Browsing by Author "Lacko, Andras G."
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Item A comparison of photophysical characteristics of rHDL encapsulated anti-cancer drug valrubicin and free valrubicin.(2015-03) Shah, Sunil Ajit; Chib, Rahul; Raut, Sangram; Bermudez, Jaclyn Y.; Sabnis, Nirupama; Duggal, Divya; Kimball, Joseph; Lacko, Andras G.; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, IgnacyNanotechnology as a channel for drug delivery is one of the rapidly developing fields in cancer therapeutics. Targeted drug delivery has the advantage of having minimal interaction with healthy tissue, thereby reducing the toxicity of the drug to the rest of the body. rHDL nanoparticles have been found to be an efficient delivery system for highly lipophilic anti-cancer drugs. This is achieved through the interaction of scavenger receptors class B type I (SR-BI), which are highly expressed on cancer cells interact with rHDL nanoparticles for effective drug delivery to the cancer cell and tumor. The drug under investigation is Valrubicin, which, apart from being an effective anti-cancer drug, also has intrinsic fluorescence. This allowed for the comparison of photophysical properties of free Valrubicin and rHDL Valrubicin via steady state and time resolved fluorescence measurements. The steady-state anisotropy of rHDL Valrubicin is higher as compared to free Valrubicin, suggesting its encapsulation in rHDL nanoparticles. A longer rotational correlation time was observed for rHDL Valrubicin in time resolved anisotropy measurements compared to free Valrubicin, further supporting steady state anisotropy data.. We also studied the cellular internalization of free Valrubicin and rHDL Valrubicin using confocal microscopy. This could help track the movement of rHDL nanoparticles within the cancer cells.Item Advancing the Role of Thyrotropin-releasing Hormone as a Central Nervous System Agent(2022-05) De La Cruz, Daniel L.; Prokai-Tatrai, Katalin; Prokai, Laszlo; Lacko, Andras G.; Forster, Michael J.Item Assessment of WIN 55,212-2 Loaded Reconstituted High-Density Lipoprotein Nanoparticles for Ocular Delivery(2024-03-21) Petty, R. Max; Ranjan, Rajiv; Sabnis, Nirupama; Fudala, Rafal; Lacko, Andras G.; Gryczynski, Zygmunt; Krishnamoorthy, Raghu; Stankowska, DorotaPurpose: Overcoming challenges in glaucoma therapy, such as biological barriers and retina delivery, led us to develop innovative reconstituted high-density lipoprotein nanoparticles (rHDL NPs) for effective drug delivery. Optic nerve head astrocytes (ONHAs) are vital in maintaining retinal ganglion cell (RGC) axon integrity. This study describes the encapsulation of WIN 55,212-2 (WIN) in rHDL NPs and investigates the delivery mechanism of these nanoparticles in ONHAs. Methods: Using a novel preparation method, a stable rHDL-payload complex was created by combining lipophilic fluorescent dye IR780 or therapeutic agent WIN with phosphatidylcholine and apolipoprotein A-I (Apo A-I). Fluorescent rHDL (rHDL-IR780) was used to assess cellular uptake in human primary ONHAs in vitro. Scavenger receptor class B1 (SR-B1) expression was confirmed in retinal cell lysates by SDS-PAGE followed by western blot analysis. Receptor-mediated payload release through SR-B1 was confirmed by receptor blocking using BLT-1 as a specific SR-B1 receptor-blocking agent. Results: Fluorescent rHDL NPs exhibited an IR780 encapsulation efficiency of 68.7% (103 M), a polydispersity index (PDI) of 0.287 ± 0.013, a size of 14.01 ± 4.36 nm, and a zeta potential of -7.44 ± 0.90 mV. Additionally, drug-loaded rHDL-WIN NPs displayed a WIN encapsulation efficiency of 44.6% (341.4 M), a PDI of 0.280 ± 0.011, a size of 62.04 ± 25.06 nm, and a zeta potential of -20.13 ± 0.86 mV. Western blot analysis on human retinal lysates, ONHA lysates, and RGC lysates indicated the expression of SR-B1 (57/82 kDa (unmodified/glycosylated)). Cellular uptake studies confirmed the ability of rHDL to deliver payloads to ONHAs and RGCs. Receptor blocking with 10 nM BLT-1 highlighted the role of SR-B1 in specific cellular uptake from rHDL to ONHAs (p < 0.01). Conclusions: Our study highlights the role of SR-B1 in facilitating the delivery of rHDL payloads to ONHAs, offering the potential for targeted drug delivery in glaucoma. We anticipate that the cellular uptake by RGCs will follow the same SR-B1-mediated pathway. Successful WIN encapsulation in rHDL NPs suggests a potential avenue for targeting therapies to treat and prevent glaucomatous damage. Further studies are needed to determine the neuroprotective effects of rHDL-WIN and develop the potential of rHDL NPs to be used as an agent to target therapies in glaucoma.Item Calcium Sensitivy of β-cell Transcription Factor Binding to an Insulin Enhancer(1998-06-01) Scott, Gary Frank; Easom, Richard; Lacko, Andras G.; Wu, Ming-ChiGary Frank Scott, Calcium Sensitivity of β-cell Transcription Factor Binding to an Insulin Enhancer. Master of Science (Biochemistry and Molecular Biology), June 1998, 104 pp., 16 illustrations, bibliography, 94 titles. Insulin is an essential hormone and is produced exclusively in endocrine pancreas β-cells for the control of glucose homeostasis in mammals. The hypothesis tested in this thesis is that increased intracellular Ca2+ ([Ca2+]i) contributes to activation of glucose-induced insulin gene transcription. Glucose-induced insulin transcription has been mapped to binding of transcription factors by β-cell sequence motifs from -197 to -247, a glucose-response-enchancer (GRE), in the rat insulin1 gene (rINS1) promoter. Using oligonucleotide probes representing this glucose-response-enhancer (GRE) in electrophorectic mobility shift assays (EMSA), we have examined the Ca2+-sensitivity of transcription factor binding to nuclear extracts from cultured rat insulinoma β-cells (INS-1). In the presence or absence of kinase inhibitors, Ca2+ chelators, and Ca2+ channel blockers, binding was assayed for the following cell conditions: 1) in situ permeabilized cells exposed to Ca2+; 2) in vitro 32p-phosphorylated nuclear extracts; and 3) in situ glucose-stimulated and K+-depolarized intact cells. Binding was Ca2+-sensitive due to activation by K+depolarization as well as inhibition by a Ca2+-chelator, a Ca2+-channel blocker, and KN-93, specific for Ca2+/calmodulin kinases, suggesting a phosphorylation-dependent mechanism. Taken together, these findings identify a role for the Ca2+ second messenger in the glucose regulation of the insulin gene which points to novel treatments for type II diabetes.Item CHARACTERIZATION AND OPTIMIZATION OF MRNA ENTRAPPED PEPTIDE NANOPARTICLES FOR TARGETED GENE DELIVERY(2014-03) Conjeevaram Nagarajan, Bhavani Saranya; Sabnis, Nirupama; Lacko, Andras G.The mRNA entrapped nanoparticles have the potential to be used in targeted gene delivery. These nanoparticles are used in treating Cancer and neurological disorders. Purpose (a): Generally, mRNA is considered to be very labile and unstable and has not been significantly used for therapeutic purposes. However, compared to DNA based gene expression, mRNA is safer as it is does not integrate with the host genome, and it does not require nuclear localization. The main aim of this project is to entrap the mRNA inside a targeted nanoparticle to increase its stability and the tissue specificity of the gene delivery. Methods (b): The particles were assembled using heat denatured mRNA and a cationic oligomer or detergent to neutralize the negative charge of the polynucleotide. Subsequently phospholipid and a protein/peptide component are added to form the stable mRNA nanoparticle. In order to minimize the size of the particles, the preparation was carried out with several cationic detergents, including Hexadecyltrimethyl ammonium bromide (HTAB), Tetrabutyl ammonium hydroxide (TBAH), and DOTAP. The peptide/protein components were 10-100µg of either Apolipoprotein A-I, or A-I mimetic peptide Myr-5A/ 5A. The incorporation efficiency of the polynucleotide is determined by separating the unincorporated mRNA using OligodT beads and lysing the particles using Trizol reagent to release the entrapped mRNA. Results (c): The yield of the entrapped mRNA analyzed using Ribogreen assay was 17-20%. Based upon the size analysis measurements made using Dynamic Light Scatterer, it was observed that the particles prepared with 5A peptide (10µg) and DOTAP (10µg) resulted in 48% of 268nm particles. Conclusions (d): Further optimization of this formulation may be achieved to produce more homogenous nanoparticles with higher mRNA incorporation efficiency, using DOTAP as the neutralizer and 5A as the peptide.Item Characterization and Optimization of Nanoparticles for Polynucleotide delivery(2015-05-01) Conjeevaram Nagarajan, Bhavani Saranya; Lacko, Andras G.; Cistola, David P.; Krishnamoorthy, Raghu R.Nucleic acid therapeutics involves the use of polynucleotides (DNA, RNA) as novel therapeutic agents for the treatment of a wide range of diseases including cancer and several metabolic and genetic disorders. However, the highly unstable nature of RNA molecules necessitates the use of drug carriers to prevent them from nuclease degradation and facilitate targeted delivery in vivo. Hence, this study was conducted to optimize the preparation of nanoparticle carriers in order to improve the stability of the polynucleotides (siRNA and mRNA). Additionally, as heterogeneity and stability of nanoparticle formulations are major issues preventing the clinical approval of therapeutic formulations this study was also focused on improving the homogeneity and the stability of the nanoparticles. In the siRNA study, reconstituted high density lipoprotein (rHDL) nanoparticles were used as the delivery vector. Optimization of siRNA-rHDL formulation was attempted with respect to homogeneity, size of the nanoparticle and entrapment efficiency of siRNA. The results showed that the inclusion of the lyophilization step in the preparation of nanoparticles resulted in a marginal increase in the homogeneity. The size analysis of siRNA rHDL nanoparticles using AFM and TEM imaging revealed the presence of spherical nanoparticles in the range of 10-16nm. Optimization studies with mRNA peptide nanoparticle formulation were conducted using a combination of cationic detergents and peptides at varied concentrations. The particle size analysis via Dynamic Light Scattering (DLS) detector revealed the presence of 268 nm diameter particles as the major component of the mRNA nanoparticle formulation that involved the combination of DOTAP (neutralizer) and Myr-5A (Apo A-I mimetic peptide). Further optimization of this formulation will be required to improve the homogeneity of the nanoparticles.Item Characterization of rHDL Nanoparticles as a Delivery Vehicle for Glioblastoma Multiforme Chemotherapy(2021) Mathew, Ezek; Sabnis, Nirupama; Dossou, Akpedje; Dickerman, Rob; Lacko, Andras G.; Fudala, RafalPurpose: Glioblastoma Multiforme (GBM), is a brain tumor that presents with a very poor prognosis; new approaches are needed to improve patient outcomes. Design of an effective therapeutic approach must include a suitable delivery vehicle, which can cross the blood-brain barrier, and can selectively target GBM tumors. Our project uses reconstituted high-density lipoprotein (rHDL) nanoparticles (NPs), which possess the above characteristics, amplifying efficacy of chemotherapy. To target the PI3K/mTOR pathway involved in the pathophysiology of GBM, we chose to encapsulate PI-103 in preliminary studies. Methods: After encapsulation and purification, the drug-containing rHDL NPs will be characterized with regard to their physical/chemical properties. We will assess drug loading, entrapment efficiency, stability, particle diameter, homogeneity and molecular weight. Afterwards, cytotoxicity against human GBM cells will be compared to normal human astrocytes. Because the scavenger receptor B type 1 (SR-B1) is known to interact with circulating HDL and the rHDL NPs, we will compare the cytotoxic efficiency of the drug transporting rHDL NPs against a high SR-B1 expressing GBM line (LN229) versus a low SR-B1 expressing GBM line (U87MG). SR-B1 levels will be assessed for all cell lines. Results: In this work we will demonstrate that after encapsulation of PI-103 into rHDL and characterization, we will observe cytotoxic effect against GBM cell lines, not normal astrocytes. Conclusion: If successful, future spheroid and mice studies, in addition to combination therapy studies, will advance the proof of concept of this therapy, allowing translation toward clinical applications.Item Cholesterol Metabolism in High Risk Neuroblastoma. Contributions by the SR-B1 Receptor and Cholesterol Ester Accumulation(2017-03-14) Spolitu, Stefano; Deligia, Stefania; Batetta, Barbara; Lacko, Andras G.; Panchoo, MarlynBackground: Cancer cells promote their survival by reprogramming metabolic pathways. Alterations in cholesterol metabolism have been observed as one of these mechanisms, including reduced levels of high-density lipoprotein (HDL) cholesterol in cancer patients. In high-risk neuroblastoma (HRNB), an extra cranial pediatric cancer, activation of genes associated with cholesterol synthesis has been reported. However, it is unclear whether modification in exogenous sources of cholesterol also occur in HRNB. An external source of cholesterol is from lipoproteins via the scavenger receptor class B type 1 (SR-B1) lipoprotein receptor that mediates the selective uptake of cholesteryl esters (CE) into the cell. In neuroblastoma high expression of SR-B1 correlates with poor prognosis. Objective: Our goal was to examine whether the accumulation of cholesteryl ester occurs in HRNB cells and whether this process is correlated with SR-B1 expression. Methods and Results: Human neuroblastoma cell lines expressing wild type p53; SH-SY-5Y, SMS-KCNR, and mutated p53; SK-N-BE (2), and BE (2) C were cultured. Western blot analysis confirmed the presence of the HDL receptor, SR-B1, and the multidrug resistant protein 1 (MDR1). Fluorescence staining of lipid droplets was performed using Nile Red. The neutral lipid content in SH-SY-5Y and SMS-KCNR, was higher at 24 hours than 72 hours suggesting that at 24 hours either increased accumulation or synthesis of neutral lipids occurred. To examine cholesterol esterification, cells were incubated with 14C-oleate. Cells were collected, lipids extracted with cold acetone and neutral lipids separated by thin layer chromatography. The results indicated notable reduction in cholesterol esterification in p53-mutated cells as compared to the wild type cells. However, triglyceride synthesis seems unaffected by p53 mutation when compared with the wild type. Conclusions: We confirm that there is accumulation of cholesteryl esters and high expression of SR-B1 in HRNB. These findings present a more in depth understanding of molecular mechanisms that drive progression of HRNB and may provide unique molecular targets to combat HRNB and other cancers. Future plans will include investigating cholesteryl ester accumulation and SR-B1 expression as a function of cell proliferation in HRNB. Additionally, human neuroblastoma tissue samples will be used to investigate the association between overexpression of SR-B1 and tumor aggressiveness.Item Compact NMR Relaxometry of Human Blood and Living Tissues(2018-08) Patel, Vipulkumar R.; Cistola, David P.; Gryczynski, Ignacy; Berg, Rance E.; Lacko, Andras G.; Park, InWooMetabolic syndrome (MetS) is a cluster of metabolic abnormalities. The designation of MetS requires three or more of five clinical criteria: central obesity, high triglycerides, low HDL cholesterol, elevated blood pressure and high blood glucose. The main purpose of the MetS diagnosis is to prevent diabetes. However, the clinical criteria of MetS are poorly calibrated and fail to detect early metabolic abnormalities essential for diabetes prevention. Additionally, the MetS definition lacks a measure of chronic inflammation, an important driver of metabolic dysregulation. Our lab has shown that plasma and serum water T2, measured using benchtop nuclear magnetic resonance (NMR) relaxometry, are better metabolic health indicators and inclusive of inflammation. In Chapter 2 of this dissertation, we describe a broad-based, unbiased proteomic search for new biomarkers that predict plasma and serum water T2. Using a multistep statistical approach, we identified five circulatory proteins that are strongly implicated in metabolic health. In Chapter 3, we investigated whether whole blood T2 can provide similar metabolic information. Mixed blood yielded a single T2, whereas settled blood gave rise to two distinct T2 values for the cell pellet (T2P) and plasma supernatant (T2S). Supernatant T2S showed strong correlations with red blood cell count and hematocrit, and this association was due to paramagnetic relaxation enhancement. In contrast, the pellet T2P exhibited strong correlations with metabolic biomarkers. Hemoglobin glycation (HbA1C, a marker of metabolic health) is responsible for this association, as it provides water binding sites that lead to faster T2 relaxation because of increased binding and chemical exchange. The T2 value for mixed blood revealed strong associations with red blood cell count and hemoglobin. In Chapter 4, we investigated the feasibility of acquiring T2 data non-invasively from living human tissue using a custom-build NMR relaxometry device equipped with a magnet configuration to accommodate the human fingertip. Using healthy volunteers, we showed that three T2 components, corresponding primarily to different mobility domains of adipose tissue, can be measured reproducibly, with significant subject-to-subject biological variation. We propose that the source of variation is adipose tissue fluidity, which varies with lipid composition and the state of connective tissue matrix.Item Copper Tolfenamic Acid as a Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cell Growth via Downregulating Sp1 and Sp3 Transcription Factors(2018-08) Hurtado, Myrna L.; Basu, Alakananda; Lacko, Andras G.; Mathew, Porunelloor A.; Cheng, Yi-Qiang; Ma, RongDespite medical advancements, PaCa unfortunately still remains a lethal malignancy. Patients are typically diagnosed once the cancer is advanced and metastatic, making them ineligible for surgery. This cancer is usually aggressive at the time of diagnosis, so chemo and radiation offer little benefit. Therefore, PaCa urgently requires more effective and sensitizing agents for treatment. Two targets of interest for PaCa have been transcription factors Sp1 and Sp3. Both Sp1 and Sp3 are involved with regulating cell proliferation, differentiation and apoptosis. Their overexpression has been found to contribute to the progression, advancement, and poor prognosis of many types of cancers, including pancreatic. Survivin, an inhibitor of apoptosis protein, is a gene known to be regulated by both these Sp proteins. Survivin has also been found to be overexpressed in various tumor types and adds to the cancer's resistance to cytotoxic therapies. For these reasons, Sp1, Sp3, and survivin have been targets of interest for PaCa and researchers have becoming interested in finding drugs that inhibit their expression. Tolfenamic Acid (TA) is a generic migraine medicine sold in Europe. The small molecule TA has been gaining popularity for its anti-cancer properties such as inhibition of cell growth and induction of apoptosis in various tumor models. TA has been shown to work by downregulation of Sp proteins and survivin. TA has also been demonstrated to sensitize PaCa cells to radiation treatment. Although the results seen with TA thus far seem promising, its IC50 value is slightly high. Recently, it has been suggested that a copper(II) complex of TA (Cu-TA) can produce a higher therapeutic response; however, its efficacy has yet to be tested in gastro-intestinal cancers. Here, we used human PaCa cell lines (MIA PaCa-2 and Panc1) to evaluate the therapeutic efficacy of Cu-TA in an effort to increase TA's efficacy. This project contains three specific aims. Aim 1: Characterization of Cu-TA and determining its stability and anti-proliferative activity in human pancreatic cancer cell lines. Aim 2: Evaluate the effect of Cu- TA on potential markers associated with PaCa cell growth. Aim 3: Use gene expression analysis to precisely elucidate the underlying mechanisms of Cu-TA's anticancer activity in PaCa cells. Cu-TA was found to have an anti-proliferative effect in PaCa cells and its IC50 value was half that of TA's. Characterization of Cu-TA demonstrated its stability as a compound and its biological stability. Importantly, treatment of Cu-TA on cardiomyocytes did not affect cell viability. This is significant since NSAIDs can potentially cause cardiotoxicity. Cu-TA also downregulates expression of Sp proteins and survivin, molecular markers involved with PaCa growth and progression. Additionally, Cu-TA induces apoptosis and cell cycle arrest in PaCa. Finally, RNA sequencing and subsequent pathway analysis of treatment revealed Cu-TA affects pathways involved with cancer progression and metastasis. Thus, these results demonstrate Cu-TA as a potential anti-cancer agent for PaCa.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 Developing a stable drug delivery system for the mesoionic compound MIH 2.4Bl using reconstituted high-density lipoproteins (rHDL) nanoparticles(2021) Petty, Roland; Sabnis, Nirupama; Debnath, Dipti; Mathis, James; Zhang, Jinmin; Lacko, Andras G.; Fudala, RafalPurpose: In recent years, mesoionic compounds have displayed promising results as an anticancer agent due to their unique structure and properties. Specifically, the 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH2.4Bl, has been shown to inhibit oxidative phosphorylation in mitochondria, induce expression of autophagy-related proteins (Beclin-1 and ATG5), and promote cell cycle arrest at the G2/M phase in breast cancer cells. However, due to the hydrophobic nature of the drug, the cellular uptake could be affected. The rHDL drug delivery technology has been successful in solubilizing several drugs. In the present study, we produced a stable rHDL-MIH2.4BI formulation for efficient drug delivery to breast cancer cells. Methods: The rHDL-MIH2.4Bl nanoparticles were prepared using the cholate dialysis protocol enhanced by thermocycling. The size, polydispersity index, and zeta potential of the formulation were determined using a light scattering analysis instrument (Zetasizer, Malvern Panalytical Ltd). Anisotropy was determined by spectrofluorometry, and the entrapment efficiency was determined using the absorbance of MIH2.4Bl at 480nm. Results: rHDL-MIH2.4Bl formulations were successfully prepared with an entrapment efficiency of 20.3%, an average size of 38.7 ± 12.14nm, and a zeta potential of -14.13 ± 0.41mV. After 26 days of storage at 4℃, this formulation retained 97.6% of the drug with an increased anisotropy measurement from 0.189 on day1 to 0.323 on day 26. Conclusions: While additional studies need to be performed to optimize the current formulation, the rHDL-MIH2.4Bl nanoparticle appears suitable to enhance the solubility and bio-availability of MIH2.4Bl.Item Enhancing the Solubility of Valrubicin via Albumin and TPGS Formulations(2018-08) Dossou, Akpedje; Lacko, Andras G.; Basu, Alakananda; Jones, Harlan P.; Ranjan, AmalenduHuman serum albumin (HSA) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) are versatile biocompatible materials used in drug formulation. Due to its lipophilicity, the anticancer drug valrubicin (VALSTAR) has been solubilized with Cremophor EL, a solvent known for its systemic toxicity. While valrubicin is less toxic than its widely used hydrophilic anthracycline analogues, its clinical use is currently restricted to intravesical route for bladder cancer treatment. Because preliminary studies have shown a strong affinity of valrubicin for HSA and TPGS micelles, this study was aimed to explore the potential of reduced HSA (rHSA) or TPGS as excipients for valrubicin. In an aqueous environment, valrubicin solubility was enhanced from 0.1 to 85.4% using rHSA while it was dependent upon TPGS concentration. With appropriate formulation approaches, rHSA or TPGS could serve as valrubicin transporters and could thus, enable its systemic administration and extended use beyond bladder cancer to other cancer types.Item Evaluating reconstituted high density lipoprotein nanoparticles as target specific Doxorubicin carriers using fluorescence spectroscopy.(2016-03-23) Requena, Sebastian; Sabnis, Nirupama; Chib, Rahul; Lacko, Andras G.; Borejdo, Julian; Gryczynski, Zygmunt; Gryczynski, Ignacy; Shah, Sunil AjitDoxorubicin, also known as Adriamycin, is an anthracycline antibiotic which first gained clinical prominence in the early 1970’s as an effective antitumor agent. It is still used today to treat a spectrum of cancers like lymphoma, bladder, stomach, lung, breast, ovarian, and several others. Due to its production of free radicals to attack tumor cells, Doxorubicin interferes with mitochondrial phosphorylation and also induces cardiotoxicity. Thus, efficient and biocompatible delivery methods are needed for targeted drug delivery to overcome systemic toxicity. To maintain a high level of growth, tumor cells overexpress Scavenger receptors type B-1 (SR-B1). This cellular characteristic can be used to target and selectively deliver doxorubicin to tumor cells by packing it in reconstituted high density lipoprotein (rHDL) nanoparticles, which bind selectively to SR-B1 receptors. Nanoparticles as target-specific drug delivery agents are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Several different formulations of rHDL nanoparticles to incorporate doxorubicin were synthesized. Doxorubicin’s intrinsic fluorescence was used to photophysically characterize the properties of loaded rHDL nanoparticles including absorption, emission, excitation, steady-state and time resolved anisotropy measurements, and quenching to study drug shielding by nanoparticles. Overall the fluorescence properties of the rHDL: doxorubicin complex may reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.Item EVALUATION OF A "TROJAN HORSE" STRATEGY TO COMBAT NEUROBLASTOMA(2014-03) Vitug, Gaile; Sabnis, Nirupama; Shi, Yi; Di Pasqua, Anthony; Bowman, W. Paul; Lacko, Andras G.Neuroblastoma (NB) is the most common type of cancer in children less than a year old and stems from early neural cells that fail to differentiate into neurons or adrenal medulla cells. Upon initial diagnosis, 67% of cases show metastases to the lymph nodes or other organs; therefore, chemotherapy effectiveness is of particular concern. Currently, there are no FDA approved treatments or designs specifically available for NB patients and most are designed for different types of adult cancers with dose-limiting toxicities as a significant concern. Present study evaluates the therapeutic potential of a novel drug delivery system utilizing reconstituted high density lipoprotein (rHDL) containing hydrophobic analogue of Cisplatin (CisX) nanoparticles that act as a “Trojan horse” towards tumor cells. The characterization studies of CisX reveal sizes of the particles ranging from 14 nm to 164.9 nm with a mean diameter of 69.46 nm. Encapsulation efficiency was observed to be 26.34%. The cytotoxicity studies of free CisX vs rHDL-CisX towards NB cell line SJ-N-KP show 5.1 times lower IC50 value for the rHDL-CisX. This study reveals the potential of rHDL as a novel delivery method for chemotherapeutic drugs in the treatment of NB and warrants more investigation. Purpose (a): Neuroblastoma (NB) is the most common type of cancer in children less than a year old and stems from early neural cells that fail to differentiate into neurons or adrenal medulla cells. Upon initial diagnosis, 67% of cases show metastases to the lymph nodes or other organs; therefore, chemotherapy effectiveness is of particular concern. Currently, there are no FDA approved treatments or designs specifically available for NB patients and most are designed for different types of adult cancers with dose-limiting toxicities as a significant concern. Present study evaluates the therapeutic potential of a novel drug delivery system utilizing reconstituted high density lipoprotein (rHDL) containing hydrophobic analogue of Cisplatin (CisX) nanoparticles that act as a “Trojan horse” towards tumor cells. Methods (b): rHDL-CisX nanoparticles was prepared by lyophilization followed by cholate dialysis. Chemical composition of rHDL-CisX nanoparticles was determined using BCA protein assay kit and enzymatic reagent assay kits (phospolipid C and cholesterol E). Encapsulation efficiency of CisX was determined by Inductively Coupled Plasma Mass Spectrometry (ICPMS). Size estimations were determined using dynamic light scattering. The Neuroblastoma cell line, SJ-N-KP, was plated in 96 well plates and incubated at 5% CO2 at 37ºC for 72 hours. Comparative cytotoxicity was examined by CCK-8 assay (Dojindo) using Free CisX and rHDL-CisX nanoparticles at various concentrations. Results (c): The characterization studies of CisX reveal sizes of the particles ranging from 14 nm to 164.9 nm with a mean diameter of 69.46 nm. An encapsulation efficiency was observed to be 26.34%. The cytotoxicity studies of free CisX vs rHDL-CisX towards NB cell line SJ-N-KP show 5.1 times lower IC50 value for the rHDL-CisX. Conclusions (d): This study reveals the potential of rHDL as a novel delivery method for chemotherapeutic drugs in the treatment of NB and warrants more investigation. Additional studies using rHDL towards normal cells are also needed to further evaluate their therapeutic potential.Item Evaluation of Lipoprotein Mimetic Anthracycline Formulation for the Treatment of Ewing Sarcoma(2020) Kvinta, Ryan; Dossou, Akpedje; Saranya Conjeevaram Nagarajan, Bhavani; Sabnis, Nirupama; Raut, Sangram; Lacko, Andras G.; Ahmed, NadiaPurpose: Anthracyclines are effective in treating many types of cancer, including pediatric cancers such as Ewing Sarcoma. Currently, their use is limited due to cardiotoxicity, but a novel drug delivery method using lipoprotein-based technologies could mitigate this via selective delivery of its payload to cancer cells using a receptor-mediated mechanism. Methods: A soluble formulation of HDL-mimetic peptide (Myr-5A)-Valrubicin was fabricated using Nanoassemblr. Payload efficiency was characterized using spectrophotometric measurements. Size and zeta potential were measured with Zetasizer. Homogeneity was measured using FPLC. Hemolysis assays were performed by incubating Rabbit Red Blood Cells with Myr-5A-Valrubicin at different time intervals and measuring the absorbance of the supernatant at 550nm after centrifugation for 5 minutes. Cytotoxicity assays were performed on Ewing sarcoma cell lines TC205, TC32, CHLA10 and rat cardiomyocytes H9C2 using Cell counting kit (CCK 8). Results: The Myr-5A-Valrubicin nanoparticle formulation was observed to be stable, non-leaky and homogeneous with payload efficiency of 65%, size 87.75 ± 28.4nm, zeta potential -2.89 ± 9.81mV. The percentage hemolysis for Myr-5A-Valrubicin formulation was 5 and 2 times less compared to the free valrubicin formulation at 3.25 and 32.5�g/mL Valrubicin concentration, respectively. The cytotoxicity assays revealed Myr-5A-Valrubicin is effective in protecting cardiomyocytes. Conclusion: These studies indicate the potential of this novel drug delivery platform as an alternative or adjuvant therapy for combating Ewing Sarcoma. More experiments with tumor bearing model are needed to evaluate the efficacy of this formulation before reaching clinic.Item Evaluation of Reconstituted High Density Lipoprotein as an Anticancer Drug Delivery Platform(2009-12-01) Mooberry, Linda; Lacko, Andras G.Item Fluorescence Characterization and Cellular Localization of the Mesoionic Compound MIH 2.4Bl(2021) Mathis, James; Debnath, Dipti; Lacko, Andras G.; Souza, Helivaldo; Filho, Petrônio; Fudala, Rafal; Zhang, JinminBreast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide, making this disease a critical public health problem. Mesoionic compounds, which possess a 5-membered heterocyclic aromatic ring associated with a sextet of electrons, have shown remarkable promise as anti-cancer agents due to their unique structure and reaction properties. We previously reported the synthesis of a new 1,3-thiazolium-5-thiolate derivative of the mesoionic class (MIH 2.4Bl) and characterization of its selective cytotoxicity in a panel of breast cancer cell lines. Our studies suggest that treatment with MIH 2.4Bl mediates apoptotic death in breast cancer cells through mitochondrial dysfunction. To advance potential translational studies toward therapeutic applications, we have begun studies of the fluorescence properties of MIH 2.4Bl, using steady-state and time-resolved fluorescence techniques. Our preliminary steady-state measurements showed that the absorption spectrum of the drug is similar in different tested solvents. All samples, dissolved in various solvents, exhibited maximum absorbance between 440 and 480 nm; excitation at 480 nm elicited the highest emission at approximately 615 nm. These results may allow for future detection and localization of MIH 2.4Bl in vitro and in vivo. Follow-up studies utilizing fluorescence confocal microscopy are anticipated to reveal the site(s) of drug accumulation in situ and how cytotoxicity is induced in cancer cells. In addition, fluorescence lifetime measurements will be conducted to provide assessments of changes in the macromolecular conformational and experimental dynamic range of the drug.Item Formulating adjuvant therapy of rHDL nanoparticles with saquinavir to combat high-risk neuroblastoma.(2015-03) Sheikh, Irtiza; Sabnis, Nirupama; Lacko, Andras G.Purpose: Despite major advances in pediatric cancer research, there has been only modest progress in the survival of children with high risk neuroblastoma (HRNB). Current chemotherapy regimens have a serious limitation due to off target toxicity. The purpose of our project is to evaluate the effectiveness of a drug delivery platform with reconstituted/synthetic high density lipoprotein nanoparticles (rHDL) using rHDL-saquinavir formulation for the treatment of HRNB. It is anticipated that upon establishing an improved chemotherapeutic regimen for HRNB, the rHDL technology could be extended to enhance the chemotherapy for other pediatric cancers. Materials and Methods: The rHDL-Saquinavir nanoparticles were prepared by cholate dialysis method. The entrapment efficiency of Saquinavir was determined by Fluorimetric measurements. The chemical composition of rHDL/Saquinavir particles was estimated by standard kits. The average size of the particles was measured by DeLsa Nano particle size analyzer. The stability of particles was estimated by dialyzing the particles at 37°C, for 48 hours at pH 7.4. The cytotoxic effectiveness of the formulation was tested against two HRNB cell lines (SJNKP and IMR-5 obtained from Dr F. Temius, Regina Margherita Children’s Hospital, Turin, Italy)) as compared to that of the free Saquinavir using CCK-8 kit. The Inhibitory concentration to kill 50% of the cells (IC50) was determined. Results: The entrapment efficiency of the rHDL-SAQ particles was determined to be 70%. The chemical composition study indicated that the rHDL-SAQ nanoparticles were composed of 60% phospholipids, 24% protein, 9% cholesterol, and 7% of Saquinavir. The average diameter of the particle was 7.3 nm. The stability of the nanoparticle formulation measured as retention of the drug under experimental conditions indicated that 71% of the drug was preserved. When testing the survival of the IMR-5 cell lines in presence of Free and rHDL-Saquinavir, it was found that the rHDL particles were 10 times more effective than free Saquinavir. The effect on the SJNKP cells was observed to be 2 fold greater when using the rHDL particles compared to the free drug. Moreover, the rHDL-SAQ particles were both able to achieve 100% killing while the free SAQ did not achieve 100% killing effect in the given range. Conclusions: The rHDL-Saquinavir nanoparticles were successfully formulated. The particles appeared to be small, stable and non-leaking. In vitro survival studies suggested that rHDL-Saquinavir formulation is more effective than the free saquinavir. Thus, these studies support the potential of this novel drug delivery platform for treating HRNB. These studies could be extended to other types of cancers as well.Item Functional characterization of a macrophage-polarizing agent loaded in reconstituted high-density lipoprotein nanoparticles for cancer immunotherapy(2020) Lacko, Andras G.; Sabnis, Nirupama; Dossou, AkpedjePurpose: The murine stimulator of interferon genes agonist 5,6-dimethylxanthenone-4-acetic acid (DMXAA) has been successfully used to modulate the function of macrophages from an immunosuppressive phenotype (M2) to a proinflammatory phenotype (M1). Drugs, such as DMXAA, that facilitate this phenotype reversal are particularly relevant in cancer therapy as the immunosuppressive phenotype of tumor-associated macrophages (TAMs) contributes to tumor progression. This functional reprogramming of TAMs by drugs, including DMXAA, has been reported to facilitate sustained tumor regression. However, drug targeting strategies are needed to specifically deliver reversal agents to TAMs and avoid off-target effects. In this study, we investigated the physicochemical characteristics, as well as phenotype reversal capability of DMXAA encapsulated in the biocompatible reconstituted high-density lipoprotein (rHDL) nanoparticles. Methods: The DMXAA-loaded rHDL (rHDL-DMXAA) was prepared using an enhanced microfluidics apparatus. Dynamic light scattering (DLS) and fast protein liquid chromatography (FPLC) were used to assess particle size and preparation homogeneity. M1 and M2 markers expression by murine macrophages were assessed via western blot and ELISA. Results: While DLS indicated particles of 33.9 ± 14 nm diameter size, the FPLC profile of preparation indicated a homogeneous preparation with an estimated particle molecular weight of 132 kDa. Upon treatment with rHDL-DMXAA, the macrophages showed increased M1 marker expression. Conclusion: rHDL-DMXAA induced an M1 phenotype in macrophages and its characteristics suggest a potential for intratumoral accumulation. Thus, the rHDL-DMXAA represents a functional proof-of-concept strategy to target macrophages with implications in cancer immunotherapy.
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