Poulomi Sengupta

Cholesterol-tethered platinum II-based supramolecular nanoparticle increases antitumor efficacy and reduces nephrotoxicity

Nanoscale drug delivery vehicles have been harnessed extensively as carriers for cancer chemotherapeutics. However, traditional pharmaceutical approaches for nanoformulation have been a challenge with molecules that exhibit incompatible physicochemical properties, such as platinum-based chemotherapeutics. Here we propose a paradigm based on rational design of active molecules that facilitate supramolecular assembly in the nanoscale dimension. Using cisplatin as a template, we describe the synthesis of a unique platinum (II) tethered to a cholesterol backbone via a unique monocarboxylato and O→Pt coordination environment that facilitates nanoparticle assembly with a fixed ratio of phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000]. The nanoparticles formed exhibit lower IC50 values compared with carboplatin or cisplatin in vitro, and are active in cisplatin-resistant conditions. Additionally, the nanoparticles exhibit significantly enhanced in vivo antitumor efficacy in murine 4T1 breast cancer and in K-RasLSL/+/Ptenfl/fl ovarian cancer models with decreased systemic- and nephro-toxicity. Our results indicate that integrating rational drug design and supramolecular nanochemistry can emerge as a powerful strategy for drug development. Furthermore, given that platinum-based chemotherapeutics form the frontline therapy for a broad range of cancers, the increased efficacy and toxicity profile indicate the constructed nanostructure could translate into a next-generation platinum-based agent in the clinics.

Mechanistic Studies of Gemcitabine-Loaded Nanoplatforms in Resistant Pancreatic Cancer Cells

BackgroundPancreatic cancer remains the deadliest of all cancers, with a mortality rate of 91%. Gemcitabine is considered the gold chemotherapeutic standard, but only marginally improves life-span due to its chemical instability and low cell penetrance. A new paradigm to improve Gemcitabine’s therapeutic index is to administer it in nanoparticles, which favour its delivery to cells when under 500 nm in diameter. Although promising, this approach still suffers from major limitations, as the choice of nanovector used as well as its effects on Gemcitabine intracellular trafficking inside pancreatic cancer cells remain unknown. A proper elucidation of these mechanisms would allow for the elaboration of better strategies to engineer more potent Gemcitabine nanotherapeutics against pancreatic cancer.MethodsGemcitabine was encapsulated in two types of commonly used nanovectors, namely poly(lactic-co-glycolic acid) (PLGA) and cholesterol-based liposomes, and their physico-chemical parameters assessed in vitro. Their mechanisms of action in human pancreatic cells were compared with those of the free drug, and with each others, using cytotoxity, apoptosis and ultrastructural analyses.ResultsPhysico-chemical analyses of both drugs showed high loading efficiencies and sizes of less than 200 nm, as assessed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), with a drug release profile of at least one week. These profiles translated to significant cytotoxicity and apoptosis, as well as distinct intracellular trafficking mechanisms, which were most pronounced in the case of PLGem showing significant mitochondrial, cytosolic and endoplasmic reticulum stresses.ConclusionsOur study demonstrates how the choice of nanovector affects the mechanisms of drug action and is a crucial determinant of Gemcitabine intracellular trafficking and potency in pancreatic cancer settings.

Sequential application of a cytotoxic nanoparticle and a PI3K inhibitor enhances antitumor efficacy

Nanomedicines that preferentially deploy cytotoxic agents to tumors and molecular targeted therapeutics that inhibit specific aberrant oncogenic drivers are emerging as the new paradigm for the management of cancer. While combination therapies are a mainstay of cancer chemotherapy, few studies have addressed the combination of nanomedicines and molecular targeted therapeutics. Furthermore, limited knowledge exists on the impact of sequencing of such therapeutics and nanomedicines on the antitumor outcome. Here, we engineered a supramolecular cis-platinum nanoparticle, which induced apoptosis in breast cancer cells but also elicited prosurvival signaling via an EGF receptor/phosphoinositide 3-kinase (PI3K) pathway. A combination of mathematical modeling and in vitro and in vivo validation using a pharmacologic inhibitor of PI3K, PI828, demonstrate that administration of PI828 following treatment with the supramolecular cis-platinum nanoparticle results in enhanced antitumor efficacy in breast cancer as compared with when the sequence is reversed or when the two treatments are administered simultaneously. This study addresses, for the first time, the impact of drug sequencing in the case of a combination of a nanomedicine and a targeted therapeutic. Furthermore, our results indicate that a rational combination of cis-platinum nanoparticles and a PI3K-targeted therapeutic can emerge as a potential therapy for breast cancer.

P2Y12 receptor inhibition augments cytotoxic effects of cisplatin in breast cancer

Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, renal carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this study, we characterize the expression of P2Y12 receptor in breast cancer cell lines and evaluate its role in enhancing the cytotoxic effects of cisplatin. We observed a significant upregulation in P2Y12 expression in 4T1 breast cancer cell line with cisplatin treatment. Co-administration of P2Y12 inhibitor with cisplatin resulted in significantly higher cytotoxic response in 4T1 cancer cell line. This was mediated by HIF1α-dependent upregulation of cellular apoptotic pathways. These findings identify P2Y12 receptor as a potential target to enhance antitumor efficacy of chemotherapeutic agents like cisplatin.

Anti-platelet agents augment cisplatin nanoparticle cytotoxicity by enhancing tumor vasculature permeability and drug delivery.

Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can augment delivery of nano-formulation based chemotherapeutic agents which depend on enhanced permeability and retention for tumor penetration. Here, we evaluated the role of Clopidogrel, a well-known inhibitor of platelet aggregation, in potentiating the tumor cytotoxicity of cisplatin nano-formulation in a murine breast cancer model. In vivo studies in murine syngeneic 4T1 breast cancer model showed a significant greater penetration of macromolecular fluorescent nanoparticles after clopidogrel pretreatment. Compared to self-assembling cisplatin nanoparticles (SACNs), combination therapy with clopidogrel and SACN was associated with a 4 fold greater delivery of cisplatin to tumor tissue and a greater reduction in tumor growth as well as higher survival rate. Clopidogrel enhances therapeutic efficiency of novel cisplatin based nano-formulations agents by increasing tumor drug delivery and can be used as a potential targeting agent for novel nano-formulation based chemotherapeutics.

Cancer, Signal Transduction and Nanotechnology

Understanding the mechanisms underlying different cellular signaling pathways implicated in the pathogenesis of cancer are leading to the identification of novel drug targets as well as novel drug candidates. Multiple targeted therapeutics that modulate aberrant molecular pathways have already reached the clinic. However, targeted therapeutics can exert mechanism-driven side effects as a result of the implication of the molecular target in normal physiological functions besides tumorigenesis. We hypothesize that targeted therapeutics can be optimized by merging them with nanotechnology, which offers the potential for preferential targeting to the tumor, resulting in increased intratumoral concentrations of the active agent with reduced distribution to other parts of the body. This review will address some of the emerging concepts that integrate these two disciplines to engineer novel nanovectors that target different signaling pathways.

Hyaluronic Acid Layered Chimeric Nanoparticles: Targeting MAPK-PI3K Signaling Hub in Colon Cancer Cells

Colon cancer has emerged as one of the most devastating diseases in the whole world. Mitogen-activated protein kinase (MAPK)-phosphatidylinsitol-3-kinase (PI3K) signaling hub has gained lots of attention due to its deregulation in colon cancer cells. However, selective targeting of oncogenic MAPK-PI3K hub in colon cancer has remained highly challenging, hence it has mostly been unexplored. To address this, we have engineered a hyaluronic acid layered lipid-based chimeric nanoparticle (HA-CNP) consisting of AZD6244 (MAPK inhibitor), PI103 (PI3K inhibitor), and cisplatin (DNA impairing drug) ratiometrically in a single particle. Electron microscopy (field emission scanning electron microscopy and atomic force microscopy) and dynamic light scattering were utilized to characterize the size, shape, morphology, and surface charge of the HA-CNPs. Fluorescent confocal laser scanning microscopy and flow cytometry analysis confirmed that HA-CNPs were taken up by HCT-116 colon cancer cells by merging of clathrin and CD44 receptor-mediated endocytosis along with macropinocytosis to home into acidic organelles (lysosomes) within 1 h. A gel electrophoresis study evidently established that HA-CNPs simultaneously inhibited MAPK-PI3K signaling hub with DNA damage in HCT-116 cells. These HA-CNPs stalled the cell cycle into G0/G1 phase, leading to induction of apoptosis (early and late) in colon cancer cells. Finally, these HA-CNPs exerted remarkable cytotoxicity in HCT-116 colon cancer cells at 24 h compared to that of the free triple drug cocktail as well as HA-coated dual drug-loaded nanoparticles without showing any cell death in healthy L929 fibroblast cells. These HA-coated CNPs have potential to be translated into clinics as a novel platform to perturb various oncogenic signaling hubs concomitantly toward next-generation targeted colon cancer therapy.

Abstract 2892: Enhancing anti cancer activity of PI103, a dual PI3K/mTOR inhibitor, by designing a self assembled Nanoformulation

The phosphoinositide 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) pathway plays a pivotal role in the growth and survival of various cancers. Small molecule inhibitors targeting PI3K/AKT/mTOR pathway (eg. Wortmannin, LY294002, PI-103, PI828) are promising anti cancer agents. Major drawbacks of these drugs are their poor water solubility and off-target toxicity. Nanoscale drug delivery systems have been harnessed as drug carriers to target tumours through enhanced permeation and retention effect. In this study, we developed a nanoformulation of PI103, a potent dual PI3K/mTOR inhibitor, to improve delivery and reduce side effects. PI103 was encapsulated in a homogeneous self-assembled mixture of phosphatidylcholine and cholesterol. DSPE-PEG protective shell was introduced to increase its circulation time. Thin-film-hydration technique followed by extrusion through 200 nm porous membranes developed nanoparticles of 110-150 nm size with 0.15-0.20 mM drug loading. Release kinetics study confirmed the stability of this formulation in PBS for several days as well as a sustained release property in in-vitro Cell Lysate system. In-vitro cell proliferation assay demonstrated improved cytotoxicity of nanoformulated PI103 in murine breast cancer cell line 4T1 and K-ras upregulated and PTEN suppressed ovarian cancer model cells. Fluorescent Activated Cell Sorting (FACS) analysis with FITC tagged Annexin-V, an early apoptosis marker, showed increased apoptosis with the nanoformulation than the free drug. Western blot analysis expressed more potent inhibition of PI3 kinase/Akt pathway by the nanoformulated PI103. These results indicate that nanoformulation can enhance the potential of PI103. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2892. doi:1538-7445.AM2012-2892

Abstract 1951: Targeting cancer by a chimeric nanoparticle formulation of Cisplatin and PI828, a PI3 kinase inhibitor

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Combination chemotherapy is the mainstay of cancer regimes. However, additive off target effect of the drugs is still a major drawback of combination therapy. Nanoscale drug delivery systems, through enhanced permeation and retention effect, target the tumour more efficiently while reducing side effects. Combination of Cisplatin, a widely used platinum based chemotherapeutic with PI3 kinase/mTOR inhibitor has shown synergism. In this study, we designed a chimeric nanoparticle of Cisplatin and PI828, a PI3 kinase inhibitor. PI828 was chemically conjugated to cholesterol by a carbamate linkage. Cisplatin was coordinated to a cholesterol-succinic acid derivative via a unique monocarboxylato and OαPt bond. The chimeric nanoparticle was formulated by mixing both the conjugates in definite proportions with phosphatidylcholine and DSPE-PEG. The chimeric naoparticle had a consistent size between 100 to 130 nm determined by dynamic light scattering. The in vitro release kinetics experiment showed slow and sustained release of both the drugs over a time period of 96 hours. In vitro cell proliferation assay expressed cytotoxicity of the chimeric nanoformulation in 4T1, a murine breast cancer cell line and in KRAS upregulated and PTEN suppressed ovarian cancer model cells. We have evaluated the activity of chimeric nanoparticle in a K-Ras(LSL/+)/PTEN(fl/fl) ovarian cancer model, with a luciferase reporter system. The mice treated with chimeric nanoparticle exhibited suppressed tumour growth as well as reduced toxicity as compared to the free drug combination and vehicle treated mice. These results indicate that chimeric nanoformulation can better the outcome of Cisplatin and PI828 combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1951. doi:1538-7445.AM2012-1951

Abstract 1945: A novel self assembled taxane nanoparticles for breast cancer chemotherapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer is the most frequently diagnosed form of cancer and a second leading cause of cancer deaths among women in the United States, with an estimated 207,090 new cases and 39,840 deaths since the beginning of 2010. Taxane-based chemotherapeutic agents are extensively used as a treatment for breast cancer and have shown important clinical benefits in both adjuvant and metastatic settings. However, the therapeutic efficacy of these drugs is hampered by limitations such as poor water solubility, narrow therapeutic indices and significant off-target toxicities which limit its dose escalations and treatment frequency. Also, current drug delivery strategies often suffer from constraints such as lower potency, low loading, non-biocompatibility/ non-biodegradability and uncontrolled release of the drug. Clearly, there is an urgent need for a paradigm shift in the strategies for the development of drug delivery vehicles for breast cancer management. We propose a novel approach in the design of nanomedicine where an active molecule can self-assemble into a nanoparticle resulting in increased loading, improved antitumor outcome and reduced off-target side effects. Different taxane analogs were attached to cholesterol using an acid labile linker. The self assembly of taxane-cholesterol conjugate was facilitated by phosphatidylcholine and DSPE-PEG to form well defined nano-sized particles. The loading of 10-15 μg/μL of drugs was obtained and sustained release of drugs was observed at pH 5.5 over 48h. In a preliminary cell viability studies, the taxane nanoparticles showed comparable efficacy to drug alone. Next, in order to achieve tumor specific homing of the nanoparticles, we plan to incorporate multiple copies of iRGD peptide on the nanoparticle surface that will bind to αvβ3 integrins overexpressed on the endothelium of tumor vessels and help in effective internalization of nanoparticles. The optimum density of peptides on the nanoparticle surface will be achieved by conjugating the peptide to DSPE-PEG. We believe that this study will not only uncover a novel and most effective nanotechnology-based approach for breast cancer treatment but will also eventually lead to an improvement in the quality of life of patients at minimum cost. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1945. doi:1538-7445.AM2012-1945