Sara Movassaghian

Applications of polymer micelles for imaging and drug delivery

Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers, are widely considered as convenient nano-carriers for a variety of applications, such as diagnostic imaging, and drug and gene delivery. They have demonstrated a variety of favorable properties including biocompatibility, longevity, high stability in vitro and in vivo, capacity to effectively solubilize a variety of poorly soluble drugs, changing the release profile of the incorporated pharmaceutical agents, and the ability to accumulate in the target zone based on the enhanced permeability and retention effect. Moreover, additional functions can be imparted to the micelle-based delivery systems by engineering their surface for specific applications. Various targeting ligands can be attached for cell or intracellular accumulation at a site of interest. Also, the chelation or incorporation of imaging moieties into the micelle structure enables in vivo biodistribution studies. Moreover, pH-, thermo-, ultrasound-, enzyme- and light-sensitive block-copolymers allow for controlled micelle dissociation and triggered drug release in response to the pathological environment-specific stimuli and/or externally applied signals. The combination of these approaches can further improve specificity and efficacy of micelle-based drug delivery to promote the development of smart multifunctional micelles.

Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors

Multidrug resistance (MDR) mediated by P-glycoprotein overexpression in solid tumors is a major factor in the failure of many forms of chemotherapy. Here we evaluated phospholipid-modified, low-molecular-weight polyethylenimine (DOPE-PEI) nanocarriers for intravenous delivery of anti-P-pg siRNA to tumors with the final goal of modulating MDR in breast cancer. First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model. Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation. Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts. Weekly injection of siRNA nanopreparations and Dox for up to 5 weeks sensitized the tumors to otherwise non-effective doses of Dox and decreased the tumor volume by threefold vs controls. This therapeutic improvement in response to Dox was attributed to the significant, sequence-specific P-gp downregulation in excised tumors mediated by the DOPE-PEI formulations.

Dendrosome-dendriplex inside liposomes: as a gene delivery system.

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.

Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes.

The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques.

Screening nylon-3 polymers, a new class of cationic amphiphiles, for siRNA delivery.

Amphiphilic nucleic acid carriers have attracted strong interest. Three groups of nylon-3 copolymers (poly-β-peptides) possessing different cationic/hydrophobic content were evaluated as siRNA delivery agents in this study. Their ability to condense siRNA was determined in SYBR Gold assays. Their cytotoxicity was tested by MTT assays, their efficiency of delivering Alexa Fluor-488-labeled siRNA intracellularly in the presence and absence of uptake inhibitors was assessed by flow cytometry, and their transfection efficacies were studied by luciferase knockdown in a cell line stably expressing luciferase (H1299/Luc). Endosomal release was determined by confocal laser scanning microscopy and colocalization with lysotracker. All polymers efficiently condensed siRNA at nitrogen-to-phosphate (N/P) ratios of 5 or lower, as reflected in hydrodynamic diameters smaller than that at N/P 1. Although several formulations had negative zeta potentials at N/P 1, G2C and G2D polyplexes yielded >80% uptake in H1299/Luc cells, as determined by flow cytometry. Luciferase knockdown (20–65%) was observed after transfection with polyplexes made of the high molecular weight polymers that were the most hydrophobic. The ability of nylon-3 polymers to deliver siRNA intracellularly even at negative zeta potential implies that they mediate transport across cell membranes based on their amphiphilicity. The cellular uptake route was determined to strongly depend on the presence of cholesterol in the cell membrane. These polymers are, therefore, very promising for siRNA delivery at reduced surface charge and toxicity. Our study identified nylon-3 formulations at low N/P ratios for effective gene knockdown, indicating that nylon-3 polymers are a new, promising type of gene delivery agent.

Post-Transcriptional Regulation of the GASC1 Oncogene with Active Tumor-Targeted siRNA-Nanoparticles

Basal-like breast cancer (BLBC) accounts for the most aggressive types of breast cancer, marked by high rates of relapse and poor prognoses and with no effective clinical therapy yet. Therefore, investigation of new targets and treatment strategies is more than necessary. Here, we identified a receptor that can be targeted in BLBC for efficient and specific siRNA mediated gene knockdown of therapeutically relevant genes such as the histone demethylase GASC1, which is involved in multiple signaling pathways leading to tumorigenesis. Breast cancer and healthy breast cell lines were compared regarding transferrin receptor (TfR) expression via flow cytometry and transferrin binding assays. Nanobioconjugates made of low molecular weight polyethylenimine (LMW-PEI) and transferrin (Tf) were synthesized to contain a bioreducible disulfide bond. siRNA complexation was characterized by condensation assays and dynamic light scattering. Cytotoxicity, transfection efficiency, and the targeting specificity of the conjugates were investigated in TfR positive and negative healthy breast and breast cancer cell lines by flow cytometry, confocal microscopy, RT-PCR, and Western blot. Breast cancer cell lines revealed a significantly higher TfR expression than healthy breast cells. The conjugates efficiently condensed siRNA into particles with 45 nm size at low polymer concentrations, showed no apparent toxicity on different breast cancer cell lines, and had significantly greater transfection and gene knockdown activity on mRNA and protein levels than PEI/siRNA leading to targeted and therapeutic growth inhibition post GASC1 knockdown. The synthesized nanobioconjugates improved the efficiency of gene transfer and targeting specificity in transferrin receptor positive cells but not in cells with basal receptor expression. Therefore, these materials in combination with our newly identified siRNA sequences are promising candidates for therapeutic targeting of hard-to-treat BLBC and are currently further investigated regarding in vivo targeting efficacy and biocompatibility.

Long-Circulating Therapies for Cancer Treatment

Abstract In the emerging field of nanotechnology, nanoparticles hold promise for overcoming challenges associated with many cancer drug delivery systems. They possess enormous potential by providing a means of modifying the fate of an active substance in vivo and of increasing drug concentration in the target tissue, thereby improving pharmacological efficacy and reducing side-effects related to systemic toxicity. However, their applications are limited by rapid clearance from circulation by the reticuloendothelial system. Characteristics that prolong circulation must be taken into account in the design of an optimized cancer delivery system. Considerable progress has been made over the past several decades to address this problem with surface treatment (alteration/modification) strategies in the development of nanoparticles with an acceptable blood circulation time. The objective of this chapter is to outline the current research investigating long-circulating nanoparticles and to discuss the physicochemical properties of nanoparticles (mainly liposomes) that have a significant impact on the fate of nanocarriers. We highlight how manipulation of these parameters influences the pharmacokinetics of the delivery system, focus on PEG modification of nanoparticle as the current gold standard in the field of so-called stealth nanoparticles, and address advantages and disadvantages of PEG-modified nanoparticles. Finally, other biomaterials and methods that have been introduced as alternatives in designing drug delivery systems that can be disguised as “self” and remain unrecognizable to immune system are discussed.

Chapter 8.1:Overcoming Biological Barriers with Parenteral Nanomedicines: Physiological and Mechanistic Issues

Nanotechnology has brought deep impacts and new possibilities to our lives, particularly, in the area of drug development and its application. Like other materials, a medicine’s physical and chemical properties as well as its biological properties can be altered dramatically at the nanoscopic scale....