Browsing by Author "Kapic, Ammar"
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Item Developing a nanoparticle platform for selective delivery of the anti-cancer drug MIH 2.4Bl to breast cancer cells(2023) Debnath, Dipti; Petty, Roland; Kapic, Ammar; Sabnis, Nirupama; Lacko, Andras G.; da Silva Souza, Helivaldo Diógenes; de Athayde Filho, Petrônio Filgueiras; Mathis, J. MichaelBased on data from the World Health Organization, breast cancer is the most common cancer among women, accounting for about 15% of all cancer-related deaths. Thus, new treatment options are urgently needed to decrease this mortality rate. In recent years, mesoionic compounds have shown promising potential as anti-cancer agents due to their unique structure and reaction properties. We reported that a 1,3-thiazolium-5-thiolate mesoionic compound (MIH 2.4Bl) inhibited the growth of most of the breast cancer cell lines tested compared with normal human mammary epithelial cells. Treatment of MCF-7 breast cancer cells with MIH 2.4Bl resulted in alterations in cell cycle distribution with an increased proportion of cells in the G2/M phase compared with untreated cells. MCF-7 cells treated with MIH 2.4Bl also showed morphological changes consistent with apoptotic cell death. In addition, treating MCF-7 cells with MIH 2.4Bl resulted in a significant reduction in all mitochondrial respiratory parameters compared with the control cells, indicative of an overall decrease in mitochondrial membrane potential. These findings suggest that MIH 2.4Bl is a promising candidate for treating breast cancer. However, cancer therapy's primary challenge is the selective destruction of malignant cells while sparing normal cells to preserve tissue integrity. The development and use of drug delivery systems is a recognized approach to improving the efficacy of chemotherapy agents. However, drug delivery systems have been unexplored in mesoionic compounds. The reconstituted high-density lipoprotein (rHDL) nanoparticles have several advantages, including enhanced safety, efficacy, and biocompatibility. The payload, which is contained in the core of the HDL particle, is taken up by SR-B1 receptors, making this method particularly useful for targeted cancer chemotherapy. The upregulation of the SR-B1 receptor by tumor cells and tissues might be helpful in cancer treatment by specifically delivering drug-loaded nanoparticles to the tumors. In this preliminary work, we present an improved delivery strategy of a newly developed formulation of MIH 2.4Bl compound with rHDL nanoparticles as the delivery agent. Initial synthesis, optimization, physicochemical characterization, drug loading, and drug release assessment of the nanoparticles were performed. These studies support the potential therapeutic use of MIH 2.4Bl in treating breast cancer. To advance potential translational studies for monitoring in vitro drug delivery and colocalization of the drug in the cells, we have begun studies of the fluorescence properties of MIH 2.4Bl, using steady-state and time-resolved fluorescence techniques. The fluorescence characteristics of free MIH 2.4Bl was evaluated using UV/VIS and fluorescence spectroscopy. The steady-state and time-resolved measurements were designed to understand the optical properties of MIH 2.4Bl in solution for monitoring in vitro drug delivery and cellular colocalization. All samples, dissolved in various solvents, exhibited maximum absorbance between 440 and 480 nm; excitation at 440 nm elicited the highest emission at approximately 580 nm in methanol. These results may allow future detection and localization of MIH 2.4Bl in vitro and in vivo. Follow-up studies utilizing fluorescence confocal microscopy are anticipated to reveal drug accumulation's site(s) in situ and how cytotoxicity is induced in cancer cells.Item Discovery-driven Label-free Quantitative Proteomics Study to Understand Estradiol-mediated Neuronal Processes in the Hippocampus and its Implication in Alzheimer’s Disease in Ovariectomized Rats(2024-03-21) Neagu, George; Zaman, Khadiza; Nguyen, Vien; Kapic, Ammar; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that affects millions of people globally. Studies report an increased susceptibility to the development of AD in post-menopausal women. There is renewed interest in utilizing estrogen therapies due to its neuroprotective effects on the brain; however, the mechanisms of these neuroprotective effects are poorly understood. The hippocampus is involved in memory formation and is a critical region where early damage in AD is often seen. This discovery-driven proteomics study elucidates several candidate proteins and biological pathways mediated via E2 and implicated in neurological signaling in the hippocampus. Methods: Ovariectomized female Sprague-Dawley rats were treated with daily subcutaneous injections of either vehicle or 50μg/kg E2 for five days before the rats were sacrificed with the hippocampus collected for proteomics. Protein extracts were taken from centrifugated hippocampal tissue and prepared via a series of steps including urea incubation, disulfide bond reduction, carbamidomethylation of thiol groups, and digestion via trypsin with subsequent quenching. The digested proteins were dried, reconstituted in solvent, and processed via nano-LC-MS/MS. The MS/MS spectra were searched against a Rattus norvegicus proteome database for peptide fragment and protein identification via ProteomeDiscover (Thermo Fisher Scientific) using Mascot as a search engine and validated using Scaffold (Proteome Software). Bioinformatic analysis using Ingenuity Pathway Analysis (Quiagen) allowed the construction of associative and predicted protein networks. Results: The processed MS/MS data proteins revealed several candidates for future targeted validation. Among those proteins, calcium/calmodulin-protein kinase II implicated in memory and learning processes, such as long-term signal potentiation in the hippocampus, was more abundant in the treatment group (p = 0.00052) with fold changes in the protein cluster abundances ranging from 1.3 to 1.5 versus control. Other proteins, such as microtubule-associated protein tau, implicated in AD, also had a marked fold change of –2.5 abundance in treatment versus control (p = 0.00014). There substantial overall difference in protein abundances for neurological disease pathways, including AD, identified in Ingenuity Pathway Analysis (p = 8.9e-7) as well as in pathways involving nervous system development and function (p= 4.57e-7) between E2-treated and vehicle-treated rats. Conclusion: This dataset analysis aims to evaluate the effects of E2 on the proteome of the hippocampus in ovariectomized rats. The evident increased fold changes in calcium-dependent and calcium-associated proteins in the context of neuronal processes suggest increased downstream modulation of synaptic signaling, which could be further examined by microdialysis assay of neurotransmitters. Future studies utilizing microdialysis may examine E2’s estrogen receptor-mediated effects on cholinergic neuronal signaling in the hippocampus, which could further understand AD in the context of cholinergic neuron loss.Item LABEL-FREE QUANTITATIVE PROTEOMICS REVEAL PROTEIN NETWORKS AND ASSOCIATED BIOLOGICAL PROCESSES IN ANDROGEN DEPRIVED MOUSE SEMINAL VESICLES IN RESPONSE TO 17-β-ESTRADIOL (E2) TREATMENT(2023) Kapic, Ammar; Zaman, Khadiza; Nguyen, Vien; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: Androgen deprivation therapy (ADT) remains the primary treatment strategy for inhibiting prostate cancer progression. However, systemic ablation of androgen-mediated signaling induces various metabolic disorders, cognitive decline, and osteoporosis. Therefore, like in menopausal women, 17-β-estradiol (E2) supplementation has been suggested as a treatment to reduce side effects associated with ADT. A recent clinical trial utilizing transdermal estrogen patches reported reduced osteoporosis markers and hot flashes. Estrogen receptors ER) are expressed in the male reproductive system and play a role alongside androgens in maintaining function and growth. Under normal physiological conditions, increased E2 concentrations induce an inhibitory effect on the size of the male reproductive organs, including the seminal vesicles (SV); however, under androgen depletion, E2 supplementation has been reported to reduce the atrophy of the SV in mice. In this study, we report for the first time a discovery-driven proteomic analysis of E2’s effects on the SV in mice under the conditions of surgical castration to model patients undergoing ADT. Methods: Surgically castrated mice (n=4) were subcutaneously injected with E2 (treated group) or vehicle (control) daily for five days and sacrificed to obtain SV. Proteins were extracted, reduced, alkylated, and digested with trypsin for analyses using data-dependent microflow liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) on LTQ Orbitrap Velos ProTM (Thermo Fisher Scientific). MS/MS data was searched against the UniProt Mouse protein database using Sequest in Proteome Discoverer (Thermo Fisher Scientific) and MaxQuant (Max Planck Institute). Validation of protein and label-free quantification (LFQ), combining spectral counting and total TIC, were performed using Scaffold (Proteome Software) to identify significantly affected proteins. Post-hoc t-test was performed to identify differences in protein abundances between groups. Regulated proteins we mapped to protein interaction networks and biological functions employing Ingenuity Pathway Analysis® (IPA®, Qiagen). Targeted proteomics- sed validation of significant candidate proteins is ongoing, and data will be analyzed using Skyline TM (MacCoss Lab, University of Washington). Results: Our discovery-driven LC–ESI-MS/MS analyses identified 7000 proteins with high confidence from the SV of E2-treated and control mice. IPA®-based bioinformatics of the E2-regulated proteins showed molecular and cellular functions-associated enrichment of carbohydrate metabolism, DNA replication, recombination, and repair, as well as free radical scavenging. The topmost regulated protein interaction network represented cell cycle, cell signaling, and small molecule biochemistry. Enhanced activation of the estrogen receptor β (ESR2) was implicated by the molecule activity predictor (MAP) tool of IPA®. Additionally, MAP predicted that the protein interaction represented within this network might impact disease and physiological processes associated with the proliferation of prostate cancer and regulation of gonadal cells. Furthermore, we were able to screen several preclinical biomarkers that participate in androgen receptor activity, modulating ER-mediated transcription and reproductive system development and function. Targeted proteomics-based validation of these biomarkers is ongoing. Conclusion: Our study aims to provide an in-depth account of the alterations occurring at the protein level in the SVs in response to E2 supplementation during ADT and to select and validate preclinical biomarkers for prognostic and therapeutic applications.Item Neuroprotective Effects of DHED Eyedrops Protects Visual Function Despite Elevated IOP in an Ocular Hypertension Animal Model(2024-03-21) Kapic, Ammar; Neagu, George; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, KatalinPurpose: Glaucoma remains the second leading cause of irreversible blindness and is often associated with chronically elevated intraocular pressure (IOP) leading to ocular hypertension (OHT). All of the currently accepted therapies attempt to reduce the elevated IOP. However, despite intervention, studies show progressive neuronal damage continues in the retina and may extend to the rest of the visual system, leading to additional neuropathologies. Interest in utilizing 17β-estradiol (E2) for its neuroprotective effects has become increasingly recognized; however, due to its side effects, such as cancer risk and feminization in males, its application as a therapy is limited. Our lab has developed the estrogen prodrug, 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), which remains inactive until its CNS-specific metabolism via short-chain reductase into the active compound E2. This study aims to elucidate the pleiotropic effects of E2 derived from DHED as a potential therapy for preserving the visual system under OHT. We hypothesize that topical application of DHED will prevent the neurodegenerative effects of chronic OHT on the retina and maintain visual function. Methods: OHT was induced in 8 to 10-month-old male Brown Norway rats via hypertonic saline injection into an episcleral vein. IOP was measured via a tonometer (Tonolab) to confirm sustained elevated IOP post-surgery and throughout the treatment period. DHED was topically delivered through eyedrops (20% 2-hydroxylpropyl-beta-cyclodextrin, 0.1% DHED, and saline) once per day. Visual acuity (VA) and contrast sensitivity (CS) were measured using the OptoMotry system with the OptoMotry 1.7 software (Cerebral Mechanics Inc). VA and CS were assessed using the "Rat" preset, and gratings were adjusted using a simple staircase progression. A fixed frequency of 0.272 c/D was chosen for the CS based on prior studies. Observers for the OptoMotry tests were blinded. The eyes and optic nerves were collected and fixed for RGC and axon counts, respectively. Seminal vesicles were collected and weighed to assess peripheral estrogenic effects. Results: The IOP was elevated by 53 % ± 15% and was sustained in both vehicle and DHED-treated groups with no differences between treatment groups. The vehicle-treated group gradually lost visual function, retaining only 60% ± 5% and 30% ± 4% of their VA and CS, respectively, by the end of the treatment period. However, the DHED-treated group maintained significantly better visual performance, retaining 91% ± 3% and 75% ± 7% of their VA and CS compared to the baseline. No differences in the mass of the seminal vesicles between treatment groups. Comparison of RGC and axon counts in the optic nerve are ongoing. Conclusion: This study demonstrates the neuroprotective effects of DHED-derived E2 on the visual system without peripheral side effects. Despite sustained OHT, the VA and CS of the topically administered DHED reduced the impact of injury compared to the vehicle control group. Future studies will investigate DHED administration's impact on the retina and visual cortex proteome.Item Optimization and characterization of mannose-decorated lipoprotein nanoparticles for the targeting of tumor-associated macrophages(2021) Dossou, Akpedje; Kapic, Ammar; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras G.Purpose: Reconstituted high-density lipoprotein nanoparticles (rHDL NPs) have been shown to accumulate into tumors. This effect is achievable because of their small size and their affinity toward the scavenger receptor SR-B1 which is overexpressed in most tumors. Thus, rHDL NPs appear suitable to target tumor-associated macrophages (TAMs) which express SR-B1 as well as the mannose receptor CD206. While cancer cells promote an immunosuppressive (M2) phenotype in TAMs, it has been shown that a re-orientation of TAMs toward an immunostimulating (M1) phenotype results in rapid tumor regression. Because a generalized immunostimulating effect is not desirable, the targeted approach via rHDL NPs offers a safer alternative via selective delivery of an M2-to-M1 reversal agent to TAMs. Vadimezan (also called DMXAA) is such an agent. Consequently, the goal of this study is to develop and characterize a formulation of DMXAA encapsulated in mannose-decorated rHDL NPs. Methods: Six formulations of DMXAA with mannose-decorated rHDL NPs with increasing concentration of the mannose moiety were prepared via microfluidics and characterized via dynamic light scattering, fast protein liquid chromatography, and colorimetric assays for their contents. Results: The formulations decorated with the mannose moiety, displayed larger particle diameters compared to the control rHDL NPs and achieved a drug incorporation efficiency of about 60%. Conclusion: The characteristics of these particles show that the mannose moiety was stably incorporated into the rHDL NPs. Consequently, this formulation is anticipated to selectively target TAMs and, thus, achieve an enhanced therapeutic effect.Item Reprogramming of B16-F10 melanoma-educated macrophages by STING agonists loaded in mannose-decorated reconstituted high density lipoprotein nanoparticles.(2022) Dossou, Akpedje; Kapic, Ammar; Mamo, Lois; Sabnis, Nirupama; Lacko, Andras G.; Fudala, RafalPurpose: Representing a large portion of tumor-infiltrating cells, tumor-associated macrophages (TAMs) have the potential to mediate an immune response against the tumor. Instead, they are educated by the tumor microenvironment (TME) to display an immunosuppressive (M2) phenotype that favors tumor progression. The utilization of stimulator of interferon genes (STING) agonists to re-program TAMs to an immunostimulatory (M1) phenotype leads to tumor regression. However, the whole-body distribution of macrophages, the complex TME architecture, and low bioavailability at the TME are challenges to this therapeutic approach. The selective delivery of STING agonists by suitable nanoparticles can help address these challenges. Reconstituted high-density lipoprotein nanoparticles (rHDL NPs) are biocompatible, can penetrate the TME, and interact readily with macrophages. The scavenger receptor class B type 1 (SR-B1) mediates the intracellular delivery of rHDL NPs' payload. Since TAMs highly express scavenger receptors and the mannose receptor CD206, we hypothesize that mannose-decorated rHDL NPs will efficiently deliver STING agonists to TAMs for their repolarization to an M1 phenotype. Thus, the purpose of this study is to assemble and characterize mannose-decorated rHDL NPs and assess the ability of the formulation to deliver STING agonists and polarize B16F10 melanoma-conditioned macrophages to an M1 phenotype. Methods: DSPE-PEG-Mannose (DPM) was used to introduce a mannose moiety onto the rHDL NPs. Two STING agonists (DMXAA, MSA-2) were loaded separately in the rHDL-DPM to form rHDL-DPM-DMXAA and rHDL-DPM-MSA-2. Dynamic light scattering, absorbance- and fluorescence-based measurements were used to evaluate the chemical composition and characterize the formulations. Murine macrophages incubated in B16-F10 melanoma-conditioned media served as an in vitro TAM model. Lipopolysaccharide + interferon-gamma- and interleukin-4 -treated macrophages respectively served as M1 and M2 macrophage references. Western blots and ELISA were used to assess the expression of M1 markers (CXCL10, HLA-DR) and M2 markers (CD36). Results: Similar characteristics, including size, were found for rHDL-DPM-DMXAA and rHDL-DPM-MSA-2. In addition to CD206 expression, B16F10-conditioned macrophages show expression of M2 markers. M2 macrophages and B16-F10-conditioned macrophages showed a higher SR-B1 expression and higher uptake of the payload from rHDL-DPM NPs than M1 macrophages. Treatment with STING agonist-loaded rHDL-DPM diminished CD36 expression and induced HLA-DR and CXCL10 expression in B16F10-conditioned macrophages. Conclusions: The above findings show that the rHDL-DPM NPs can serve as a delivery vehicle for both DMXAA and MSA-2 and potentially can be extended to other TAM-repolarizing drugs. The expression of SR-B1 and payload uptake by the B16-F10-conditioned macrophages validate the utility of rHDL-DPM NPs to efficiently target TAMs. In addition, the induced expression of CXCL10 could be beneficial for the recruitment of CD8+ T-cells when rHDL-DPM NPs are used in combination with T-cell-based immunotherapies to improve treatment outcomes for cancer patients.Item Targeted Delivery of [alpha]-Mangostin to Prostate Cancer Cells Utilizing Reconstituted High-Density Lipoprotein Nanoparticles(2022-08) Kapic, Ammar; Berg, Rance E.; Basha, Riyaz; Ranjan, Amalendu P.Item Targeted delivery of α-Mangostin to Prostate Cancer Cells Utilizing Reconstituted High-Density Lipoprotein Nanoparticles(2022) Kapic, Ammar; Dossou, Akpedje; Lacko, Andras G.; Sabnis, Nirupama; Petty, Roland; Fudala, RafalPurpose: Androgen deprivation therapy remains the primary treatment for inhibiting the progression of prostate cancer (PCa). However, depletion of systemic androgens enhances the development of androgen independence resulting in the highly malignant castration-resistant phenotype. Interest has grown in utilizing cell cycle checkpoint inhibitors to induce cellular arrest and apoptosis. A natural compound, α-Mangostin, possesses selective anti-cancer effects against PCa by inhibiting CDK4-CyclinD complex activity, thus restricting the progression of the cell cycle. However, the employment of α-Mangostin as a chemotherapeutic is limited due to its poor oral bioavailability and hydrophobicity, making delivery difficult. Reconstituted High-Density Lipoproteins nanoparticles (rHDL-NPs) are biocompatible targeted nanoparticles capable of encapsulating various compounds. Drug delivery is mediated through a non-endocytic mechanism via the Scavenger Receptor Class B Type-1 (SR-B1). Significant overexpression of SR-B1 has been documented in PCa, which enhances cholesterol accumulation thus fuels growth, proliferation, and intertumoral androgen synthesis. This increased expression of the SR-B1 makes it a primary target for the rHDL-NPs. Because of its lipophilic characteristics, we hypothesize that the α-Mangostin can be successfully encapsulated in the rHDL-NPs, retain its biological effects, and be delivered via the SR-B1. Methods: The rHDL formulations were prepared using a modified protocol utilizing sodium cholate and sonication. The physical characteristics of the rHDL were determined using Dynamic Light Scattering (DLS), which include the polydispersity index (PDI) Zeta Potential particle diameter. The absorbance of the samples was measured using a spectrophotometer for the concentration of α-Mangostin and used to calculate the encapsulation efficiency. The anisotropy was calculated to compare the degree of molecular rotation between the free and encapsulated drug. The fluorescence lifetime (FLT) was used to detect changes in the local environment of the drug. Cytotoxicity studies were conducted using three cell lines: PZ-HPV as the normal cell line, DU145, and 22RV1 as the PCa cell lines. Different concentrations of free and rHDL encapsulated α-Mangostin were administered to 2D cell cultures to determine if there was a difference in cytotoxicity. Vehicle and empty particles were used as controls. A CCK-8 assay was used to determine the cell viability after administering the treatments. Results: The rHDL nanoparticles were produced to meet the standards of the NIH criteria for lipid-based nanoparticles. The drug was found in the same fraction as the rHDL, with an average encapsulation efficiency of 55%. There was a significant increase in anisotropy when compared to the free drug. The FLT decreased by more than half. The drug loading study found the maximum amount of drug encapsulated without decreasing the particle quality. Finally, the α-Mangostin rHDL-NPs continued to produce a cytotoxic effect comparable to the free drug. Conclusion: This study produced and characterized a stable α-Mangostin rHDL-NP formulation. The changes in the anisotropy and FLT suggest that encapsulation of the α-Mangostin has occurred. Furthermore, the encapsulation of the α-Mangostin did not block the cytotoxic effects against the PCa cell lines and decreased cell viability.