Acetalated dextran is a chemically and biologically tunable material for particulate immunotherapy.

Materials that combine facile synthesis, simple tuning of degradation rate, processability, and biocompatibility are in high demand for use in biomedical applications. We report on acetalated dextran, a biocompatible material that can be formed into microparticles with degradation rates that are tunable over 2 orders of magnitude depending on the degree and type of acetal modification. Varying the degradation rate produces particles that perform better than poly(lactic-co-glycolic acid) and iron oxide, two commonly studied materials used for particulate immunotherapy, in major histocompatibility complex class I (MHC I) and MHC II presentation assays. Modulating the material properties leads to antigen presentation on MHC I via pathways that are dependent or independent of the transporter associated with antigen processing. To the best of our knowledge, this is the only example of a material that can be tuned to operate on different immunological pathways while maximizing immunological presentation.

Mannosylated dextran nanoparticles: a pH-sensitive system engineered for immunomodulation through mannose targeting.

Biotherapeutic delivery is a rapidly growing field in need of new materials that are easy to modify, are biocompatible, and provide for triggered release of their encapsulated cargo. Herein, we report on a particulate system made of a polysaccharide-based pH-sensitive material that can be efficiently modified to display mannose-based ligands of cell-surface receptors. These ligands are beneficial for antigen delivery, as they enhance internalization and activation of APCs, and are thus capable of modulating immune responses. When compared to unmodified particles or particles modified with a nonspecific sugar residue used in the delivery of antigens to dendritic cells (DCs), the mannosylated particles exhibited enhanced antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. This represents the first demonstration of a mannosylated particulate system that enables enhanced MHC I antigen presentation by DCs in vitro. Our readily functionalized pH-sensitive material may also open new avenues in the development of optimally modulated vaccine delivery systems.

In vitro analysis of acetalated dextran microparticles as a potent delivery platform for vaccine adjuvants.

Toll-like receptor (TLR) agonists induce potent innate immune responses and can be used in the development of novel vaccine adjuvants. However, access to TLRs can be challenging as exemplified by TLR 7, which is located intracellularly in endosomal compartments. To increase recognition and subsequent stimulatory effects of TLR 7, imiquimod was encapsulated in acetalated dextran (Ac-DEX) microparticles. Ac-DEX, a water-insoluble and biocompatible polymer, is relatively stable at pH 7.4, but degrades rapidly under acidic conditions, such as those found in lysosomal vesicles. To determine the immunostimulatory capacity of encapsulated imiquimod, we compared the efficacy of free versus encapsulated imiquimod in activating RAW 264.7 macrophages, MH-S macrophages, and bone marrow derived dendritic cells. Encapsulated imiquimod significantly increased IL-1 beta, IL-6, and TNF-alpha cytokine expression in macrophages relative to the free drug. Furthermore, significant increases were observed in classic macrophage activation markers (iNOS, PD1-L1, and NO) after treatment with encapsulated imiquimod over the free drug. Also, bone marrow derived dendritic cells produced significantly higher levels of IL-1 beta, IL-6, IL-12p70, and MIP-1 alpha as compared to their counterparts receiving free imiquimod. These results suggest that encapsulation of TLR ligands within Ac-DEX microparticles results in increased immunostimulation and potentially better protection from disease when used in conjunction with vaccine formulations.

Chemoselective ligation in the functionalization of polysaccharide-based particles.

Despite the promise of precisely targeted or otherwise functionalized polymeric particulate drug delivery vehicles, typical biocompatible particles are generally not amenable to facile and selective surface modification. Herein, we report the development of a simple, mild, and chemoselective strategy for the conjugation of biologically active molecules to the surface of dextran-based microparticles. Alkoxyamine-bearing reagents were used to form stable oxime conjugates with latent aldehyde functionality present in reducing carbohydrate chain ends. We demonstrate the functionalization of dextran-based microparticles with a fluorophore as well as a cell-penetrating peptide sequence, which facilitated the delivery of cargo to nonphagocytic cells leading to a 60-fold increase in the expression of a reporter gene when plasmid DNA-loaded particles were used.

Microfabricated devices for enhanced bioadhesive drug delivery: attachment to and small-molecule release through a cell monolayer under flow.

The development of a novel microfabricated device for oral drug delivery that overcomes many of the common barriers present in the gastrointestinal tract is reported. Specifically, the attachment of targeting ligands, subsequent device binding, and small molecule release from the microdevices in flow are investigated. A diffusion chamber that permits the simultaneous study of particle binding and small-molecule release under physiologically relevant shear conditions is developed. It is observed that once the particles bind to the cell surface, they remain attached. A small fraction of the devices detach in flow; however, most of these devices readily reattach to the cell layer in a new location. This steady-state density of microdevices is most likely the result of larger order microdevice clusters releasing their loose interactions with nearby microdevices, shifting slightly downstream, and subsequently reattaching to the cell monolayer. The release of a model small molecule from microdevices over time is roughly linear and approximately ten times greater than that observed with the small molecule alone. Overall, the preparation and characterization of an oral drug-delivery microdevice system capable of both targeting and asymmetric release in flow is reported.

T-cell activation by antigen-loaded pH-sensitive hydrogel particles in vivo: the effect of particle size.

Polymeric carriers designed to encapsulate protein antigens have great potential for improving the efficacy of vaccines and immunotherapeutics for diseases such as cancer. We recently developed a carrier system based on polyacrylamide hydrogel microparticles cross-linked with acid-labile moieties. After being phagocytosed by antigen-presenting cells, the protein encapsulated within the carrier is released and processed for subsequent presentation of antigenic epitopes. To understand the impact of particle size on the activation of T-cells following uptake by antigen-presenting cells, particles with mean diameters of 3.5 microm and 35 nm encapsulating a model protein antigen were synthesized by emulsion and microemulsion based polymerization techniques, respectively. In vivo tests demonstrated that both sizes of particles were effective at stimulating the proliferation of T-cells and were capable of generating an antigen-specific cytotoxic T-cell response when coadministered with immunostimulatory DNA. Contrary to previous reports in the literature, our results suggest that there is no significant difference in the magnitude of T-cell activation for the two sizes of particles used in these experiments. This disparity in findings may be related to fundamental differences in material properties of the carriers used in these studies, such as the hydrophilicity of the polyacrylamide particles described here versus the hydrophobic nature of carriers investigated by other groups.

Acetal-derivatized dextran: an acid-responsive biodegradable material for therapeutic applications.

Dextran, a biocompatible, water-soluble polysaccharide, was modified at its hydroxyls with acetal moieties such that it became insoluble in water but freely soluble in common organic solvents enabling its use in the facile preparation of acid-sensitive microparticles. These particles degrade in a pH-dependent manner: FITC-dextran was released with a half-life at 37 degrees C of 10 h at pH 5.0 compared to a half-life of approximately 15 days at pH 7.4. Both hydrophobic and hydrophilic cargoes were successfully loaded into these particles using single and double emulsion techniques, respectively. When used in a model vaccine application, particles loaded with the protein ovalbumin (OVA) increased the presentation of OVA-derived peptides to CD8+ T-cells 16-fold relative to OVA alone. Additionally, this dextran derivative was found to be nontoxic in preliminary in vitro cytotoxicity assays. Owing to its ease of preparation, processability, pH-sensitivity, and biocompatibility, this type of modified dextran should find use in numerous drug delivery applications.

Fully acid-degradable biocompatible polyacetal microparticles for drug delivery.

A library of polyurethanes and polyureas with different hydrophobicities containing the same acid-degradable dimethyl ketal moiety embedded in the polymer main chain have been prepared. All polymers were synthesized using an AA-BB type step-growth polymerization by reaction of bis(p-nitrophenyl carbamate/carbonate) or diisocyanate monomers with an acid-degradable, ketal-containing diamine. These polymers were designed to hydrolyze at different rates in mildly acidic conditions as a function of their hydrophobicity to afford small molecules only with no polymeric byproduct. The library of polymers was screened for the formation of microparticles using a double emulsion technique. The microparticles that were obtained degraded significantly faster at acidic pH (5.0) than at physiological pH (7.4) with degradation kinetics related to the hydrophobicity of the starting polymer. In vitro studies demonstrated the ability of the FITC-BSA loaded microparticles to be phagocytosed by macrophages resulting in a 10-fold increase in the protein uptake compared to a free protein control; in addition, the microparticles were found to be nontoxic at the concentrations tested of up to 1 mg/mL. The ease of preparation of the polymers coupled with the ability to tune their hydrophobicity and the high acid sensitivity of the microparticles identify this new class of materials as promising candidates for the delivery of bioactive materials.

In vivo studies on the effect of co-encapsulation of CpG DNA and antigen in acid-degradable microparticle vaccines.

Protein-based vaccines have been explored as a safer alternative to traditional weakened or killed whole organism based vaccination strategies and have been investigated for their ability to activate the immune system against certain cancers. For optimal stimulation of T lymphocytes, protein-based vaccines should deliver protein antigens to antigen presenting cells in the context of appropriate immunostimulatory signals, thus mimicking actual pathogens. In this report, we describe the synthesis, characterization, and biological evaluation of immunostimulatory acid-degradable microparticles, which are suitable delivery vehicles for use in protein-based vaccines and cancer immunotherapy. Using a 3' conjugation strategy, we optimized the attachment of immunostimulatory CpG DNA to our vaccine carriers and demonstrated that under acidic conditions similar to those found in endosomal compartments, these new particles were capable of simultaneously releasing a model protein antigen and a CpG DNA adjuvant. We found in an in vivo cytotoxicity assay that the co-encapsulation of ovalbumin, a model antigen, and immunostimulatory agent in the same particle led to superior cytotoxic T lymphocyte activity compared to particles coadministered with adjuvant in an unbound form. In addition, we investigated the ability of these acid-degradable particles to induce protective immunity in the MO5 murine melanoma model and found that they were effective until tumor escape, which appeared to result from a loss of antigen expression by the cancer cells due to in vivo selection pressure.

Acid-degradable polyurethane particles for protein-based vaccines: biological evaluation and in vitro analysis of particle degradation products.

Acid-degradable particles containing a model protein antigen, ovalbumin, were prepared from a polyurethane with acetal moieties embedded throughout the polymer, and characterized by dynamic light scattering and transmission electron microscopy. The small molecule degradation byproduct of the particles was synthesized and tested in vitro for toxicity indicating an LC 50 of 12,500 microg/mL. A new liquid chromatography-mass spectrometry technique was developed to monitor the in vitro degradation of these particles. The degradation byproduct inside RAW macrophages was at its highest level after 24 h of culture and was efficiently exocytosed until it was no longer detectable after 4 days. When tested in vitro, these particles induced a substantial increase in the presentation of the immunodominant ovalbumin-derived peptide SIINFEKL in both macrophages and dendritic cells. In addition, vaccination with these particles generated a cytotoxic T-lymphocyte response that was superior to both free ovalbumin and particles made from an analogous but slower-degrading acid-labile polyurethane polymer. Overall, we present a fully degradable polymer system with nontoxic byproducts, which may find use in various biomedical applications including protein-based vaccines.

Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity.

The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.