Poly(diaminosulfide) Microparticle-Based Vaccine for Delivery of Leptospiral Antigens.

Leptospirosis is a debilitating infectious disease that detrimentally affects both animals and humans; therefore, disease prevention has become a high priority to avoid high incidence rates of disease in the herd and break the transmission cycle to humans. Thus, there remains an important unmet need for a prophylactic vaccine that can provide long-term immunity against leptospirosis in cattle. Herein, a novel vaccine formulation was developed where poly(diaminosulfide) polymer was employed to fabricate microparticles encapsulating the antigen of Leptospira borgpetersenii serovar Hardjo strain HB15B203 (L203-PNSN). A prime-boost vaccination with a L203-PNSN microparticle formulation increased the population of L203-specific CD3+ T cells and CD21+ B cells to levels that were significantly higher than those of cattle vaccinated with L203-AlOH or the vehicle control (empty PNSN microparticles and blank AlOH). In addition, L203-PNSN was demonstrated to stimulate durable humoral immune responses as evidenced by the increases in the antibody serum titers following the vaccination. It was also found that cattle vaccinated with L203-PNSN produced higher macroscopic agglutinating titers than cattle in other groups. Thus, it can be concluded that L203-PNSN is a novel first-in-class microparticle-based Leptospira vaccine that represents a powerful platform with the potential to serve as a prophylactic vaccine against leptospiral infection in cattle.

The absence of CpG in plasmid DNA-chitosan polyplexes enhances transfection efficiencies and reduces inflammatory responses in murine lungs.

Chitosan polyplexes containing plasmid DNA (pDNA) have significant potential for pulmonary gene delivery applications. However, prior to using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. In this study, we formulated 200-400 nm CSpp with amine to phosphate (N/P) ratios that ranged from 1 to 100. We compared two types of plasmids within CSpp: pDNA that was free of CpG sequences (CpG(-)) and pDNA that contained CpG sequences (CpG(+)). Both forms of CSpp showed low cytotoxicity when cultured with A549 and HEK293 cell lines in vitro. CSpp(CpG(-)) generated higher luciferase expression both in vitro, for A549 cells, and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(-)) elicited milder inflammatory responses in mice one day subsequent to nasal instillation, as determined by proinflammatory cytokine levels within the bronchoalveolar lavage fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the preclinical safety assessments of CSpp in pulmonary gene delivery systems.

Preparation and Characterization of a Liver Targeted, Poly(amidoamine) Based, Gene Delivery System.

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease that is considered a major cause of liver cirrhosis and hepatocellular carcinoma. NASH is characterized by multiple underlying genetic mutations, with no approved cure to date. Gene therapies that target those genetic mutations may play a major role in treating this disease, once delivered specifically to the hepatocytes. In this chapter we present, in detail, the synthesis and the characterization of an efficient gene delivery system capable of targeting hepatocytes by exploiting the overexpression of asialoglycoprotein receptors on their cell surface. The targeting ligand, galactose derivative, lactobionic acid (Gal), is first conjugated to bifunctional poly(ethylene glycol) (PEG), and then the formed PEG-Gal is further conjugated to the positively charged polymer, poly(amidoamine) (PAMAM) to form a PAMAM-PEG-Gal construct that can complex and deliver genetic material (e.g., pDNA, siRNA, mRNA) specifically to hepatocytes. We first synthesize PAMAM-PEG-Gal using carbodiimide click chemistry. The synthesized conjugate is characterized using 1H NMR spectroscopy and mass spectrometry. Next, nanoplexes are prepared by combining the positively charged conjugate and the negatively charged genetic material at different nitrogen to phosphate (N/P) ratios; then the size, charge, electrophoretic mobility, and surface morphology of those nanoplexes are estimated. The simplicity of complexing our conjugate with any type of genetic material, the ability of our delivery system to overcome the current limitations of delivering naked genetic material, and the efficiency of delivering its payload specifically to hepatocytes, makes our formulation a promising tool to treat any type of genetic abnormality that arises in hepatocytes, and specifically NASH.

The MEK 1/2 inhibitor PD98059 exhibits synergistic anti-endometrial cancer activity with paclitaxel in vitro and enhanced tissue distribution in vivo when formulated into PAMAM-coated PLGA-PEG nanoparticles.

Endometrial cancer is the most common gynecological cancer that affects the female reproductive organs. The standard therapy for EC for the past two decades has been chemotherapy and/or radiotherapy. PD98059 is a reversible MEK inhibitor that was found in these studies to increase the cytotoxicity of paclitaxel (PTX) against human endometrial cancer cells (Hec50co) in a synergistic and dose-dependent manner. Additionally, while PD98059 arrested Hec50co cells at the G0/G1 phase, and PTX increased accumulation of cells at the G2/M phase, the combination treatment increased accumulation at both the G0/G1 and G2/M phases at low PTX concentrations. We recently developed poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) modified with polyethylene glycol (PEG) and coated with polyamidoamine (PAMAM) (referred to here as PGM NPs) which have favorable biodistribution profiles in mice, compared to PD98059 solution. Here, in order to enhance tissue distribution of PD98059, PD98059-loaded PGM NPs were prepared and characterized. The average size, zeta potential, and % encapsulation efficiency (%EE) of these NPs was approximately 184 nm, + 18 mV, and 23%, respectively. The PD98059-loaded PGM NPs released ~ 25% of the total load within 3 days in vitro. In vivo murine studies revealed that the pharmacokinetics and biodistribution profile of intravenous (IV) injected PD98059 was improved when delivered as PD98059-loaded PGM NPs as opposed to soluble PD98059. Further investigation of the in vivo efficacy and safety of this formulation is expected to emphasize the potential of its clinical application in combination with commercial PTX formulations against different cancers.

Combining Doxorubicin-Loaded PEGylated Poly(Lactide-co-glycolide) Nanoparticles with Checkpoint Inhibition Safely Enhances Therapeutic Efficacy in a Melanoma Model.

Doxorubicin (DOX) has been widely used for the treatment of various cancers, however, the use of soluble DOX is limited by its low therapeutic index and improved formulations are therefore sought. Aside from its tumoricidal properties, DOX has also been shown to cause an immunogenic form of cell death, however, it is becoming abundantly clear that in situ immune stimulation alone is insufficient to cause significant immune based antitumor activity and that immune checkpoint modulation is also required. In this study, DOX-loaded nanoparticles were made by nanoprecipitation of DOX with a PEGylated poly(lactide-co-glycolide) copolymer (DOX-PLGA-PEG NPs) and were then tested in combination with immune checkpoint blockade (antiprogrammed death (anti-PD-1)) in a murine melanoma model to enhance antitumor effectiveness. Results showed the prepared particles to be approximately 134 nm in diameter (zeta potential -22 mV) with a loading of 1.75 µg DOX/mg NPs. In vitro release studies (of DOX) revealed the NPs to exhibit a 12 h burst release phase, followed by a slower release phase for up to 200 h. Survival studies of mice challenged with B16.F10 melanoma cells, revealed 60% of mice treated with the combination of DOX-PLGA-PEG NPs plus anti-PD-1 were tumor-free at the completion of the study. This combination therapy demonstrated higher antitumor efficacy in vivo compared to control, soluble DOX, and monotherapy of DOX-PLGA-PEG NPs or anti-PD-1 solution (p < 0.05). Moreover, in vivo safety studies (mouse weight/histopathological/toxicity) were investigated and results suggested that the combination therapy was safe. In conclusion, this study demonstrates the successful fabrication of DOX-loaded NPs by a nanoprecipitation method, and when combined with checkpoint inhibition could provide significant therapy in a murine melanoma model, suggesting that the DOX-PLGA-PEG NPs may be generating immune stimulation in situ and that benefit from this combination may be obtained in a clinical setting in the future.

Tissue Engineering for the Temporomandibular Joint.

Tissue engineering potentially offers new treatments for disorders of the temporomandibular joint which frequently afflict patients. Damage or disease in this area adversely affects masticatory function and speaking, reducing patients' quality of life. Effective treatment options for patients suffering from severe temporomandibular joint disorders are in high demand because surgical options are restricted to removal of damaged tissue or complete replacement of the joint with prosthetics. Tissue engineering approaches for the temporomandibular joint are a promising alternative to the limited clinical treatment options. However, tissue engineering is still a developing field and only in its formative years for the temporomandibular joint. This review outlines the anatomical and physiological characteristics of the temporomandibular joint, clinical management of temporomandibular joint disorder, and current perspectives in the tissue engineering approach for the temporomandibular joint disorder. The tissue engineering perspectives have been categorized according to the primary structures of the temporomandibular joint: the disc, the mandibular condyle, and the glenoid fossa. In each section, contemporary approaches in cellularization, growth factor selection, and scaffold fabrication strategies are reviewed in detail along with their achievements and challenges.

Antitumor Efficacy and Toxicity of 5-Fluorouracil-Loaded Poly(Lactide Co-glycolide) Pellets.

The aim of this study was to formulate a biodegradable implant capable of imparting local antitumor activity through the sustained release of the chemotherapeutic agent, 5-fluorouracil (5-FU). Thus, injectable pellets (<1.2 mm diameter) made from poly(lactide co-glycolide) (PLGA) and loaded with 5-FU at varying drug:polymer ratios were fabricated using hot-melt extrusion and tested for their ability to provide sustained release of 5-FU in in vitro and in vivo settings. In addition, these formulations were compared against soluble 5-FU for their antitumor activity in vivo as well as for their toxicity. It was demonstrated that the release rate of 5-FU from PLGA pellets was directly related to the percentage of 5-FU in the pellets. PLGA pellets loaded with 50% w/w 5-FU exhibited comparable, and significantly enhanced, antitumor activity (as measured by tumor volumes and survival) in vivo in a thymoma and colon cancer model, respectively, when compared to an equivalent bolus dose (120 mg/kg) of soluble 5-FU. We concluded that 5-FU-loaded PLGA pellets were more effective and specifically less erythrotoxic than 5-FU bolus injections and therefore may prove to be of benefit as an intraoperative adjunct therapy for patients with cancers that are sensitive to 5-FU and who are undergoing tumor resection.

Combining ultrasound and intratumoral administration of doxorubicin-loaded microspheres to enhance tumor cell killing.

Melanoma is an incurable disease for which alternative treatments to chemotherapy alone are sought. Here, using a melanoma model, we investigated the antitumor potential of combining ultrasound (US) with poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with doxorubicin (DOX). The aim was to achieve synergistic tumoricidal activity through direct and indirect US-mediated damage of tumor cells combined with sustained and potentially controllable release (when combined with US) of DOX from microspheres. An in vitro release assay demonstrated an ability of US to affect the release kinetics of DOX from DOX-loaded PLGA microspheres by inducing a 12% increase in the rate of release. In vitro viability assays demonstrated that combining US with DOX-loaded PLGA microspheres resulted in synergistic tumor cell (B16-F10 melanoma cells) killing. Melanoma-bearing mice were treated intratumorally with DOX (8 µg)-loaded microspheres and subjected to US treatment at the tumor site. This treatment could significantly extend survival (mean survival (MS) = 22.1 days) compared to untreated mice (MS = 10.4 days) and most other treatments, such as blank microspheres plus US (MS = 11.5 days) and DOX (8 µg)-loaded microspheres alone (MS = 13 days). The findings that immune checkpoint blockade did not significantly extend survival of mice treated with DOX (8 µg)-loaded microspheres plus US, and that tumor-free ("cured") mice were not protected from subsequent tumor rechallenge suggests minimal involvement of the adaptive immune response in the observed antitumor activity. Nevertheless, the synergistic increase in survival of melanoma-challenged mice treated with the combination of US and DOX-loaded microspheres implicates such a treatment methodology as a promising additional tool for combatting otherwise currently incurable cancers.

Surface engineering tumor cells with adjuvant-loaded particles for use as cancer vaccines.

Cell surface engineering is an expanding field and whilst extensive research has been performed decorating cell surfaces with biomolecules, the engineering of cell surfaces with particles has been a largely unexploited area. This study reports on the assembly of cell-particle hybrids where irradiated tumor cells were surface engineered with adjuvant-loaded, biodegradable, biocompatible, polymeric particles, with the aim of generating a construct capable of functioning as a therapeutic cancer vaccine. Successfully assembled cell-particle hybrids presented here comprised either melanoma cells or prostate cancer cells stably adorned with Toll-like receptor-9 ligand-loaded particles using streptavidin-biotin cross-linking. Both cell-particle assemblies were tested in vivo for their potential as therapeutic cancer vaccines yielding promising therapeutic results for the prostate cancer model. The ramifications of results obtained for both tumor models are openly discussed.

Surface-modified particles loaded with CaMKII inhibitor protect cardiac cells against mitochondrial injury.

An excess of calcium (Ca2+) influx into mitochondria during mitochondrial re-energization is one of the causes of myocardial cell death during ischemic/reperfusion injury. This overload of Ca2+ triggers the mitochondrial permeability transition pore (mPTP) opening which leads to programmed cell death. During the ischemic/reperfusion stage, the activated Ca2+/calmodulin-dependent protein kinase II (CaMKII) enzyme is responsible for Ca2+ influx. To reduce CaMKII-related cell death, sub-micron particles composed of poly(lactic-co-glycolic acid) (PLGA), loaded with a CaMKII inhibitor peptide were fabricated. The CaMKII inhibitor peptide-loaded (CIP) particles were coated with a mitochondria targeting moiety, triphenylphosphonium cation (TPP), which allowed the particles to accumulate and release the peptide inside mitochondria to inhibit CaMKII activity. The fluorescently labeled TPP-CIP was taken up by mitochondria and successfully reduced reactive oxygen species (ROS) caused by Isoprenaline (ISO) in a differentiated rat cardiomyocyte-like cell line. When cells were treated with TPP-CIP prior to ISO exposure, they maintained mitochondrial membrane potential. The TPP-CIP protected cells from ISO-induced ROS production and decreased mitochondrial membrane potential. Thus, TPP-CIP has the potential to be used in protection against ischemia/reperfusion injury.

Production of Adjuvant-Loaded Biodegradable Particles for Use in Cancer Vaccines.

Immune adjuvants, such as ligands for pathogen-associated molecular patterns (PAMPs), have been showing promise in boosting immune responses to tumor associated antigens, and delivering these adjuvants as discrete packages is considered advantageous over delivery in soluble form. Here we describe in detail, methods for independently loading a range of adjuvants into polymer-based biodegradable particles. We also describe the means by which to characterize these particles with respect to adjuvant loading and release kinetics as well as in terms of particle size, shape, and zeta-potential. These adjuvant-loaded particles have the potential to be used in dendritic cell-based uptake experiments performed in vitro or to be used in preclinical cancer vaccine research applications where they can be co-delivered with antigen-loaded particles or some other vaccine component comprising antigenic material.

Development and Evaluation of Biodegradable Particles Coloaded With Antigen and the Toll-Like Receptor Agonist, Pentaerythritol Lipid A, as a Cancer Vaccine.

Immune adjuvants are important components of current and prospective cancer vaccines. In this study, we aimed at evaluating the use of a synthetic lipid A derivative, pentaerythritol lipid A (PET lipid A), loaded into poly(lactic-co-glycolic acid) particles, as a potential cancer vaccine adjuvant. Poly(lactic-co-glycolic acid) particles (size range: 250-600 nm) were successfully formulated to include PET lipid A and/or the model tumor antigen, chicken ovalbumin (OVA). It was shown that particulated PET lipid A had a distinct advantage at promoting secretion of the immune potentiating cytokine, IL-12p70, and upregulating key costimulatory surface proteins, CD86 and CD40, in murine dendritic cells in vitro. In a murine tumor model, involving prophylactic vaccination with various permutations of soluble versus particulated formulations of OVA with or without PET lipid A, modest benefit was observed in terms of OVA-specific cell-mediated immune responses when PET lipid A was delivered in particles. These findings translated into a corresponding trend toward increased survival of mice challenged with OVA-expressing tumor cells (E.G7). In terms of translation of safe adjuvants into the clinic, these results promote the concept of delivering toll-like receptor-4 agonists in particles because doing so improves their adjuvant properties, while decreasing the chances of adverse effects due to off-target uptake by nonphagocytic cells.

Diaminosulfide based polymer microparticles as cancer vaccine delivery systems.

The aim of the research presented here was to determine the characteristics and immunostimulatory capacity, in vivo, of antigen and adjuvant co-loaded into microparticles made from a novel diaminosulfide polymer, poly(4,4'-trimethylenedipiperdyl sulfide) (PNSN), and to assess their potential as cancer vaccine vectors. PNSN microparticles co-loaded with the antigen, ovalbumin (OVA), and adjuvant, CpG 1826, (PNSN(OVA + CpG)) were fabricated and characterized for size (1.64 µm diameter; PDI=0.62), charge (-23.1 ± 0.3), and loading efficiencies of antigen (7.32 µg/mg particles) and adjuvant (0.95 µg/mg particles). The ability of PNSN(OVA + CpG) to stimulate cellular and humoral immune responses in vivo was compared with other PNSN microparticle formulations as well as with poly(lactic-co-glycolic acid)(PLGA)-based microparticles, co-loaded with OVA and CpG (PLGA(OVA + CpG)), an adenovirus encoding OVA (Ad5-OVA), and OVA delivered with incomplete Freund's adjuvant (IFA(OVA)). In vivo OVA-specific IgG1 responses, after subcutaneous prime/boosts in mice, were similar when PNSN(OVA + CpG) and PLGA(OVA + CpG) were compared and the presence of CpG 1826 within the PNSN microparticles demonstrated significantly improved responses when compared to PNSN microparticles loaded with OVA alone (PNSN(OVA)), plus or minus soluble CpG 1826. Cellular immune responses to all particle-based vaccine formulations ranged from being negligible to modest with PNSN(OVA + CpG) generating the greatest responses, displaying significantly increased levels of OVA-specific CD8+ T lymphocytes compared to controls and IFA(OVA) treated mice. Finally, it was shown that of all vaccination formulations tested PNSN(OVA + CpG) was the most protective against subsequent challenge with an OVA-expressing tumor cell line, E.G7. Thus, microparticles made from poly(diaminosulfide)-based macromolecules possess promising potential as vaccine vectors and, as demonstrated here, may have impact as cancer vaccines in particular.

Exploiting the tumor phenotype using biodegradable submicron carriers of chemotherapeutic drugs.

Tumor tissues possess characteristics that distinguish them from healthy tissues and make them attractive targets for submicron carriers of chemotherapeutic drugs (CTX). CTX are generally administered systemically in free form to cancer patients resulting in unwanted cytotoxic effects and placing limitations on the deliverable CTX dose. In an effort to raise the therapeutic index of CTX there are now liposome-based CTX formulations in clinical use that are more tumor specific than the free form of CTX. However, progression to liposome-based chemotherapy in the clinic has been slow and there have been no approved formulations introduced in the last decade. Alternative carrier systems such as those made from the biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) have been investigated in preclinical settings with promising outcomes. Here we review the principle behind biodegradable submicron carriers as CTX delivery vehicles for solid tumors with a specific focUS on liposomes and PLGA-based carriers, highlighting the strengths and weaknesses of each system.

Production of antigen-loaded biodegradable nanoparticles and uptake by dendritic cells.

Particle-based cancer vaccines prepared from biodegradable polymers are a potentially attractive way of delivering antigen alone, or in combination with adjuvant molecules, to dendritic cells (DC). Non-viral modes of vaccination must ensure the activation of cellular immune responses in addition to humoral responses if an effective antitumor immune response is to be initiated. Here we describe in detail a method for manufacturing poly(D,L-lactide-co-glycolide) (PLGA) particles loaded with purified protein/antigen. We also describe a method for generating bone marrow-derived DC in vitro. These DC can be subsequently used to measure the adjuvant properties of the particle formulations.

Applying biodegradable particles to enhance cancer vaccine efficacy.

One of the primary goals of our group and our collaborators here at the University of Iowa is to develop therapeutic cancer vaccines using biodegradable and biocompatible polymer-based vectors. A major advantage of using discretely packaged immunogenic cargo over non-encapsulated vaccines is that they promote enhanced cellular immunity, a key requirement in achieving antitumor activity. We discuss the importance of co-encapsulation of tumor antigen and adjuvant, with specific focus on the synthetic oligonucleotide adjuvant, cytosine-phosphate-guanine oligodeoxynucleotides. We also discuss our research using a variety of polymers including poly(α-hydroxy acids) and polyanhydrides, with the aim of determining the effect that parameters, such as size and polymer type, can have on prophylactic and therapeutic tumor vaccine formulation efficacy. Aside from their role as vaccine vectors per se, we also address the research currently underway in our group that utilizes more novel applications of biodegradable polymer-based particles in facilitating other types of immune-based therapies.

Mitochondria-targeting particles.

Mitochondria are a promising therapeutic target for the detection, prevention and treatment of various human diseases such as cancer, neurodegenerative diseases, ischemia-reperfusion injury, diabetes and obesity. To reach mitochondria, therapeutic molecules need to not only gain access to specific organs, but also to overcome multiple barriers such as the cell membrane and the outer and inner mitochondrial membranes. Cellular and mitochondrial barriers can be potentially overcome through the design of mitochondriotropic particulate carriers capable of transporting drug molecules selectively to mitochondria. These particulate carriers or vectors can be made from lipids (liposomes), biodegradable polymers, or metals, protecting the drug cargo from rapid elimination and degradation in vivo. Many formulations can be tailored to target mitochondria by the incorporation of mitochondriotropic agents onto the surface and can be manufactured to desired sizes and molecular charge. Here, we summarize recently reported strategies for delivering therapeutic molecules to mitochondria using various particle-based formulations.

Biodegradable particles as vaccine delivery systems: size matters.

Poly(lactide-co-glycolide) (PLGA) particles have strong potential as antigen delivery systems. The size of PLGA particles used to vaccinate mice can affect the magnitude of the antigen-specific immune response stimulated. In this study, we fabricated and characterized 17 µm, 7 µm, 1 µm, and 300 nm PLGA particles coloaded with a model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG ODN). PLGA particles demonstrated a size-dependent burst release followed by a more sustained release of encapsulated molecules. PLGA particles that were 300 nm in size showed the highest internalization by, and maximum activation of, dendritic cells. The systemic antigen-specific immune response to vaccination was measured after administration of two intraperitoneal injections, 7 days apart, of 100 µg OVA and 50 µg CpG ODN in C57BL/6 mice. In vivo studies showed that 300 nm sized PLGA particles generated the highest antigen-specific cytotoxic T cell responses by days 14 and 21. These mice also showed the highest IgG2a:IgG1 ratio of OVA-specific antibodies on day 28. This study suggests that the smaller the PLGA particle used to deliver antigen and adjuvants the stronger the antigen-specific cytotoxic T cell response generated.

Biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses.

There is a need for both new and improved vaccination formulations for a range of diseases for which current vaccines are either inadequate or non-existent. Biodegradable polymer-based vaccines fulfill many of the desired properties in achieving effective long-term protection in a manner that is safe, economical, and potentially more practicable on a global scale. Here we discuss some of the work performed with micro/nanoparticles made from either synthetic (poly[lactic-co-glycolic acid] [PLGA] and polyanhydrides) or natural (chitosan) biodegradable polymers. Our attention is focused on, but not limited to, the generation of antitumor immunity where we stress the importance of particle size and co-delivery of antigen and adjuvant.

Characterizing the antitumor response in mice treated with antigen-loaded polyanhydride microparticles.

Delivery of vaccine antigens with an appropriate adjuvant can trigger potential immune responses against cancer leading to reduced tumor growth and improved survival. In this study, various formulations of a bioerodible amphiphilic polyanhydride copolymer based on 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and 1,6-bis(p-carboxyphenoxy) hexane (CPH) with inherent adjuvant properties were evaluated for antigen-loading properties, immunogenicity and antitumor activity. Mice were vaccinated with 50:50 CPTEG:CPH microparticles encapsulating a model tumor antigen, ovalbumin (OVA), in combination with the Toll-like receptor-9 agonist, CpG oligonucleotide 1826 (CpG ODN). Mice treated with OVA-encapsulated CPTEG:CPH particles elicited the highest CD8(+) T cell responses on days 14 and 20 when compared to other treatment groups. This treatment group also displayed the most delayed tumor progression and the most extended survival times. Particles encapsulating OVA and CpG ODN generated the highest anti-OVA IgG(1) antibody responses in mice but these mice did not show significant tumor protection. These results suggest that antigen-loaded CPTEG:CPH microparticles can stimulate antigen-specific cellular responses and could therefore potentially be used to promote antitumor responses in cancer patients.