Acetalated Dextran Microparticles for Codelivery of STING and TLR7/8 Agonists.

Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.

In Vivo and Cellular Trafficking of Acetalated Dextran Microparticles for Delivery of a Host-Directed Therapy for Salmonella enterica Serovar Typhi Infection.

Previously we have encapsulated host-directed therapy AR-12 into acetalated dextran (Ace-DEX) microparticles (MPs) to mitigate drug toxicity and passively target phagocytic host cells. Herein, we have improved upon our initial emulsion-based formulation of Ace-DEX MPs encapsulating AR-12 (AR-12/MPs) by improving the drug encapsulation efficiency, evaluating sterilization processes for manufacturing, and understanding cellular and in vivo trafficking of the MPs. By using an alternative solvent system, ethyl acetate, we report an increased encapsulation efficiency of AR-12 while maintaining the pH-responsive degradation kinetics of Ace-DEX MPs. To better manufacture this novel antimicrobial formulation, we sterilized AR-12/MPs by gamma irradiation or ethylene oxide and evaluated their efficacy against intracellular Salmonella enterica serovar Typhi. Sterilized AR-12/MPs resulted in a significant reduction in intracellular bacterial burden compared to Blank/MPs. We also characterized intracellular trafficking of Ace-DEX MPs encapsulating fluorophores, which demonstrated internalization of MPs in endo/lysosomal compartments and time and degradation-rate dependent lysosomal escape into cytosolic compartments. Additionally, in vivo toxicity was mitigated following encapsulation of AR-12, where the maximum tolerated dose of AR-12 was increased compared to soluble treatment via intranasal, intravenous, and intraperitoneal administration routes. Following in vivo trafficking of Ace-DEX MPs via the same routes, intranasal administration demonstrated the highest accumulation in the lungs, liver, and kidneys, which persisted out to 240 h. Overall, we have advanced the formulation of this host-directed therapy and broadened the understanding of Ace-DEX MP delivery.

Electrosprayed Myocet-like Liposomes: An Alternative to Traditional Liposome Production.

PURPOSE: Although doxorubicin (DXR) has been on the market for many years as an anti-cancer drug, a number of serious dose-limiting toxicities hinder its widespread use. To reduce the known toxicities of soluble DXR, various liposomes have been designed including Doxil, Caelyx, and Myocet. Myocet, a non-PEGylated liposomal formulation containing DXR, was found to reduce the toxicities associated with soluble DXR and has been used in Europe and Canada (but not the US) as a first line therapy. While regarded as successful, Myocet does have some formulation drawbacks including stability, drug release, and an arduous formulation and remote loading method for preparation. METHODS: Our lab has developed a liposomal electrospray process in which formulation and remote loading occurs continuously in one step, cutting down on the total time of production and increasing the drug retention in the liposomes with respect to more conventional methods. Electrosprayed Myocet-like liposomes were then tested in vitro for release kinetics and cytotoxicity with respect to a more conventional formulation method. RESULTS: Myocet-like liposomes manufactured via electrospray had similar DXR loadings, hydrodynamic diameters, morphologies, and cytotoxic profiles as their thin-film hydration counterparts, but their release profiles were drastically prolonged. CONCLUSIONS: Our findings indicate that electrospray is a viable manufacturing procedure to scalably produce Myocet-like liposomes that appear to be more stable than those formulated through thin-film hydration.

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

Francisella tularensiscauses 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 activityin vitroagainstSalmonella entericaserovar Typhimurium andF. 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 controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin 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 ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

One Step Encapsulation of Small Molecule Drugs in Liposomes via Electrospray-Remote Loading.

Resiquimod is a Toll-like receptor (TLR) 7/8 agonist that has previously been used as a vaccine adjuvant, as a topical treatment of viral lesions and skin cancer, and as an antiviral treatment. We report on the combined application of remote loading and electrospray to produce liposomal resiquimod, with the broader goals of improving drug encapsulation efficiency and scalability of liposome production methods. Drug loading in liposomes increased from less than 1% to greater that 3% by mass when remote loading was used, whether the liposomes were generated by thin-film hydration or electrospray methods. Dynamic light scattering (DLS) determined mean vesicle diameters of 137 ± 11 nm and 103 ± 4 for the thin-film and electrospray methods, respectively. Transmission electron microscopy (TEM) images showed spherical vesicles with sizes consistent with the DLS measurements. In vitro drug release profiles found that most of the drug remained within the liposomes at both pH 5.5 and 7.4. The in vitro bioactivity of the liposomal drug was also demonstrated by the increase in nitrite production when RAW macrophages were exposed to the drug. Our findings indicate that the remotely loaded liposomes formed via the scalable electrospray method have characteristics comparable to those produced via conventional batch methods. The methods discussed here are not limited to the enhanced delivery of resiquimod. Rather, they should be readily adaptable to other compounds compatible with remote loading.

Saquinavir Loaded Acetalated Dextran Microconfetti - a Long Acting Protease Inhibitor Injectable.

PURPOSE: Since the adoption of highly active antiretroviral therapy, HIV disease progression has slowed across the world; however, patients are often required to take multiple medications daily of poorly bioavailable drugs via the oral route, leading to gastrointestinal irritation. Recently, long acting antiretroviral injectables that deliver drug for months at a time have moved into late phase clinical trials. Unfortunately, these solid phase crystal formulations have inherent drawbacks in potential dose dumping and a greater likelihood for burst release of drug compared to polymeric formulations. METHODS: Using electrospinning, acetalated dextran scaffolds containing the protease inhibitor saquinavir were created. Grinding techniques were then used to process these scaffolds into injectables which are termed saquinavir microconfetti. Microconfetti was analyzed for in vitro and in vivo release kinetics. RESULTS: Highly saquinavir loaded acetalated dextran electrospun fibers were able to be formed and processed into saquinavir microconfetti while other polymers such as poly lactic-co-glycolic acid and polycaprolactone were unable to do so. Saquinavir microconfetti release kinetics were able to be tuned via drug loading and polymer degradation rates. In vivo, a single subcutaneous injection of saquinavir microconfetti released drug for greater than a week with large tissue retention. CONCLUSIONS: Microconfetti is a uniquely tunable long acting injectable that would reduce the formation of adherence related HIV resistance. Our findings suggest that the injectable microconfetti delivery system could be used for long acting controlled release of saquinavir and other hydrophobic small molecule drugs.

Progesterone and calcitriol attenuate inflammatory cytokines CXCL1 and CXCL2 in ovarian and endometrial cancer cells.

Cytokines/chemokines are key players in cancer-related inflammation. Increasing evidence suggests that chemokines produced by tumor cells are the mediators of metastasis. Thus, agents that can downregulate chemokines expression have potential against cancer metastasis. We have previously shown inhibition of ovarian and endometrial cancer cell growth with progesterone and calcitriol. In the present study, we evaluated the effect of these two agents on the expression of inflammatory genes. Using a RT-PCR array of inflammatory cytokines/chemokines and their receptors, we found a marked attenuation of CXCL1 and CXCL2 (GRO-α and -ß) in cancer cells by both treatments. Knockdown of NFκB resulted in a reduced expression of CXCL1 and CXCL2 and the inhibitory effect of progesterone and calcitriol on the expression of chemokines was abrogated in NFκB-silenced cancer cells. Silencing of IκBα increased the expression of CXCL1 and CXCL2 in cancer cells, which can be attributed to the increased activation of NFκB-p65, caused by the lack of its inhibitor. Progesterone and calcitriol-induced inhibition was abolished in IκBα-knockdown cells. Our results demonstrate that suppression of IκBα phosphorylation by progesterone and calcitriol contributes to the reduced expression of CXCL1 and CXCL2. Downregulation of CXCL1 and CXCL2 was associated with a marked inhibition of metastasis-promoting genes. Overall, our results indicate that progesterone and calcitriol inhibit IκBα phosphorylation, NFκB activation, and the expression of NFκB regulated metastasis promoting genes. These results provide attractive data for the possible use of progesterone and calcitriol in the management of endometrial and ovarian tumors.

Tunable degradation of acetalated dextran microparticles enables controlled vaccine adjuvant and antigen delivery to modulate adaptive immune responses.

Subunit vaccines are often poorly immunogenic, and adjuvants and/or delivery vehicles, such as polymeric microparticles (MPs), can be used to enhance immune responses. MPs can also be used to understand cell activation kinetics and the significant impact antigen and adjuvant release has on adaptive immune responses. By controlling antigen and adjuvant release, we can determine if it is important to have precise temporal control over release of these elements to optimize the peak and duration of protective immunity and improve vaccine safety profiles. In order to study the effect of tunable adjuvant or antigen delivery on generation of adaptive immunity, we used acetalated dextran (Ace-DEX) MPs. Ace-DEX MPs were used because their tunable degradation can be controlled based on polymer cyclic acetal coverage (CAC). Ace-DEX MPs of varying degradation profiles were used to deliver murabutide or ovalbumin (OVA) as a model adjuvant or antigen, respectively. When murabutide was encapsulated within Ace-DEX MPs to test for controlled adjuvant delivery, fast-degrading MPs exhibited higher humoral and cellular responses in vivo at earlier time points, while slow-degrading MPs resulted in stronger responses at later time points. When OVA was encapsulated within Ace-DEX MPs to test for controlled antigen delivery, fast-degrading MPs induced greater antibody and cytokine production throughout the length of the experiment. This differential response suggests the need for distinct, flexible control over adjuvant or antigen delivery and its impact on immune response modulation.

Injectable long-acting human immunodeficiency virus antiretroviral prodrugs with improved pharmacokinetic profiles.

While highly active antiretroviral therapy (HAART) has significantly reduced mortality rates in patients with human immunodeficiency virus type 1 (HIV-1), its efficacy may be impeded by emergence of drug resistance caused by lack of patient adherence. A therapeutic strategy that requires infrequent drug administration as a result of sustained release of antiretroviral drugs would put less burden on the patient. Long-acting antiretroviral prodrugs for HIV therapy were synthesized through modification of the active drugs, emtricitabine (FTC) and elvitegravir (EVG), with docosahexaenoic acid (DHA) in one-step, one-pot, high-yielding reactions. The in vitro drug release profiles of these synthetic conjugates demonstrated sustained and controlled release of the active drug over a period of 3-4 weeks attributable to the hydrolysis of the chemical linker in conjunction with the hydrophilicity of the parent drug. Both conjugates exhibited superior antiviral activities in tissue culture models of HIV replication as compared to those of the free drugs, strengthening their role as potent prodrugs for HIV therapy. Pharmacokinetic analysis in CD1 mice further confirmed the long-acting aspect of these conjugates with released drug concentrations in plasma detected at their respective IC90/IC95 values over a period of 2 weeks and discernable amounts of active drug even at 6 weeks. Our findings suggest that the injectable small molecule conjugates could be used as long-acting controlled release of FTC and EVG in attempts to mitigate adherence-related HIV resistance.

A robust microparticle platform for a STING-targeted adjuvant that enhances both humoral and cellular immunity during vaccination.

Most FDA-approved adjuvants for infectious agents boost humoral but not cellular immunity, and have poorly-understood mechanisms. Stimulator of interferon genes (STING, also known as MITA, MPYS, or ERIS) is an exciting adjuvant target due to its role in cyclic dinucleotide (CDN)-driven anti-viral immunity; however, a major hindrance is STING's cytosolic localization which requires intracellular delivery of its agonists. As a result, STING agonists administered in a soluble form have elicited suboptimal immune responses. Delivery of STING agonists via particle platforms has proven a more successful strategy, but the opportunity for improved formulations and bioactivity remains. In this study we evaluated the adjuvant activity of the potent STING agonist, CDN 3'3'-cGAMP (cGAMP), encapsulated in acid-sensitive acetalated dextran (Ace-DEX) polymeric microparticles (MPs) which passively target antigen-presenting cells for intracellular release. This formulation was superior to all particle delivery systems evaluated and maintained its bioactivity following a sterilizing dose of gamma irradiation. Compared to soluble cGAMP, the Ace-DEX cGAMP MPs enhanced type-I interferon responses nearly 1000-fold in vitro and 50-fold in vivo, caused up to a 104-fold boost in antibody titers, increased Th1-associated responses, and expanded germinal center B cells and memory T cells. Furthermore, the encapsulated cGAMP elicited no observable toxicity in animals and achieved protective immunity against a lethal influenza challenge seven months post-immunization when using CDN adjuvant doses up to 100-fold lower than previous reports. For these reasons, Ace-DEX MP-encapsulated cGAMP represents a potent vaccine adjuvant of humoral and cellular immunity.

Chemically modified inulin microparticles serving dual function as a protein antigen delivery vehicle and immunostimulatory adjuvant.

To develop a new subunit vaccine adjuvant, we chemically modified a naturally-occurring, immunostimulatory inulin polysaccharide to produce an acid-sensitive biopolymer (acetalated inulin, Ace-IN). Various hydrophobic Ace-IN polymers were formed into microparticles (MPs) by oil-in-water emulsions followed by solvent evaporation These Ace-IN MPs possessed tunable degradation characteristics that, unlike polyesters used in FDA-approved microparticulate formulations, had only pH-neutral hydrolytic byproducts. Macrophages were passively targeted with cytocompatible Ace-IN MPs. TNF-α production by macrophages treated with Ace-IN MPs could be altered by adjusting the polymers' chemistry. Mice immunized with Ace-IN MPs encapsulating a model ovalbumin (OVA) antigen showed higher production of anti-OVA IgG antibody levels relative to soluble antigen. The antibody titers were also comparable to an alum-based formulation. This proof-of-concept establishes the potential for chemically-modified inulin MPs to simultaneously enable dual functionality as a stimuli-controlled antigen delivery vehicle and immunostimulatory adjuvant.

Degradation of acetalated dextran can be broadly tuned based on cyclic acetal coverage and molecular weight.

Microparticles (MPs) derived from acid-sensitive biopolymers enable rapid degradation and cargo release under acidic conditions, such as at tumor microenvironments, within lysosomal/phagosomal compartments inside phagocytic cells, or at sites of inflammation. One such acid-sensitive biopolymer, acetalated dextran (Ace-DEX), has tunable degradation rates and pH-neutral degradation byproducts consisting of dextran, acetone, and ethanol. By studying the degradation profiles of Ace-DEX MPs with varying cyclic acetal coverage (CAC) and dextran molecular weight (MW), we concluded that MPs composed of low CAC or high MW polymer degraded the fastest at both pH 7.4 and 5.0. To further understand the properties of this unique polymer, we encapsulated a model drug resiquimod, which is a toll-like receptor (TLR) 7/8 agonist, into Ace-DEX MPs of different polymer CAC and dextran MW. It was observed that resiquimod was released faster from MPs of lower CAC or higher MW. By evaluating the activation of RAW macrophages cultured with different types of resiquimod-loaded Ace-DEX MPs, we found that MPs of lower CAC or higher MW promoted greater nitrite production and resulted in more robust cell activation. Our results indicate we can precisely control the degradation profile, release kinetics, and bioactivity of encapsulated cargos by altering CAC and MW, furthering Ace-DEX MPs' novelty as a drug carrier.

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.

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.