro culture experiments. CONCLUSIONS FDA-approved resiquimod, in a liposomal formulation, displays promising results in treating visceral leishmaniasis.

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.

Delivery of host cell-directed therapeutics for intracellular pathogen clearance.

Intracellular pathogens present a major health risk because of their innate ability to evade clearance. Their location within host cells and ability to react to the host environment by mutation or transcriptional changes often enables survival mechanisms to resist standard therapies. Host-directed drugs do not target the pathogen, minimizing the potential development of drug resistance; however, they can be difficult to deliver efficiently to intracellular sites. Vehicle delivery of host-mediated response drugs not only improves drug distribution and toxicity profiles, but can reduce the total amount of drug necessary to clear infection. In this article, we will review some host-directed drugs and current drug delivery techniques that can be used to efficiently clear intracellular infections.

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.

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.