lipofectamine and Neoplasms

Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles.

Topics: Antigens, Surface; Bioengineering; Biomimetic Materials; Calcium Chloride; Diabetes Mellitus; Drug Compounding; Drug Delivery Systems; Electroporation; Extracellular Vesicles; Gene Expression; Humans; Lipids; Lysosomal-Associated Membrane Protein 2; Milk Proteins; Neoplasms; Recombinant Fusion Proteins; Sepsis; Sonication; Tetraspanins

A novel and efficient branch PCR strategy has been used to construct a TP53 gene nanovector based on a pair of trimers as primers, which showed unique advantages compared to other existing systems for gene delivery and effective potential cancer therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; DNA; Gene Transfer Techniques; Genes, p53; Genetic Therapy; Humans; Lipids; Mice, Inbred BALB C; Nanoparticles; Neoplasms; Particle Size; Polymerase Chain Reaction; Tumor Suppressor Protein p53; Up-Regulation

Various non-viral delivery systems for small interfering RNAs (siRNA) have been developed. Such delivery systems generally exhibit tightly formed spherical structures. While such carriers have demonstrated good transfection activity in mono-layered cell systems, effects against solid tumors are often less apparent and difficult to demonstrate, likely due to the rigid structures of the carriers, which may prevent penetration to deeper regions within tumor tissue. Herein, we developed a flexible nanocarrier (FNC) system that is able to penetrate to deeper regions within tumor tissue. Specifically, we employed previously found flexible polyplexes comprised of siRNA and poly-l-lysine as wick structures for the preparation of FNCs. FNCs were constructed by coating the wick structures with lipids using a liposomal membrane fusion method. The diameters of the resulting FNCs were ca. 170nm, and the shapes were non-spherical. Lipid coating was confirmed using a nuclease resistance assay. Furthermore, FNCs showed significant RNA interference effects, comparable to Lipofectamine 2000, in a mono-layered cell system. To accelerate tumor penetration, the FNC surface was modified with polyethylene glycol (PEG) and the tight junction opener peptide AT1002. Surface-modified FNCs demonstrated effective penetrability into a cancer spheroid. Thus, we developed a novel and unique tumor-penetrable siRNA FNC system.

Topics: Animals; Cell Line, Tumor; Gene Transfer Techniques; Lipids; Mice; Nanoparticles; Neoplasms; Particle Size; Polyethylene Glycols; RNA Interference; RNA, Small Interfering; Tissue Distribution; Transfection

MicroRNAs (miRNAs) are a class of post-transcriptional gene regulators involved in various physiological processes including carcinogenesis, and they have emerged as potential targets for tumor theranostics. However, the employment of antisense oligonucleotides, termed anti-miRs, for antagonizing miRNA functions in vivo has largely been impeded by a lack of effective delivery carriers. Here, we describe the development of polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG)-functionalized nanographene oxide (NGO) conjugate (NGO-PEG-dendrimer) for the efficient delivery of anti-miR-21 into non-small-cell lung cancer cells. To monitor the delivery of anti-miR-21 into cells and tumors, we also constructed an activatable luciferase reporter (Fluc-3xPS) containing three perfectly complementary sequences against miR-21 in the 3' untranslated region (UTR) of the reporter. Compared with bare dendrimer and Lipofectamine 2000 (Lipo2000), NGO-PEG-dendrimer showed considerably lower cytotoxicity and higher transfection efficiency. As demonstrated by in vitro bioluminescence imaging and Western blotting assays, NGO-PEG-dendrimer effectively delivered anti-miR-21 into the cytoplasm and resulted in the upregulation of luciferase intensity and PTEN target protein expression in a dose-dependent manner. Moreover, transfection with anti-miR-21 by NGO-PEG-dendrimer led to stronger inhibition of cell migration and invasion than did bare dendrimer or Lipo2000 transfection. The intravenous delivery of anti-miR-21 via NGO-PEG-dendrimer induced a significant increase in the bioluminescence signal within the Fluc-3xPS reporter-transplanted tumor areas. These results suggest that NGO-PEG-dendrimer could be an efficient and a potential nanocarrier for delivering RNA oligonucleotides. In addition, the strategy of combining NGO-PEG-dendrimer with an activatable luciferase reporter allows the image-guided monitoring of the delivery process, which can provide insights into the RNA-based cancer treatments.

Topics: Animals; Cell Line, Tumor; Cell Movement; Dendrimers; Gene Expression Regulation, Neoplastic; Gene Transfer Techniques; Graphite; Humans; Lipids; Mice; MicroRNAs; Molecular Imaging; Nanoparticles; Neoplasms; Oligonucleotides, Antisense; Polyethylene Glycols; PTEN Phosphohydrolase; Xenograft Model Antitumor Assays

Tumor-associated macrophages (TAMs) are critically important in the context of solid tumor progression. Counterintuitively, these host immune cells can often support tumor cells along the path from primary tumor to metastatic colonization and growth. Thus, the ability to transform protumor TAMs into antitumor, immune-reactive macrophages would have significant therapeutic potential. However, in order to achieve these effects, two major hurdles would need to be overcome: development of a methodology to specifically target macrophages and increased knowledge of the optimal targets for cell-signaling modulation. This study addresses both of these obstacles and furthers the development of a therapeutic agent based on this strategy. Using ex vivo macrophages in culture, the efficacy of mannosylated nanoparticles to deliver small interfering RNA specifically to TAMs and modify signaling pathways is characterized. Then, selective small interfering RNA delivery is tested for the ability to inhibit gene targets within the canonical or alternative nuclear factor-kappaB pathways and result in antitumor phenotypes. Results confirm that the mannosylated nanoparticle approach can be used to modulate signaling within macrophages. We also identify appropriate gene targets in critical regulatory pathways. These findings represent an important advance toward the development of a novel cancer therapy that would minimize side effects because of the targeted nature of the intervention and that has rapid translational potential.

Topics: Animals; Bone Marrow Cells; Cell Line, Tumor; Chemokine CXCL9; Female; Glycosylation; Lipids; Macrophages; Mice, Knockout; Mice, Transgenic; Nanomedicine; Nanoparticles; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Ovarian Neoplasms; RNA, Small Interfering; Signal Transduction; Tumor Necrosis Factor-alpha

Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.. We studied the antitumor efficacy of a PEI (polyethylenimine)-PEG (polyethylene glycol) copolymer carrying the HSVtk gene combined in one vector with granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA. The system HSVtk-GM-CSF/PEI-PEG was tested in vitro in various mouse and human cell lines, ex vivo and in vivo using mouse models.. We showed that the HSVtk-GM-CSF/PEI-PEG system effectively inhibited the growth of transplanted human and mouse tumors, suppressed metastasis and increased animal lifespan.. We demonstrated that appreciable tumor shrinkage and metastasis inhibition could be achieved with a simple and low toxic chemical carrier - a PEI-PEG copolymer. Our data indicate that combined suicide and cytokine gene therapy may provide a powerful approach for the treatment of solid tumors and their metastases.

Topics: Animals; Cations; Cell Line, Tumor; Cell Proliferation; Ganciclovir; Genetic Therapy; Genetic Vectors; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Internal Ribosome Entry Sites; Lipids; Lymph Nodes; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms; Polyethylene Glycols; Polyethyleneimine; Polymers; Simplexvirus; Thymidine Kinase

Clinical drug resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. One mechanism by which a living cell can achieve multidrug resistance (MDR) is via the active efflux of a broad range of anti-cancer drugs through the cellular membrane by MDR proteins. Over-expression of multidrug resistance-associated protein 1 (MRP1) is one of the important MDR phenotypes. RNA interference (RNAi) is a fundamental cellular mechanism for silencing gene expression that can be harnessed for the development of new drugs. In our study, by using lipofectamin(TM) 2000 (Invitrogen, Carlsbad, CA, USA) in vitro and electric pulse in vivo to delivery siRNA, we successfully inhibited MRP1 both at mRNA and protein level as determined by reverse transcription-PCR and western blot or immunohistochemistry. Furthermore, the efficacy of chemotherapeutic drugs (epirubicin) to tumour cells dramatically improved both in vivo and in vitro. These studies demonstrate that through efficient delivery siRNA, MRP1-mediated MDR can be reversed and siRNA can be used for further study in clinical cancer therapy.

Topics: Animals; Cell Line, Tumor; Drug Delivery Systems; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electroporation; Female; Gene Expression Regulation, Neoplastic; Humans; Lipids; Male; Mice; Mice, Nude; Multidrug Resistance-Associated Proteins; Neoplasms; RNA Interference; RNA, Messenger; RNA, Small Interfering; Xenograft Model Antitumor Assays

The development of new vectors to deliver DNA into cells for therapy of cancers or genetic diseases has been a major area of research for many years. However, the clinical application of this technology requires the development of efficient, reliable and sterile vectors enabling the transfer of genes in vivo. Non viral, polymer or lipid-based vectors offer a new impetus to gene therapy because they are less toxic than viral vectors (no endogenous recombination, fewer immunological reactions, easy production and delivery of large-sized plasmid). The aim of this study is to develop a new tool for DNA delivery composed of methacrylic polymeric (Eudragit RS and RL) nanoparticles. These nanoparticles were prepared by two methods: nanoprecipitation and double emulsion. The nanoparticles were characterized by their size, zeta potential and amount of DNA adsorption. Cytotoxicity tests based on mitochondrial activity (MTT test) revealed that the nanoparticles had limited cytotoxicity and that this depended on both the cell type and the nanoparticle concentration. Transgene expression was observed using the Green Fluorescence Protein gene as reporter gene, and was evaluated by flow cytometry in FaDu, MDA-MB 231 and MCF-7 cell lines. The results showed that transfection rates ranging between 4 and 7% were achieved in FaDu and MDA-MB 231 cells with nanoparticles prepared by the nanoprecipitation method. In MCF-7 cells transfected with nanoparticles prepared by either the double emulsion or the nanoprecipitation method, the transfection efficiency was between 2 and 4%. Nanoparticles prepared by nanoprecipitation were slightly more efficient than nanoparticles prepared from a double emulsion. Particle size was not an important factor for transfection, since no significant difference was observed with size between 50 and 350 nm. We showed that Eudragit RS and RL nanoparticles could introduce the transgene into different types of cells, but were generally less effective than the lipofectamine control.

Topics: Adsorption; Cell Line, Tumor; DNA; Drug Delivery Systems; Genetic Vectors; Humans; Lipids; Methacrylates; Mitochondria; Nanoparticles; Nanotechnology; Neoplasms; Plasmids; Polymers; Polymethacrylic Acids; Recombination, Genetic; Tetrazolium Salts; Thiazoles; Transfection

Degradable polymers were synthesized that self-assemble with DNA to form particles that are effective for gene delivery. Small changes to polymer synthesis conditions, particle formulation conditions, and polymer structure led to significant changes to efficacy in a cell-type dependent manner. Polymers presented here are more effective than Lipofectamine 2000 or polyethylenimine for gene delivery to cancerous fibroblasts or human primary fibroblasts. These materials may be useful for cancer therapeutics and regenerative medicine.

Topics: Animals; Biodegradation, Environmental; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Drug Carriers; Fibroblasts; Gene Transfer Techniques; Genetic Therapy; Humans; Lipids; Neoplasms; Polyethyleneimine; Polymers; Regenerative Medicine

Our current understanding of how the unique tumour microenvironment influences the efficacy of gene delivery is limited. The current investigation systematically examines the efficiency of several non-viral gene transfer agents to transfect multicellular tumour spheroids (MCTS), an in vitro model that displays a faithful three-dimensional (3D) representation of solid tumour tissue.. Using a luciferase reporter assay, gene transfer to MCTS was optimised for 22 kDa linear and 25 kDa branched polyethyleneimine (PEI), the cationic lipids Lipofectamine(trade mark) and DCChol : DOPE, and the physical approach of tissue electroporation. Confocal microscopy was used to take optical tissue slices to identify the tissue localisation of green fluorescent protein (GFP) reporter gene expression and the distribution of fluorescently labelled complexes. A MCTS model of quiescent tumour regions was used to establish the influence of cellular proliferation status on gene transfer efficiency.. Of the polyplexes tested, 22 kDa linear PEI provided optimal gene delivery, with gene expression peaking at 46 h. Despite being the optimal vector tested, PEI-mediated transfection was limited to cells at the MCTS periphery. Using fluorescent PEI, it was found that complexes could only penetrate the outer 3-5 proliferating cell layers of the MCTS, sparing the deeper quiescent cells. Gene delivery in an MCTS model comprised entirely of quiescent cells demonstrated that in addition to being inaccessible to the vector, quiescent tumour regions are inherently less susceptible to PEI-mediated transfection than proliferating regions. This 'resistance' to transfection observed in quiescent cells was overcome through the use of electroporation. Despite the improved efficacy of electroporation in quiescent tissue, the gene expression was still confined to the outer regions of MCTS. The results suggest that limited access to central regions of an MCTS remain a significant barrier to gene delivery.. This data provides new insights into tumour-specific factors affecting non-viral gene transfer and highlights the difficulties in delivering genes to avascular tumour regions. The MCTS model is a useful system for the initial screening of future gene therapy strategies for solid tumours.

Topics: Cell Line, Tumor; Cell Proliferation; Electroporation; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Green Fluorescent Proteins; Humans; Lipids; Neoplasms; Polyethyleneimine; Recombinant Proteins; Spheroids, Cellular; Transfection