Boosting In-Vivo Anti-Tumor Immunity with an Oral Microparticulate Breast Cancer Vaccine and Low-Dose Cyclophosphamide.

Tumor cells express antigens that should induce immune-mediated rejection; however, spontaneous rejection of established tumors is rare. Recent evidence suggests that patients suffering from cancer exhibit an elevation in regulatory T cells population, a subset of CD4+ T cells, which suppress tumor recognition and elimination by cytotoxic T cells. This study investigates immunotherapeutic strategies to overcome the immunosuppressive effects exerted by regulatory T cells. A novel immunotherapeutic strategy was developed by simultaneous administration of oral microparticulate breast cancer vaccines and cyclophosphamide, a regulatory T cell inhibitor. Breast cancer vaccine microparticles were prepared by spray drying, and administered orally to female mice inoculated with 4TO7 murine breast cancer cells in combination with a low dose of intraperitoneally administered cyclophosphamide. Mice receiving the combination of vaccine microparticles and cyclophosphamide exhibited maximal tumor regression and the highest survival rate compared with the control groups. This study highlights the importance of cancer vaccination along with regulatory T cell depletion in cancer therapy, and suggests that a low dose of cyclophosphamide that specifically and significantly depletes regulatory T cells may be a highly effective immunotherapeutic strategy for the treatment of cancer.

Preclinical evaluation of a microparticle-based transdermal vaccine patch against metastatic breast cancer.

Breast cancer is the number one cause of cancer-related deaths among females. Current chemotherapy targets both tumor and normal cells, leading to pronounced side effects. Therefore, therapeutic vaccines acting against specific cancer cells would be the choice of treatment. We prepared microparticles entrapping the antigens obtained from a murine metastatic breast cancer cell line, 4 T1 using the spray drying technology. These microparticles were incorporated into microneedle patches to deliver to the animals for the efficacy study. An antineoplastic drug, cyclophosphamide, in a very low dose has been found to inhibit the immunosuppressive regulatory T cells (Treg) (Le and Jaffee, 2012). In-vivo efficacy of the microparticulate vaccine given along with a low dose of cyclophosphamide was evaluated in a murine breast cancer model. Animals immunized with vaccine microparticles showed considerably slower tumor growth than animals that did not receive the vaccine. The results of the study showed that the Tumor-Associated Antigens (TAAs) within the microparticles were responsible for the delayed tumor growth in vaccinated animals. Vaccinated animals also showed an increase in the population of CD4 and CD8 T cells. Overall, our results demonstrated that immunotherapy with vaccine microparticles encapsulating TAA's could potentially be an effective treatment for metastatic breast cancer.

Enhanced Immunogenicity of Adjuvanted Microparticulate HPV16 Vaccines Administered via the Transdermal Route.

Human papillomavirus (HPV) causes cervical cancer among women and is associated with other anogenital cancers in men and women. Prophylactic particulate vaccines that are affordable, self-administered and efficacious could improve uptake of HPV vaccines world-wide. The goal of this research is to develop a microparticulate HPV16 vaccine for transdermal administration using AdminPatch® and assess its immunogenicity in a pre-clinical mouse model. HPV16 microparticles were prepared using a biocompatible polymer and characterized in terms of size, zeta potential, encapsulation efficiency and microparticle yield. Scanning and transmission electron microscopy were conducted to confirm particle image and to visualize the conformation of HPV16 vaccine particles released from microparticle formulation. In vivo studies performed to evaluate the potential of the microparticulate vaccine initiated a robust and sustained immune response. HPV16 IgG antibodies were significantly elevated in the microparticle group compared to antigen solutions administered by the transdermal route. Results show significant expansion of CD4+, CD45R, CD27 and CD62L cell populations in the vaccinated mice group, indicating the high efficacy of the microparticulate vaccine when administered via transdermal route. The findings of this study call attention to the use of minimally invasive, pain-free routes to deliver vaccine.

Formulation and Characterization of Microcapsules Encapsulating PC12 Cells as a Prospective Treatment Approach for Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurological disorder, associated with decreased dopamine levels in the brain. The goal of this study was to assess the potential of a regenerative medicine-based cell therapy approach to increase dopamine levels. In this study, we used rat adrenal pheochromocytoma (PC12) cells that can produce, store, and secrete dopamine. These cells were microencapsulated in the selectively permeable polymer membrane to protect them from immune responses. For fabrication of the microcapsules, we used a modified Buchi spray dryer B-190 that allows for fast manufacturing of microcapsules and is industrially scalable. Size optimization of the microcapsules was performed by systematically varying key parameters of the spraying device. The short- and long-term stabilities of the microcapsules were assessed. In the in vitro study, the cells were found viable for a period of 30 days. Selective permeability of the microcapsules was confirmed via dopamine release assay and micro BCA protein assay. We found that the microcapsules were permeable to the small molecules including dopamine and were impermeable to the large molecules like BSA. Thus, they can provide the protection to the encapsulated cells from the immune cells. Griess's assay confirmed the non-immunogenicity of the microcapsules. These results demonstrate the effective fabrication of microcapsules encapsulating cells using an industrially scalable device. The microcapsules were stable, and the cells were viable inside the microcapsules and were found to release dopamine. Thus, these microcapsules have the potential to serve as the alternative or complementary treatment approach for PD.

Harnessing T-cell activity against prostate cancer: A therapeutic microparticulate oral cancer vaccine.

Prostate Cancer specific immunotherapy in combination with immune stimulating adjuvants may serve as a viable strategy for facilitating tumor regression and preventing recurrence. In this study, an oral microparticulate vaccine encapsulating tumor associated antigens (TAA) extracted from a murine prostate cancer cell line, TRAMP-C2, was formulated with the help of a spray dryer. Microparticles were characterized in vitro to determine their physicochemical properties and antigenicity. Formulated microparticles had an average size of 4.92 ±â€¯0.5 µm with a zeta potential of 7.92 ±â€¯1.2 mV. In order to test our formulation for its ability to demonstrate adequate antigen presentation and co-stimulation, microparticles were tested in vitro on murine dendritic cells. In vitro biological characterization demonstrated the activation of specific immune system markers such as CD80/86, CD40, MHC-I and MHC-II. Following in vitro characterization, in vivo anti-tumor efficacy of the oral microparticulate vaccine was evaluated in C57BL/6 male mice. Combination therapy of vaccine microparticles with cyclophosphamide and granulocyte macrophage-colony stimulating factor (GM-CSF) demonstrated a five-fold reduction in tumor volume as compared to non-vaccinated mice. At the cellular level, cyclophosphamide and GM-CSF augmented the vaccine response as indicated by the reduced tumor volume and significant elevation of cytotoxic T-cell (CTL) CD8+ and (T-helper) CD4+ T-cells compared to mice receiving vaccine microparticles alone. Furthermore, our studies indicate a significant reduction in T-regulatory cells (T-regs) in mice receiving vaccine along with GM-CSF and cyclophosphamide, one of the immune escape mechanisms linked to tumor growth and progression. Thus, oral microparticulate vaccines have the potential to trigger a robust anti-tumor cellular response, and in combination with clinically relevant agents, significantly resist tumor growth and progression.

Evaluation of a Particulate Breast Cancer Vaccine Delivered via Skin.

Breast cancer impacts female population globally and is the second most common cancer for females. With various limitations and adverse effects of current therapies, several immunotherapies are being explored. Development of an effective breast cancer vaccine can be a groundbreaking immunotherapeutic approach. Such approaches are being evaluated by several clinical trials currently. On similar lines, our research study aims to evaluate a particulate breast cancer vaccine delivered via skin. This particulate breast cancer vaccine was prepared by spray drying technique and utilized murine breast cancer whole cell lysate as a source of tumor-associated antigens. The average size of the particulate vaccine was 1.5 µm, which resembled the pathogenic species, thereby assisting in phagocytosis and antigen presentation leading to further activation of the immune response. The particulate vaccine was delivered via skin using commercially available metal microneedles. Methylene blue staining and confocal microscopy were used to visualize the microchannels. The results showed that microneedles created aqueous conduits of 50 ± 10 µm to deliver the microparticulate vaccine to the skin layers. Further, an in vivo comparison of immune response depicted significantly higher concentration of serum IgG, IgG2a, and B and T cell (CD4+ and CD8+) populations in the vaccinated animals than the control animals (p < 0.001). Upon challenge with live murine breast cancer cells, the vaccinated animals showed five times more tumor suppression than the control animals confirming the immune response activation and protection (p < 0.001). This research paves a way for individualized immunotherapy following surgical tumor removal to prolong relapse episodes.

Pulmonary Administration of Microparticulate Antisense Oligonucleotide (ASO) for the Treatment of Lung Inflammation.

Targeted delivery to the lung for controlling lung inflammation is an area that we have explored in this study. The purpose was to use microparticles containing an antisense oligonucleotide (ASO) to NF-κB to inhibit the production of proinflammatory cytokines. Microparticles were prepared using the B-290 Buchi Spray Dryer using albumin as the microparticle matrix. Physicochemical characterization of the microparticles showed the size ranged from 2 to 5 µm, the charge was - 38.4 mV, and they had a sustained release profile over 72 h. Uptake of FITC-labeled ASO-loaded microparticles versus FITC-labeled ASO solution by RAW264.7 murine macrophage cells was 5-10-fold higher. After pulmonary delivery of microparticles to Sprague-Dawley rats, the microparticles were uniformly distributed throughout the lung and were retained in the lungs until 48 h. Serum cytokine (TNF-α and IL-1ß) levels of rats after induction of lung inflammation by lipopolysaccharide were measured until 72 h. Animals receiving ASO-loaded microparticles were successful in significantly controlling lung inflammation during this period as compared to animals receiving no treatment. This study was successful in proving that microparticulate ASO therapy was capable of controlling lung inflammation.

Evaluation of microparticulate ovarian cancer vaccine via transdermal route of delivery.

Ovarian cancer is the fifth most commonly occurring malignancy in women, with the highest mortality rate among all the gynecological tumors. Microparticulate vaccine can serve as an immunotherapeutic approach with a promising antigenic delivery system without a need for conventional adjuvants. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared by spray drying. Further, the effect of interleukins (ILs) such as IL-2 and IL-12 was evaluated in a separate study group by administering them with vaccine particles to enhance the immune response. The vaccine microparticles were administered to C57BL/6 female mice via transdermal alone and in combination with the oral route. The transdermal vaccine was delivered using a metallic microneedle device, AdminPen™. Orally administered microparticles also included an M-cell targeting ligand, Aleuria aurantia lectin, to enhance the targeted uptake from microfold cells (M-cells) in Peyer's patches of small intestine. In case of combination of routes, mice were given 5 transdermal doses and 5 oral doses administered alternatively, beginning with transdermal dose. At the end of vaccination, mice were challenged with live tumor cells. Vaccine alone resulted in around 1.5 times tumor suppression in case of transdermal and combination of routes at the end of 15th week when compared to controls. Inclusion of interleukins resulted in 3 times tumor suppression when administered with transdermal vaccine and around 9 times tumor suppression for the combination route of delivery in comparison to controls. These results were further potentiated by serum IgG, IgG1 and IgG2a titers. Moreover, CD8+ T-cell, CD4+ T-cell and NK (natural killer) cell populations in splenocytes were elevated in case of vaccinated mice. Thus, vaccine microparticles could trigger humoral as well as cellular immune response when administered transdermally and via combination of route of delivery. However overall, vaccine administered with interleukins, via combination of route, was found to be the most efficacious to suppress the tumor growth and lead to a protective immune response.

Evaluation of the Intestinal Colonizing Potential and Immunomodulating Capacity of Lactobacilli Microspheres.

Lactobacilli species get degraded by acidic conditions in the stomach. Thus, the objective of this study was to (1) formulate and characterize gastro-resistant Lactobacilli microspheres and (2) evaluate the ability of Lactobacilli microspheres to colonize the intestine and their capacity to have an immunomodulating effect in vivo. The product yield and the encapsulation efficiency were 45% and 100%, respectively. The average microsphere particle size was 5 µm. Lactobacilli microspheres were most stable at 4°C and showed a better suspendibility in distilled water. Without encapsulation, the viability of bacteria decreased within 30 min. In the case of Lactobacilli microspheres, no Lactobacilli were released in the first 3 h, and highest release was observed at 4 h, thus, suggesting the significance of encapsulation of Lactobacilli. Lactobacilli microspheres maintained intestinal colonization only during the dosing period, and the serum IgG, serum IgA, fecal, intestinal, nasal IgA, and the serum interleukin-1ß levels were higher in the Lactobacilli microsphere group compared with the blank microsphere and the lactobacilli solution group, suggesting that the Lactobacilli microspheres were more gastro-resistant and, hence, showed positive effects compared with the Lactobacilli solution. However, the Lactobacilli microspheres did not have a significant effect on the tumor necrosis factor-α levels.

Plasma membrane vesicles decorated with glycolipid-anchored antigens and adjuvants via protein transfer as an antigen delivery platform for inhibition of tumor growth.

Antigen delivered within particulate materials leads to enhanced antigen-specific immunity compared to soluble administration of antigen. However, current delivery approaches for antigen encapsulated in synthetic particulate materials are limited by the complexity of particle production that affects stability and immunogenicity of the antigen. Herein, we describe a protein delivery system that utilizes plasma membrane vesicles (PMVs) derived from biological materials such as cultured cells or isolated tissues and a simple protein transfer technology. We show that these particulate PMVs can be easily modified within 4 h by a protein transfer process to stably incorporate a glycosylphosphatidylinositol (GPI)-anchored form of the breast cancer antigen HER-2 onto the PMV surface. Immunization of mice with GPI-HER-2-modified-PMVs induced strong HER-2-specific antibody responses and protection from tumor challenge in two different breast cancer models. Further incorporation of the immunostimulatory molecules IL-12 and B7-1 onto the PMVs by protein transfer enhanced tumor protection and induced beneficial Th1 and Th2-type HER-2-specific immune responses. Since protein antigens can be easily converted to GPI-anchored forms, these results demonstrate that isolated plasma membrane vesicles can be modified with desired antigens along with immunostimulatory molecules by protein transfer and used as a vaccine delivery vehicle to elicit potent antigen-specific immunity.

Development of a microparticulate prostate cancer vaccine and evaluating the effect of route of administration on its efficacy via the skin.

The skin has been identified as a promising target to deliver vaccines. In this study, prostate cancer antigens were delivered in a spray-dried microparticulate carrier to a murine model via the transdermal route and the subcutaneous route. There was a significant increase in the humoral responses as determined by the total serum IgG titres (p < 0.05) and the cellular responses as determined by the T- and B-cells sub-population in spleen samples and delay in tumour growth till 8 weeks post-tumour challenge of both vaccinated groups when compared to the controls. The vaccine microparticles administered via the transdermal route induced a Th2-mediated immune response versus a mixed Th1- and Th2-mediated immune response via the subcutaneous route. Thus, the particulate vaccine delivery system proves to be a promising alternative for generation of a robust immune response against prostate cancer via the skin in a murine model.

Induction of death receptor CD95 and co-stimulatory molecules CD80 and CD86 by meningococcal capsular polysaccharide-loaded vaccine nanoparticles.

Neisseria meningitidis is a leading cause of bacterial meningitis and sepsis, and its capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. We formulated a novel nanovaccine containing meningococcal CPS as an antigen encapsulated in albumin-based nanoparticles (NPs) that does not require chemical conjugation to a protein carrier. These nanoparticles are taken up by antigen-presenting cells and act as antigen depot by slowly releasing the antigen. In this study, we determined the ability of CPS-loaded vaccine nanoparticles to induce co-stimulatory molecules, namely CD80, CD86, and CD95 that impact effective antigen presentation. Co-stimulatory molecule gene induction and surface expression on macrophages and dendritic cells pulsed with meningococcal CPS-loaded nanoparticles were investigated using gene array and flow cytometry methods. Meningococcal CPS-loaded NP significantly induced the surface protein expression of CD80 and CD86, markers of dendritic cell maturation, in human THP-1 macrophages and in murine dendritic cells DC2.4 in a dose-dependent manner. The massive upregulation was also observed at the gene expression. However, high dose of CPS-loaded NP, but not empty NP, induced the expression of death receptor CD95 (Fas) leading to reduced TNF-α release and reduction in cell viability. The data suggest that high expression of CD95 may lead to death of antigen-presenting cells and consequently suboptimal immune responses to vaccine. The CPS-loaded NP induces the expression of co-stimulatory molecules and acts as antigen depot and can spare antigen dose, highly desirable criteria for vaccine formulations.

Formulation and evaluation of drug-loaded targeted magnetic microspheres for cancer therapy.

Enhanced and targeted drug delivery using biodegradable microspheres is emerging as a promising approach for cancer therapy. The main objective of the present research was to formulate, characterize, and evaluate iron oxide (magnetic) containing a bovine serum albumin-based microsphere drug delivery system, capable of efficiently delivering sulforaphane, a histone deacetylase inhibitor, for an extended period of time in vivo. Magnetic microspheres were prepared by spray-drying and characterized for their physicochemical properties and dissolution profile. Further, they were evaluated for therapeutic efficacy in in vitro and in vivo systems. In vitro studies in B16 melanoma cells revealed that there was about 13%-16% more inhibition of cell viability when either 30 µM or 50 µM of sulforaphane was used with iron oxide in the polymeric carrier. Data from in vivo studies in C57BL/6 mice revealed that the magnetic microspheres (localized to the tumor site with the help of a strong magnet) inhibited 18% more tumor growth as compared with sulforaphane in solution. In addition, there was a 40% reduction in histone deacetylation levels in mice treated with iron oxide microspheres containing sulforaphane. Thus, magnetic microspheres are shown to be an effective drug delivery system for anticancer drugs.

Formulation development of albumin based theragnostic nanoparticles as a potential delivery system for tumor targeting.

BACKGROUND: Generally, chemotherapeutic drugs attack on both normal and tumor cells non-specifically causing life threatening side effects, necessitating targeted drug delivery to tumors. PURPOSE: The purpose of this study is to formulate albumin-based nanoparticles for tumor targeted drug delivery and noninvasive diagnosis. METHODS: Albumin based nanoparticles (NPs) were developed as a potential tumor theragnostic agent by entrapping an anti cancer drug, doxorubicin and a near infrared dye, indocyanine green. Theragnostic nanoparticles were prepared using a well established coacervation/nanoprecipitation method followed by lyophilization. The formulation was optimized by varying process parameters using full factorial design of experiments. Release of dye and drug from NPs and physical state of the drug in NPs was studied using DSC. The NPs were injected into tumor bearing mice intravenously and imaged using a bio-imager. RESULTS: The optimized nanoparticle formulation had a particle size of 125.0 ± 1.8 nm, poly dispersity index of 0.180 ± 0.057 and zeta potential of -32.7 ± 0.9 mV. The release of dye and drug from the nanoparticles was determined to be quasi-fickian diffusion mediated. Differential scanning calorimetry (DSC) studies revealed the stability of drug in the NP. The in-vivo studies showed enhanced accumulation of the dye loaded NPs at the tumor site than the dye solution, thus allowing noninvasive tumor monitoring. CONCLUSION: These results project the newly proposed and evaluated nanoparticle formulation as a potential tumor targeting and imaging delivery system.

Cancer testes antigens in breast cancer: biological role, regulation, and therapeutic applicability.

Breast cancer remains one of the leading causes of death among women across the world. The last few decades have seen significant reduction in mortality owing to earlier detection and better adjuvant treatments that were developed based on clinical staging and morphological features. As these treatments have evolved, the heterogeneity of breast cancer poses a new challenge, since there is no standard gold-therapy suitable for all tumors of the mammary gland. Therefore, contemporary management and research efforts are directed toward specific prognostic and predictive molecular signatures that can guide targeted individualized therapy. The goal of ongoing research in this field is to identify specific molecular targets for developing novel therapeutic approaches. These targets can also serve to improve screening of breast cancer. This review focuses on the role of cancer testis antigens (CTAs) in breast carcinogenesis and explores the potential for development of targeted screening and therapeutic approaches. Normally found in the testes, these antigens are highly correlative with cancers of the breast, skin, and ovaries. These implications have been further corroborated through uncovering the interaction of CTAs with genes and proteins involved in tumor suppression and homeostasis like p53. There is some evidence that these genes can be targeted for early detection in addition to being candidates for cancer immunotherapy.

Formulation and evaluation of oral microparticulate ovarian cancer vaccines.

Ovarian cancer is the fifth most leading cause of cancer related deaths in women in the US. Customized immunotherapeutic strategies may serve as an alternative method to control the recurrence or progression of ovarian cancer and to avoid severe adverse effects of chemotherapy. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared with the use of a spray dryer. These particles were designed for oral delivery using enteric polymers such as methacrylic copolymer, Eudragit(®) FS30D and hydroxyl propyl methyl cellulose acetate succinate. These particles were targeted for uptake via microfold cell (M-cell) in Peyer's patches of small intestine using M-cell targeting ligand, Aleuria aurantia lectin. The interleukins (ILs) such as IL-2 and IL-12 were added to the vaccine formulation to further enhance the immune response. The particles obtained were of 1.58±0.62 µm size with a charge of 12.48±2.32 mV. The vaccine efficacy was evaluated by administering the particles via oral route to C57BL/6 female mice. At the end of vaccination, mice were challenged with live tumor cells. Vaccinated mice showed significant (around six-fold) retardation of tumor volume in comparison to non-vaccinated animals for 3 weeks after the tumor challenge (p<0.001). The serum IgG antibody levels were found to be elevated in case of vaccinated animals in comparison to non-vaccinated group (p<0.05). Analysis of IgG1 titers (indicative of Th2 response) and IgG2a titers (indicative of Th1 response) showed a mixed Th1 and Th2 immune response in case vaccine alone and Th2 response in case of vaccine with interleukins group. Moreover, CD8+ T-cell, CD4+ T-cell and B-cell populations in different lymphatic organs were elevated in case of vaccinated mice. Thus, whole cell lysate vaccine microparticles formulated by spray drying could trigger humoral as well as cellular immune response when administered orally. Such vaccine could potentially be an effective treatment for patients with residual tumor or high tumor-relapse probability.

Formulation and evaluation of a particulate oral breast cancer vaccine.

Breast cancer being the most fatal form of cancer for female population, justifies exploration of immunotherapy as an alternative treatment. Here, we have formulated and evaluated an oral microparticulate breast cancer vaccine to provide a new line of therapy. The whole cell lysate of 4T07 murine breast cancer cells was incorporated in an aqueous polymer matrix and spray dried to formulate an enteric protected vaccine microparticle. These particles were characterized in vitro and then administered orally to female Balb/c mice in successive boosters. Serum antibody titers during the study were analyzed using enzyme-linked immunosorbent assay. Postvaccination animals were challenged with live 4T07 cells, and tumor growth was monitored. Flow cytometry studies were performed to analyze the role of T cells. Results show that the vaccine microparticles were 1-4 µm in volume diameter and neutral in charge. The particles were protected enterically and had sustained-release profile. Serum antibody titers of vaccinated animals increased significantly after boosters compared with controls (p < 0.05). Tumor challenge studies revealed that vaccinated animals developed significantly smaller tumors (p < 0.05). Significantly higher numbers of CD4(+) cells occurred in vaccinated animals (p < 0.05). Thus, we conclude that the particulate oral breast cancer vaccine was effective in providing protective immune response in the murine model.

Oral delivery of particulate prostate cancer vaccine: in vitro and in vivo evaluation.

BACKGROUND: Various approaches have been evaluated for generation of efficient immune response against tumor antigens. Our approach exploits usage of particulate delivery to generate immune response against prostate cancer antigens. PURPOSE: The aim of this study was to evaluate the efficacy of prostate cancer vaccine derived from a murine prostate cancer cell line, TRAMP C2 in murine model via oral route using aleuria aurantia lectin as a targeting ligand for M-cells in the intestinal Peyer's patches. METHODS: The whole cell lysate (WCL) was obtained from TRAMP C2 murine prostate cancer cell line and was formulated into particles using one step spray drying process. For in vivo studies, 4-6 week old C57BL/6 male mice were vaccinated orally biweekly for 10 weeks. Serum samples were analyzed at regular intervals to determine serum IgG levels. The mice were then challenged with live TRAMP C2 cells to determine efficacy of the vaccine. RESULTS: The serum IgG levels of vaccinated animals were higher compared to that of the controls. Moreover, the tumor growth was retarded significantly in the vaccinated mice compared to that of controls (p < 0.001). CONCLUSIONS: The above findings suggest that oral particulate WCL vaccine can trigger an immune response against prostate cancer antigens.

Oral delivery of microparticles containing plasmid DNA encoding hepatitis-B surface antigen.

The role of albumin-based chitosan microparticles on enhancing immune response of plasmid DNA (pDNA) to hepatitis-B surface antigen (HBsAg) vaccine after oral administration was investigated in mice. The pDNA encoding HBsAg was entrapped in albumin microparticles using a one-step spray drying technique optimized in our laboratory. The encapsulated particles were also characterized in vitro for their shape, size, encapsulation efficiency, content, and stability. Albumin microparticles could protect the DNA from nuclease degradation as confirmed in our agarose gel study. Further immune modulating effect was studied in our formulation by measuring IgG antibodies in serum as well as IgA antibodies in fecal extracts. The mice were immunized with a prime dose of 100 µg of pDNA in microparticle formulations with and without interleukins biweekly until week 7 followed by a booster dose of equivalent strength on week 33 to compare the response with the subcutaneous group. The oral immunization with the pDNA to HBsAg microparticles gave significantly higher titer level of both sIgA and IgG at week 9 and 34, respectively, in oral vaccine with interleukins group when compared with the subcutaneous group. Thus, we observed an augmentation of both humoral and cellular immune responses for prolonged periods after immunization.

Potentiation of pro-inflammatory cytokine suppression and survival by microencapsulated dexamethasone in the treatment of experimental sepsis.

Cytokine inhibiting drugs are much more effective when delivered intracellularly to phagocytic cells in the microencapsulated form. Dexamethasone is a powerful inhibitor of TNF-α cytokine through inhibition of NF-κB which is a gene regulator of multiple pro-inflammatory cytokines. We have determined the effect of microencapsulated dexamethasone in pro-inflammatory cytokine release both in in vitro using whole blood model, and in vivo using peritonitis model of septic shock. Microspheres of 1-4 µm mean size were prepared by using albumin polymer matrix in a one-step spray drying method. Microencapsulated form of dexamethasone with concentration of 10(-1), 10(-2) and 10(-3) M was compared to an equivalent concentration of solution form of dexamethasone in the in vitro whole blood model. The results show microencapsulated dexamethasone inhibited tumor necrosis factor-alpha (TNF-α) and interleukin-beta (IL-1ß) significantly in comparison with the solution form of dexamethasone. The in vivo peritonitis model also demonstrated significant inhibition of TNF-α and IL-1ß cytokines in microencapsulated form in comparison with solution form of dexamethasone. In the in vivo study, the animal survival rate after 5 days was 90%, dexamethasone in solution with gentamicin was 40% and gentamicin alone was 30%. This study demonstrates significantly improved inhibition of TNF-α and IL-1ß both in vivo and in vitro when dexamethasone was used in microencapsulated form.