Lara Milane

Development of EGFR-Targeted Polymer Blend Nanocarriers for Combination Paclitaxel/Lonidamine Delivery To Treat Multi-Drug Resistance in Human Breast and Ovarian Tumor Cells

Multi-drug resistant (MDR) cancer is a significant clinical obstacle and is often implicated in cases of recurrent, nonresponsive disease. Targeted nanoparticles were made by synthesizing a poly(D,L-lactide-co-glycolide)/poly(ethylene glycol)/epidermal growth factor receptor targeting peptide (PLGA/PEG/EGFR-peptide) construct for incorporation in poly(epsilon-caprolactone) (PCL) nanoparticles. MDR was induced in a panel of nine human breast and ovarian cancer cell lines using hypoxia. EGFR-targeted polymer blend nanoparticles were shown to actively target EGFR overexpressing cell lines, especially upon induction of hypoxia. The nanoparticles were capable of sustained drug release. Combination therapy with lonidamine and paclitaxel significantly improved the therapeutic index of both drugs. Treatment with a nanoparticle dose of 1 μM paclitaxel/10 μM lonidamine resulted in less than 10% cell viability for all hypoxic/MDR cell lines and less than 5% cell viability for all normoxic cell lines. Comparatively, treatment with 1 μM paclitaxel alone was the approximate IC₅₀ value of the MDR cells while treatment with lonidamine alone had very little effect. The PLGA/PEG/EGFR-peptide delivery system actively targets a MDR cell by exploiting the expression of EGFR. This system treats MDR by inhibiting the Warburg effect and promoting mitochondrial binding of pro-apoptotic Bcl-2 proteins (lonidamine), while hyperstabilizing microtubules (paclitaxel). This nanocarrier system actively targets a MDR associated phenotype (EGFR receptor overexpression), further enhancing the therapeutic index of both drugs and potentiating the use of lonidamine/paclitaxel combination therapy in the treatment of MDR cancer.

Label-Free Raman Spectral Imaging of Intracellular Delivery and Degradation of Polymeric Nanoparticle Systems

Novel optical imaging methods, such as Raman microspectroscopy, have been gaining recognition in their ability to obtain noninvasively the distribution of biochemical components of a sample. Raman spectroscopy in combination with optical microscopy provides a label-free method to assess and image cellular processes, without the use of extrinsic fluorescent dyes. The submicrometer resolution of the confocal Raman instrumentation allows us to image cellular organelles on the scale of conventional microscopy. We used the technique to monitor subcellular degradation patterns of two biodegradable nanocarrier systems-poly(epsilon-caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA). Our results suggest that both drug-delivery systems eventually are incorporated into Golgi-associated vesicles of late endosomes. These processes were monitored via the decrease of the molecule-characteristic peaks of PCL and PLGA. As the catabolic pathways proceed, shifts and variations in peak intensities and intensity ratios in the rendered Raman spectra unequivocally delineate their degradation patterns.

Multi-modal strategies for overcoming tumor drug resistance: hypoxia, the Warburg effect, stem cells, and multifunctional nanotechnology.

Inefficiencies in systemic drug delivery and tumor residence as well as micro-environmental selection pressures contribute to the development of multidrug resistance (MDR) in cancer. Characteristics of MDR include abnormal vasculature, regions of hypoxia, up-regulation of ABC-transporters, aerobic glycolysis, and an elevated apoptotic threshold. Nano-sized delivery vehicles are ideal for treating MDR cancer as they can improve the therapeutic index of drugs and they can be engineered to achieve multifunctional parameters. The multifunctional ability of nanocarriers makes them more adept at treating heterogeneous tumor mass than traditional chemotherapy. Nanocarriers also have preferential tumor accumulation via the EPR effect; this accumulation can be further enhanced by actively targeting the biological profile of MDR cells. Perhaps the most significant benefit of using nanocarrier drug delivery to treat MDR cancer is that nanocarrier delivery diverts the effects of ABC-transporter mediated drug efflux; which is the primary mechanism of MDR. This review discusses the capabilities, applications, and examples of multifunctional nanocarriers for the treatment of MDR. This review emphasizes multifunctional nanocarriers that enhance drug delivery efficiency, the application of RNAi, modulation of the tumor apoptotic threshold, and physical approaches to overcome MDR.

Pharmacokinetics and biodistribution of lonidamine/paclitaxel loaded, EGFR-targeted nanoparticles in an orthotopic animal model of multi-drug resistant breast cancer

UNLABELLED The aim of this study was to assess the biodistribution and pharmacokinetics of epidermal growth factor receptor (EGFR)-targeted polymer-blend nanoparticles loaded with the anticancer drugs lonidamine and paclitaxel. Plasma, tumor, and tissue distribution profiles were quantified in an orthotopic animal model of multidrug-resistant breast cancer and were compared to treatment with nontargeted nanoparticles and to treatment with drug solution. A poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-EGFR targeting peptide (PLGA-PEG-EFGR peptide) construct was synthesized for incorporation in poly(ɛ-caprolactone) particles to achieve active EGFR targeting. An isocratic high-pressure liquid chromatography method was developed to quantify lonidamine and paclitaxel in mice plasma, tumors, and vital organs. The targeted nanoparticles demonstrated a superior pharmacokinetic profile relative to drug solution and nontargeted nanoparticles, particularly for lonidamine delivery. The first target site of accumulation was the liver, followed by the kidneys, and then the tumor mass; maximal tumor accumulation occured at 3 hours after administration. Lonidamine-paclitaxel combination therapy administered via EGFR-targeted polymer-blend nanocarriers may become a viable platform for the future treatment of multidrug-resistant cancer. FROM THE CLINICAL EDITOR In this study the biodistribution and pharmacokinetics of epidermal growth factor receptor (EGFR)-targeted polymer-blend nanoparticles loaded with lonidamine and paclitaxel were assessed. The targeted nanoparticles demonstrated a superior pharmacokinetic profile relative to drug solution and nontargeted nanoparticles, paving the way to new therapeutic approaches for multidrug-resistant malignancies.

Role of hypoxia and glycolysis in the development of multi-drug resistance in human tumor cells and the establishment of an orthotopic multi-drug resistant tumor model in nude mice using hypoxic pre-conditioning

BackgroundThe development of multi-drug resistant (MDR) cancer is a significant challenge in the clinical treatment of recurrent disease. Hypoxia is an environmental selection pressure that contributes to the development of MDR. Many cancer cells, including MDR cells, resort to glycolysis for energy acquisition. This study aimed to explore the relationship between hypoxia, glycolysis, and MDR in a panel of human breast and ovarian cancer cells. A second aim of this study was to develop an orthotopic animal model of MDR breast cancer.MethodsNucleic and basal protein was extracted from a panel of human breast and ovarian cancer cells; MDR cells and cells pre-exposed to either normoxic or hypoxic conditions. Western blotting was used to assess the expression of MDR markers, hypoxia inducible factors, and glycolytic proteins. Tumor xenografts were established in the mammary fat pad of nu/nu mice using human breast cancer cells that were pre-exposed to either hypoxic or normoxic conditions. Immunohistochemistry was used to assess the MDR character of excised tumors.ResultsHypoxia induces MDR and glycolysis in vitro, but the cellular response is cell-line specific and duration dependent. Using hypoxic, triple-negative breast cancer cells to establish 100 mm3 tumor xenografts in nude mice is a relevant model for MDR breast cancer.ConclusionHypoxic pre-conditiong and xenografting may be used to develop a multitude of orthotopic models for MDR cancer aiding in the study and treatment of the disease.

Hyaluronic acid targeting of CD44 for cancer therapy: from receptor biology to nanomedicine

Abstract Cluster of differentiation-44 (CD44) is a ubiquitously present glycoprotein on the surface of mammalian cells that plays a significant role in a number of biological functions. Since the discovery that the receptor is over-expressed in a variety of solid tumors, such as pancreatic, breast and lung cancer, many studies have focused on methods for targeting CD44 in an attempt to improve drug delivery and discrimination between healthy and malignant tissue, while reducing residual toxicity and off-target accumulation. In this review, we describe CD44 receptor biology and its involvement in the different stages of tumor growth and metastasis, as well as methods currently used for targeting the receptor. Hyaluronic acid, the primary CD44 binding molecule, has proved a significant ally in developing nanocarriers that demonstrate preferential tumor accumulation and increased cell uptake. We outline a number of research approaches from the current literature that take advantage of hyaluronic acid’s targeting ability and describe the possible advantages for each approach. The value of CD44 targeting can be easily appreciated from the number of different approaches that have reached clinical trials.

Therapeutic efficacy and safety of paclitaxel/lonidamine loaded EGFR-targeted nanoparticles for the treatment of multi-drug resistant cancer.

The treatment of multi-drug resistant (MDR) cancer is a clinical challenge. Many MDR cells over-express epidermal growth factor receptor (EGFR). We exploit this expression through the development of EGFR-targeted, polymer blend nanocarriers for the treatment of MDR cancer using paclitaxel (a common chemotherapeutic agent) and lonidamine (an experimental drug; mitochondrial hexokinase 2 inhibitor). An orthotopic model of MDR human breast cancer was developed in nude mice and used to evaluate the safety and efficacy of nanoparticle treatment. The efficacy parameters included tumor volume measurements from day 0 through 28 days post-treatment, terminal tumor weight measurements, tumor density and morphology assessment through hematoxylin and eosin staining of excised tumors, and immunohistochemistry of tumor sections for MDR protein markers (P-glycoprotein, Hypoxia Inducible Factor, EGFR, Hexokinase 2, and Stem Cell Factor). Toxicity was assessed by tracking changes in animal body weight from day 0 through 28 days post-treatment, by measuring plasma levels of the liver enzymes ALT (Alanine Aminotransferase) and LDH (lactate dehydrogenase), and by white blood cell and platelet counts. In these studies, this nanocarrier system demonstrated superior efficacy relative to combination (paclitaxel/lonidamine) drug solution and single agent treatments in nanoparticle and solution form. The combination nanoparticles were the only treatment group that decreased tumor volume, sustaining this decrease until the 28 day time point. In addition, treatment with the EGFR-targeted lonidamine/paclitaxel nanoparticles decreased tumor density and altered the MDR phenotype of the tumor xenografts. These EGFR-targeted combination nanoparticles were considerably less toxic than solution treatments. Due to the flexible design and simple conjugation chemistry, this nanocarrier system could be used as a platform for the development of other MDR cancer therapies; the use of this system for EGFR-targeted, combination paclitaxel/lonidamine therapy is an advance in personalized medicine.

Mitochondrial biology, targets, and drug delivery.

In recent years, mitochondrial medicine has emerged as a new discipline resting at the intersection of mitochondrial biology, pathology, and pharmaceutics. The central role of mitochondria in critical cellular processes such as metabolism and apoptosis has placed mitochondria at the forefront of cell science. Advances in mitochondrial biology have revealed that these organelles continually undergo fusion and fission while functioning independently and in complex cellular networks, establishing direct membrane contacts with each other and with other organelles. Understanding the diverse cellular functions of mitochondria has contributed to understanding mitochondrial dysfunction in disease states. Polyplasmy and heteroplasmy contribute to mitochondrial phenotypes and associated dysfunction. Residing at the center of cell biology, cellular functions, and disease pathology and being laden with receptors and targets, mitochondria are beacons for pharmaceutical modification. This review presents the current state of mitochondrial medicine with a focus on mitochondrial function, dysfunction, and common disease; mitochondrial receptors, targets, and substrates; and mitochondrial drug design and drug delivery with a focus on the application of nanotechnology to mitochondrial medicine. Mitochondrial medicine is at the precipice of clinical translation; the objective of this review is to aid in the advancement of mitochondrial medicine from infancy to application.

Cluster of Differentiation 44 Targeted Hyaluronic Acid Based Nanoparticles for MDR1 siRNA Delivery to Overcome Drug Resistance in Ovarian Cancer

PurposeApproaches for the synthesis of biomaterials to facilitate the delivery of “biologics” is a major area of research in cancer therapy. Here we designed and characterized a hyaluronic acid (HA) based self-assembling nanoparticles that can target CD44 receptors overexpressed on multidrug resistance (MDR) ovarian cancer. The nanoparticle system is composed of HA-poly(ethyleneimine)/HA-poly(ethylene glycol) (HA-PEI/HA-PEG) designed to deliver MDR1 siRNA for the treatment of MDR in an ovarian cancer model.MethodsHA-PEI/HA-PEG nanoparticles were synthesized and characterized, then the cellular uptake and knockdown efficiency of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles was further determined. A human xenograft MDR ovarian cancer model was established to evaluate the effects of the combination of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles and paclitaxel on MDR tumor growth.ResultsOur results demonstrated that HA-PEI/HA-PEG nanoparticles successfully targeted CD44 and delivered MDR1 siRNA into OVCAR8TR (established paclitaxel resistant) tumors. Additionally, HA-PEI/HA-PEG nanoparticles loaded with MDR1 siRNA efficiently down-regulated the expression of MDR1 and P-glycoprotein (Pgp), inhibited the functional activity of Pgp, and subsequently increased cell sensitivity to paclitaxel. HA-PEI/HA-PEG/MDR1 siRNA nanoparticle therapy followed by paclitaxel treatment inhibited tumor growth in MDR ovarian cancer mouse models.ConclusionsThese findings suggest that this CD44 targeted HA-PEI/HA-PEG nanoparticle platform may be a clinicaly relevant gene delivery system for systemic siRNA-based anticancer therapeutics for the treatment of MDR cancers.

Targeted Cancer Therapy; Nanotechnology Approaches for Overcoming Drug Resistance

Recent advances in cancer molecular biology have resulted in parallel and unprecedented progress in the development of targeted cancer therapy. Targeted therapy can provide higher efficacy and lower toxicity than conventional chemotherapy for cancer. However, like traditional chemotherapy, molecularly targeted cancer therapy also faces the challenge of drug resistance. Multiple mechanisms are responsible for chemotherapy resistance in tumors, including over-expression of efflux transporters, somatic alterations of drug targets, deregulation of apoptosis, and numerous pharmacokinetic issues. Nanotechnology based approaches are proving to be efficacious in overcoming drug resistance in cancer. Combination of targeted therapies with nanotechnology approaches is a promising strategy to overcome targeted therapy drug resistance in cancer treatment. This review discusses the mechanisms of targeted drug resistance in cancer and discusses nanotechnology approaches to circumvent this resistance.