Browsing by Subject "nanoparticle"
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Item Formulation, characterization and validation of CpG functionalized PLGA bacteriomimetic nanoparticles for breast cancer immunotherapy(2015-05-01) Kokate, Rutika A.; Harlan P. JonesTargeted Immunotherapy represents a potential and innovative means to combat cancer. Cancer vaccines designed against a specific tumor antigen have been efficiently utilized to trigger immune responses against tumor cells. Despite the preliminary evidence in animal models, low immunogenicity is one of the major hurdles in the development of vaccines in humans. Several approaches including the use of an “ideal” tumor antigen, appropriate delivery techniques, immune boosting strategies with co-stimulatory molecules are being explored to surmount this obstacle. The goal of this dissertation project was to utilize polymeric nanoparticles (NPs) as a vehicle to deliver Tumor Associated Antigen (TAA) that would elicit a strong antitumor immune response. In the present study, we successfully formulated CpG surface functionalized Tag encapsulating PLGA nanoparticles (CpG-NP-Tag) and tested their efficacy using in vivo and ex vivo experimental models. Specifically, we developed and characterized NPs for physicochemical properties including particle size, surface charge, surface morphology, Polydispersity index (PDI), encapsulation efficiency and CpG ligand binding efficiency. CpGNP-Tag NPs were found be of desired size (220-230 nm) and surface charge (negative zeta potential). Particles were non agglomerated, spherical in shape and uniform in size with PDI in the range of 0.03-0.1. Due to the hydrophobic nature of the encapsulated entity (Tag), the encapsulation efficiency was limited to 30-40%. CpG ligand conjugation on the surface of NPs was confirmed using Fluorescence Correlation Spectroscopy (FCS). CpG ligand binding efficiency was found to be around 10-14%. We also found that CpG-NP-Tag NP formulation had desired properties (size, charge and morphology) for efficient uptake by phagocytic antigen presenting cells (APCs) such as dendritic cells (DCs). The major aim of our studies was to test the antitumor efficacy of NPs. Using a prophylactic syngenic Balb/c mice model, we demonstrated that CpG-NP-Tag can serve as an efficient tool to bolster antitumor immunity and thus could be used as a platform for the development of NP based immunotherapeutic interventions in future. We found that CpG-NPTag NP immunization attenuated tumor growth, proliferation, angiogenesis and induced apoptotic tumor cell death. These NPs indicated immunostimulatory potential by enhancing tumor CD4+ and CD8+ T cell infiltration as well as local IFN-γ production. Overall, from these in vivo studies we concluded that CpG-NP-Tag promotes IFN-γ secretion which possibly mediates the inhibited tumor growth, angiogenesis and enhanced T cell mediated immunity which facilitates tumor cell death via apoptosis. To understand the mechanism by which CpG-NP-Tag imparts antitumor effects we used ex vivo model of APCs. Studies were conducting using Bone Marrow Derived Dendritic Cells (BMDCs) isolated from female Balb/c mice. We demonstrated enhanced NP uptake, preferential Endosomal localization, and increased population of CD80/86 expressing BMDCs in case of CpG-NP-Tag pulsed BMDCs indicating these NPs could serve as candidates for DC based vaccines in future. In summary, both ex vivo and in vivo studies conducted with CpG-NP-Tag NPs provide insight in the development of particulate vaccines in cancer immunotherapy.Item MIMICKING INFECTION FOR IMMUNOTHERAPY AGAINST BREAST CANCER - FOOLING THE IMMUNE SYSTEM(2013-04-12) Kokate, RutikaPurpose: The purpose of this study was to develop "bacteriomimetic nanoparticles" to enhance adaptive cell-mediated immune responses (CD4+ and CD8+ T cell responses) against tumor antigen as a therapeutic option for cancer treatment. Methods: NPs were prepared by modified solid/oil/water solvent evaporation method using an ultrasonic processor UP200H system (Hielscher Ultrasonics GmbH, Germany). We used membrane preparations of the 4T1 mouse mammary cancer cell line as a tumor antigen and CpG ODN's as a "bactriomimetic" stimulant. Fourteen days before tumor challenge BALB/c female mice (6-8 weeks) were pre-immunized with CpG followed by secondary immunization using respective NPs encapsulated with the membrane antigen preparation. Subsequently, mice (n=4) were challenged with 105 tumor cells intravenously (IV). Mice were sacrificed and tumors were harvested at days 3, 7 and 14 respectively. CD4+ and CD8+ T cell responses were measured in lower respiratory node and spleen using flow cytometry. In another experimental set, following the same immunization schedule as mentioned above, mice (n=5) were challenged subcutaneously (SC) with 105 tumor cells. Primary tumor size was monitored using vernier caliper and bioluminiscence imaging (Caliper Life Sciences Inc., MA, USA). Mice were sacrificed on day sixteen after tumor challenge; spleen cells were used for flow cytometric analysis and primary tumor tissue was used to evaluate CD4+ and CD8+ T cell via immunohistochemistry. Results: We found significant reduction in progression of tumor growth in mice immunized with CpG coated NPs containing tumor antigen (CpG-NP-Tag). Cytometry analysis demonstrated increased CD4+ (helper) and CD8+ (cytotoxic) T cell response emphasizing enhanced immunogenicity against cancer cells. IHC data indicated greater CD4+ T cell infiltration of the tumor tissue for the animals immunized with CpG-NP-Tag. Conclusions: Primary tumor size, IHC and flow cytometry analysis indicate that CpG-NP-Tag NPs were successfully employed to boost the immune response against tumor cells.Item SR-B1 directed nanoparticles as a drug delivery system for the treatment of triple negative breast cancer(2016-08-01) Johnson, Rebecca A.; Lacko, Andras G.; Basu, Alakananda; Mathew, Porunelloor A.The overall goal of this research was to determine the effectiveness of reconstituted high-density lipoprotein (rHDL) nanoparticles as a drug delivery system against metastatic triple negative breast cancer (TNBC). TNBC patients have a less favorable prognosis than those with hormone positive breast cancers. TNBC does not respond to current endocrine treatment. Consequently, the five- year survival rate for patients with metastatic TNBC is [less than] 30%. The studies performed here were intended to fill a void in the treatment of metastatic TNBC with the use of targeted reconstituted high-density lipoprotein (rHDL) nanoparticles, an innovative approach. The rHDL nanoparticles are small, biocompatible, non-immunogenic complexes, targeted to the high-density lipoprotein receptor (scavenger receptor class B type 1 [SR-B1]). While most malignant cells and tumors overexpress the SR-B1 receptor, its expression levels are nearly undetectable in most normal tissues. These findings present the opportunity to exploit a key vulnerability of cancerous tumors as a “Trojan horse” therapeutic strategy and thus markedly limit the toxic impact of chemotherapy. Accordingly, we loaded rHDL nanoparticles with the anti-cancer drugs: valrubicin and lapatinib and tested their effectiveness against TNBC cells and cardiomyocytes. The outcome of these studies show that: (1) The rHDL encapsulated drugs performed significantly better than their free (un-encapsulated) counterparts, (2) The enhancement of the therapeutic effect of the drugs delivered via the rHDL nanoparticles was likely due to the overexpression of the SR-B1 receptor by the TNBC cells. This was confirmed by the enhanced uptake of valrubicin when delivered as a component of the rHDL complex. 3. We have also found that the combination of lapatinib and valrubicin may be ultimately more effective than the respective single drugs for the therapy of TNBC.