Browsing by Author "Lampe, Jana B."
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Item A Bioinformatics Approach to the Design and Engineering of Biomimetic Personalized Nanoparticle Therapy for Bone Metastatic Prostate Cancer(2017-03-21) Ranjan, Amalendu; Lin, Victor; Lampe, Jana B.; Vishwanatha, Jamboor; Gdowski, AndrewPurpose: Bone metastatic prostate cancer remains a challenge to treat clinically due to lack of therapies prolonging overall survival and off target side effects of current treatments. In this study, we employ a bioinformatics approach for target validation and design of biomimetic cancer-coated nanoparticles (CCNP) for treatment of bone metastatic prostate cancer. Our goal is to personalize this targeted therapy by utilizing a patient’s own cancer cells to coat the nanoparticles. We hypothesize that this approach will be an effective strategy to deliver drugs to the site of metastasis. Methods: A bone metastatic prostate cancer target was identified utilizing The Cancer Genome Atlas (TCGA) database from a study of 130 patients with metastatic prostate cancer who underwent next-generation sequencing of their tumors. We used this information and stimulated prostate cancer cells to increase expression of this targeted cell membrane protein. These membranes where purified and used to coat nanoparticles. Nanoparticles were characterized with TEM, DLS, and zeta potential. Membrane purification was validated with coommassie stain and western blot. Membrane orientation on nanoparticle surface was verified with an immuno-conjugation assay. Nanoparticle cancer cell uptake was quantified through immunofluorescence and flow cytometry. Cell viability was performed with MTT assay. Results: Nanoparticles were successfully coated with stimulated cancer cell membranes. Nanoparticle size and zeta potential both increased after coating with membrane. After membrane purification, only markers for cell membranes were identified. Immuno-conjugation assay demonstrated that the cell membrane coating was correctly oriented on the nanoparticle surface. Immunofluorescence results showed when nanoparticles were coated with the cell membrane, there was increased nanoparticle uptake. This was verified by flow cytometry. Stimulated nanoparticles showed decreased cell viability in MTT assay. Conclusions: We successfully engineered cancer coated nanoparticles and validated the manufacturing process. This novel approach to target identification and personalized coating of nanoparticles has tremendous potential as a strategy for treating bone metastasis in prostate cancer patients. Future experiments will study in vivo targeting of bone metastatic lesions with these biomimetic nanoparticles.Item A Drug-Loaded Nanoparticle to Target Bone-Metastatic Prostate Cancer(2022) Lampe, Jana B.; Desai, Priyanka; Tripathi, Amit K.; Ranjan, Amalendu; Vishwanatha, JamboorTreatment for localized prostate cancer (PCa) has a tremendous success rate. However, the fact that the five-year overall survival rate drops from 100% to 30.2% when tumor cells metastasize to distant sites, represents an unmet medical need. In 90% of metastatic cases, bone is the primary metastatic site. Our objective is to co-load a poly(lactic-co-glycolic) (PLGA) nanoparticle (NP) with Cabazitaxel (CBZ) and Bortezomib (BTZ) and to conjugate a bone-targeting moiety, Alendronate (ALN), to the outside of the nanoparticle to facilitate targeting to bone tumors and to ameliorate the resulting bone damage. We hypothesize that this targeted nanomedicine will affect genes and proteins that contribute to invasion and migration, anti-apoptotic signaling, and ultimately lead to tumor-cell apoptosis. Furthermore, we predict that the nano-delivery system will help ameliorate bone lesions inflicted by the tumors. Methods: Nanoparticles were engineered using an Emulsion-Diffusion-Evaporation Technique in which PLGA is dissolved in dichloromethane, 5% polyvinyl alcohol, and Bis(sulfosuccinimydyl)suberate (BS3) crosslinker. For targeting, Alendronate (ALN) is later conjugated to the outside of the nanoparticle via this crosslinker. Results: Our average NP size was around 240 nm in diameter, a PDI of < 0.2, with a Zeta Potential (ZP) of -28 mV. Our drug loading capacity (DL) for CBZ was 11.97% and for BTZ 0.9%. Encapsulation efficiency (EE) for CBZ was 25.26% and 8.9% for BTZ. The IC50 for the CBZ NPs is 5.6 nM and BTZ NPs is 15.6 nM. We have successfully shown that the gene expression for various migration and invasion markers as well as cell signaling proteins have been affected by the nanoparticles. Conclusions: Our nanoparticles have a desirable size, PDI, ZP, DL, and EE for our intended therapeutic purpose. Furthermore, we have shown alterations in the cell signaling and gene expression responsible for Epithelial-to-Mesenchymal Transition-Transcription Factors (EMT-TFs), indicating that our nanotherapeutic has significant potential to treat metastatic PCa and to mitigate the damage done by metastatic tumors.Item A Liposomal Platform Using a Microfluidic Mixing Method for Drug Delivery and Targeting of Metastatic Prostate Cancer(2019-03-05) Ranjan, Amalendu; Joshi, Rohan; Vishwanatha, Jamboor; Lampe, Jana B.Purpose: The success rate for the treatment of localized prostate cancer (PCa) is very high. However, the overall survival rate for patients with metastatic PCa drops to 28%. Bone is the primary metastatic site in 90% of PCa patients, which not only shortens survival, but also causes a significant decrease in the quality of life. The objective of the project is to develop a dual-targeted nanotherapeutic for bone metastatic PCa. We will engineer liposomes composed of two lipids, DOPE and DOTAP. A bone-targeting moiety with a high affinity to the Ca2+ in bone will be conjugated to the outside of the liposome. This liposome will be loaded with a cabazitaxel-ligand conjugate that has a high affinity for the receptors that are upregulated in 95% of PCa cells. Methods: In this formulation we have used a lipid ratio between 40% and 60%. Then the size and polydispersity were optimized by selecting the best Flow Ratio (FR) and Total Flow Rate (TFR) settings in the NanoAssemblr. We also measured the zeta potential (ZP). Cellular uptake studies were performed using the PC3 cell line and DID dye-loaded, DOPE-DOTAP liposomes, then imaged with a Zeiss LSM 510 confocal microscope. Additionally, liposomes were loaded with curcumin to determine their % drug loading (DL) and encapsulation efficiency (EE). Results: The liposomes were optimized with a 50:50 mol% ratio, a 1:1 FR, and a 6 ml/min TFR. Size (~150 nm) and polydispersity index (0.2), were measured and found to be consistent over a 7-day period to show stability. The ZP (~+40 mV) was also measured. The cellular uptake studies showed that the liposomes were increasingly taken up by the cells over time. The EE was ~93% and DL was ~5%. Conclusion: By finding a desirable ratio of lipids, FR and TFR, we have optimized our liposomal formulation based on size, PDI, and ZP. Also, we have demonstrated that our liposome can easily be taken up by cancer cells and have shown excellent DL and EE. As a result, we are prepared to continue with the next steps of the project. In the next step we will attach the bone-targeting moiety to the liposome, conjugate cabazitaxel with the targeting ligand, and load the liposomes with the cabazitaxel-ligand. Fully functional liposomes will be tested for in vitro and in vivo functionality.Item Bioengineered Liposomal Platform for Bone Metastatic Prostate Cancer(2020) Joshi, Rohan; Ranjan, Amalendu; Vishwanatha, Jamboor; Wang, Jianmei; Halbert-Hoshikawa, Jason; Desai, Priyanka; Lampe, Jana B.Purpose: Despite the success in treating early-stage prostate cancer (PCa), the outcomes for metastatic disease are far from ideal. Once cancer reaches distant sites, the five-year overall survival rate drops to 28%. For 90% of patients with advanced PCa, skeletal metastasis is the primary destination. To address the unmet medical need of bone-metastatic prostate cancer, we have engineered a liposomal platform to deliver a chemotherapeutic directly to the bone. Methods: First, a bone-targeting moiety with a high affinity for calcium of the hydroxyapatite in bone was conjugated to one of the lipids and characterized using H NMR, P NMR, and FTIR. Next, we incorporated the conjugate and a chemotherapeutic cargo into the liposome using a microfluidic, nanoprecipitation method. We characterized our nanoparticles using dynamic light scattering, transmission electron microscopy, and nanotracking analysis. Results : Our average nanoparticle size was 150 nm ± 2.50 with a polydispersity index (PDI) of less than 0.2 ± 0.22 and a zeta potential of 28.18 ± 1.71. Drug loading and encapsulation efficiency will be determined using LC/MS. Other characterization includes in vitro uptake studies, cytotoxicity studies in C4-2B and mPC3 cell lines, and in vivo biodistribution studies. Conclusions: We hypothesize that our liposome will ameliorate bone damage caused by metastatic PCa, inhibit PCa differentiation and proliferation, and stimulate apoptosis of PCa cells. Our overall objective is to improve the quality of life by reducing off-target side-effects and extend the overall survival rate of bone-metastatic prostate cancer patients.Item Bioengineered Mesenchymal Stem Cell Exosome-Coated Polymeric Nanoparticles for the Treatment of Triple Negative Breast Cancer(2019-03-08) Lampe, Jana B.; Ranjan, Amalendu; Vishwanatha, Jamboor; Joshi, RohanBackground: Metastasis is the leading cause of death in breast cancer worldwide. Although there have been many new agents approved for metastatic breast cancer, they are poorly efficacious. Mesenchymal stem cells (MSC) and their exosomes play a role in the tumor microenvironment and they may have tumor homing properties. Our goal is to bioengineer MSC exosome-coated polymeric nanoparticles (BioExoNP) to deliver a chemotherapeutic drug for targeted therapy of TNBC. Methods: To isolate the MSC exosomes, we grew MSC cells in exosome free media and used ultracentrifugation at 100,000 x g for exosome isolation. We used Dynamic Light Scattering (DLS) for size analysis, polydispersity index (PDI) and zeta potential (ZP). Western blotting was used for exosomal protein identification. PLGA polymeric nanoparticles were formulated using the microfluidic based Nanoassembler. Their size, PDI and ZP were obtained using DLS. To make the coated NP we used an extrusion method. High performance liquid chromatography was used for drug loading and encapsulation efficiency. Results: The MSC exosomes had a size of 77 nm, ZP of -14 mV, and PDI of 0.24. The NP also have similar results with a size 76 nm, and PDI of 0.2, however the ZP was -38. Our exosome sample was positive for known exosomal proteins and negative for all other extracellular vesicle markers. After extrusion, the ZP of our sample was closer to -14 mV which, tells us that our sample was coated in exosomal membrane. However, after extrusion we did obtain three populations of NPs: bare NPs, coated NPs and just exosomes. Our sample was further purified using centrifugation. Conclusion: In this study, we have demonstrated that exosome-coated polymeric nanoparticles can be successfully formulated with optimal characteristics. These hybrid nanoparticles were stable and uniform. In future applications, we will use this platform to formulate BioEXoNP and evaluate its therapeutic potential using in vitro and in vivo studies. Funding: Supported by a grant award number RP170301 from the Cancer Prevention and Research Institute of Texas (CPRIT).Item Bone Sectioning Technique for 3D Confocal Image Resolution and Capture of Dye-Loaded Nanotherapeutics(2018-03-14) Gdowski, Andrew; Ranjan, Amalendu; Vishwanatha, Jamboor; Lampe, Jana B.ABSTRACT Purpose:Capturing detailed images of bone architecture has unique challenges and conventional procedures have proved to be insufficient for molecular 3D imaging. Furthermore, traditional 2D immunohistochemistry provides limited information for assessing therapeutic localization in the bone. In addition, techniques such as thin paraffin sections visualized by immunofluorescence microscopy or transmission electron microscopy, require prolonged exposure to damaging decalcification reagents. These chemicals have destructive effects on bone morphology and limit the capture of proteins. The objective of this project was to develop an adapted protocol for bone tissue preparation prior to sectioning and immunohistochemical (IHC) staining. This method enables ultra-thick sections for enhanced Z-stacking, enables the generation of high-resolution 3D images that map the bone tissue, and provides oseo-spatial detection of our dye-loaded nanotherapeutics. Methods:Bones were decalcified then incubated in cryoprotectant before emersion in the embedding solution. Samples were frozen at -80. Ultra-thick sections were made on a Thermo Fisher Cryostar NX70 Cryostat (75 – 100 m) and placed on polar slides. Immunohistochemical staining was applied to the slides, which were imaged with a Zeiss LSM 510 confocal microscope. Our therapeutic was labeled with near fluorescent dye.Results:High-fidelity, 3D images of mouse tibia and femur were imaged. Furthermore, visualization of nuclear staining, bone epithelial cells, and the fluorescently labeled therapeutics were easily detected. Thick sectioning provided us with a more robust, tomographic image, allowing for more thorough mapping and analysis of the nanotherapeutics in the bone. Conclusions:Our modified protocol for processing and imaging bone is an effective approach to bone handling, confocal imaging, and detecting bone and dye-labeled nanotherapeutics. This approach will provide benefits for facilitating our understanding of the significance that drug localization has on the bone microenvironment and its impact on therapeutic efficacy.Item Cabazitaxel-Loaded Nanoparticles Reduce the Invasiveness in Metastatic Prostate Cancer Cells: Beyond the Classical Taxane Function(MDPI, 2023-02-26) Lampe, Jana B.; Desai, Priyanka P.; Tripathi, Amit K.; Sabnis, Nirupama A.; Chen, Zhe; Ranjan, Amalendu P.; Vishwanatha, Jamboor K.Bone-metastatic prostate cancer symbolizes the beginning of the later stages of the disease. We designed a cabazitaxel-loaded, poly (lactic-co-glycolic acid) (PLGA) nanoparticle using an emulsion-diffusion-evaporation technique. Bis (sulfosuccinimidyl) suberate (BS3) was non-covalently inserted into the nanoparticle as a linker for the conjugation of a bone-targeting moiety to the outside of the nanoparticle. We hypothesized that the nanoparticles would have the ability to inhibit the epithelial-to-mesenchymal transition (EMT), invasion, and migration in prostate cancer cells. Targeted, cabazitaxel-loaded nanoparticles attenuated the EMT marker, Vimentin, and led to an increased E-cadherin expression. These changes impart epithelial characteristics and inhibit invasive properties in cancer progression. Consequently, progression to distant sites is also mitigated. We observed the reduction of phosphorylated Src at tyrosine 416, along with increased expression of phosphorylated cofilin at serine 3. These changes could affect migration and invasion pathways in cancer cells. Both increased p-120 catenin and inhibition in IL-8 expression were seen in targeted, cabazitaxel-loaded nanoparticles. Overall, our data show that the targeted, cabazitaxel-loaded nanoparticles can act as a promising treatment for metastatic prostate cancer by inhibiting EMT, invasion, and migration, in prostate cancer cells.Item Current landscape of immunotherapy clinical trials in prostate cancer(2018-03-14) Gdowski, Andrew; Lampe, Jana B.; Ranjan, Amalendu; Vishwanatha, Jamboor; Gorman, BrendanPurpose The number of immunotherapies that have been approved in recent years has generated a lot of enthusiasm in the field of oncology. This success has come on the results of approvals for immunotherapy drugs and the expansion of indications for a variety of hematological and solid malignancies. However, very few immunotherapies have demonstrated improved overall survival in treating patients with prostate cancer. This is due to several factors including tumor heterogeneity, limited prostate tumor-associated antigens, and an immunosuppressive environment. Despite these challenges, numerous clinical trial efforts are ongoing to determine outcomes of immunotherapies in prostate cancer, and many are in the context of combination strategies. The purpose of this project was to identify and categorize the current landscape of immunotherapies that are in clinical trials for prostate cancer. Methods An extensive evaluation of the all currently registered clinical trials in the United States of immunotherapies in the setting of prostate cancer was performed utilizing www.clinicaltrials.gov on 1-20-18. The following search parameters were used: Condition/disease: “prostate cancer” Other terms: “immunotherapy”, “CAR-T cell therapy”, “monoclonal antibody”, “checkpoint inhibitors”, and “vaccine”. Results The query resulted in a total of 215 registered clinical trials. Most of these trials (84%) were in the context of vaccine therapy against prostate cancer, 12% of trials involved checkpoint inhibitors, and 4% were testing CAR-T cell therapy. Only 6% of the trials were in the phase 3 setting while 32% and 60% were in phase I or phase II, respectively (the remainder were not categorized into a phase). The majority of these trials used combination strategies. Conclusion The slow-growing nature of prostate cancer in many patients makes this cancer uniquely suitable for utilizing immunotherapies that may need time to allow for an immune response to mount against cancer cells. There is a tremendous amount of clinical trials that are currently being performed on prostate cancer with a variety of immunotherapeutic strategies. Although more research needs to be done, the potential of a durable and sustained response with immunotherapies is encouraging in the setting of prostate cancer.Item Engineering a Nanotherapeutic for Metastatic Prostate Cancer with Bone-targeting Specificity(2021) Lampe, Jana B.; Desai, Priyanka; Ranjan, Amalendu; Vishwanatha, JamboorProstate cancer (PCA) derived bone metastases account for 90% of metastatic tumors with a five-year overall survival rate of 29.5%. Our objective is to develop a clinically feasible nano-delivery system targeting bone-metastatic sites to prolong overall survival and improve quality of life. The purpose of this project is to engineer a cabazitaxel-loaded, poly-lactic(co-glycolic) acid (PLGA) nanoparticle (NP) with alendronate (ALN) coating to target and treat metastatic bone lesions. We hypothesize that a bone targeted nano-delivery system will ameliorate bone lesions and trigger tumor cell apoptosis. Methods: NPs were formulated using a water-in-oil-in solvent evaporation method. NPs were prepared by sonicating 50 mg/ml PLGA in dichloromethane (DCM), 5% polyvinyl alcohol (PVA), and Bis(sulfosuccinimydyl)suberate (BS3) linker. Later, ALN was conjugated to the NP. Results: Our average NP size was around 200 nm in diameter with a Zeta Potential (ZP) of – 28 mV. Our drug loading capacity (DL) was 12.4% and encapsulation efficiency was 25.3%. The IC50 value is 10 μM. NPs have also shown to be easily taken up by cancer cells. Conclusion: We have shown that our PLGA NPs have an optimal size, PDI, ZP, DL%, and EE%, which indicates that we have developed a NP that will function as a nanotherapeutic for bone metastatic PCa. The next steps will include spheroid cultures and in vivo studies.Item Exosome clocked hybrid nanosystem for targeted TNBC therapy(2020) Ranjan, Amalendu; Vishwanatha, Jamboor; Lampe, Jana B.; Joshi, RohanBackground: Metastasis is the leading cause of death in breast cancer worldwide. In recent years, it has been found that mesenchymal stem cells (MSC) cells and their exosomes play a role in the tumor microenvironment and they may have tumor homing properties. Using this information, our plan was to bioengineer MSC exosome-coated drug loaded polymeric nanoparticles to deliver our chemotherapeutic drug. Methods: To isolate the MSC exosomes we grew the cells in exosome free media and used ultracentrifugation with the highest speed at 100,000 g for isolation. We used Dynamic Light Scattering (DLS) for size analysis, polydispersity index (PDI) and zeta potential (ZP). PLGA polymeric nanoparticles were made using the Nanoassembler which uses microfluidics to make nanoparticles. Their size, PDI and ZP was also obtained using DLS and NTA. To make the coated NP we used the extrusion method. Results: The MSC exosomes and NP had the expected size. Our exosome sample was positive for exosomal proteins and negative for all other extracellular vesicles. After extrusion the ZP of our sample tells us that our sample was coated in exosomal membrane. Our sample was future purified using centrifugation and were loaded with Doxorubicin. Conclusion: We were able to make MSC exosome-coated drug loaded polymeric nanoparticles and show that they were stable and uniform in size. In the future we plan to do more cell studies to look at cell viability and toxicity as well as more compressive animal studies.Item Phosphorylated Annexin A2 at Tyrosine 23 Regulates Exosome Release and Biogenesis in Triple Negative Breast Cancer(2022) Desai, Priyanka P.; Tripathi, Amit K.; Donkor, Michael; Thyagarajan, Srikantha; Jones, Harlan; Van Treuren, Timothy; Lampe, Jana B.; Chaudhary, Panka J.; Vishwanatha, JamboorPurpose: Exosomes are highly involved in the progression of diverse diseases. Targeting exosome biogenesis and release is a potential strategy for the treatment of the disease like cancer which urges an improved understanding of the process. During the exosomes biogenesis, invagination of the plasma membranes forms early endosomes which mature into late endosomes and multivesicular bodies. Annexin A2 (AnxA2), a calcium dependent phospholipid binding protein, is one of the cargo proteins which gets uploading into the exosomes and impart aggressive phenotype in triple negative breast cancer (TNBC). The mechanism how AnxA2 uploads the exosomal cargo into the exosomes and releases exosomes in the tumor microenvironment remains to be unidentified. In this study, we have explored the potential mechanism for exosome biogenesis and release to target it in TNBC, which lacks the targeted based therapies. Methods: Plasmids expressing constitutive phosphomimetic (AnxA2-Y23E) and non-phosphomimetic AnxA2 (AnxA2-Y23F) mutant gene were transfected in MDA-MB-231 cells. Exosomes isolated from AnxA2-Y23E and AnxA2-Y23F mutant cells were analyzed for expression of the exosomal cargo proteins and RNAs by Western blot and RT-PCR. The number of exosomes released were analyzed by Nanotrack analysis (NTA). Mutant cells treated with Rapamycin, mTORC1(Mammalian Target of Rapamycin Complex 1) inhibitor, were analyzed for the cargo and exosomal secretion. Mutant cells were injected in nude mice to generate tumors. Serum exosomes were isolated and analyzed for cargo and number of exosome release by NTA. Results: In this study, we found that phosphorylated Annexin A2 at tyrosine 23 increases exosome secretion. It loads proteins like AnxA2, CD9 (Cluster of Differentiation 9), LC3B, and Tsg101(Tumor susceptibility gene 101), and AnxA2 and mTOR mRNA into the exosomes. Moreover, secretion and loading of cargo into the exosomes is regulated by increased phosphorylation of AnxA2 and reduced downstream mTORC1 activity. Conclusions: Phosphorylation of AnxA2 at tyrosine 23 regulates exosome secretion and cargo loading into the exosomes in TNBC.Item Short Peptides based on the conserved regions of MIEN1 protein exhibit anti-cancer activity by targeting the MIEN1 Signaling Pathway(Elsevier B.V., 2024-01-26) Tripathi, Amit K.; Desai, Priyanka P.; Tyagi, Antariksh; Lampe, Jana B.; Srivastava, Yogesh; Donkor, Michael; Jones, Harlan P.; Dzyuba, Sergei V.; Crossley, Eric; Williams, Noelle S.; Vishwanatha, Jamboor K.Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved ITAM and prenylation motifs within MIEN1, we identified potent anti-cancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and EMT pathways, concurrently suppressing EGF-induced NF-kappaB nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and circular dichroism spectroscopy indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high peptide doses of up to 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anti-cancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.