P53 Gene Delivery Research Articles

A cancer cell membrane coated nanoparticles-based gene delivery system for enhancing cancer therapy

Gene therapy is a superior therapeutic means in cancer therapy. However, the instability of nucleic acid and the lack of suitable delivery carrier greatly restricts its further development and application. Herein, we coupled low molecular weight polyethyleneimine (LMW PEI) through disulfide bonds, then modified it with manganese dioxide (MnO2) nanosheets and nuclear localization signal peptide (NLS), as a p53 gene carrier, and finally coated it with B16F10 cell membrane to construct a novel gene-carrier system CM@MnO2-PEI-NLS-ss/p53 (M@MPNs/p53). Tumor cell membrane coating endows nanoparticles with homotypic targeting and immune escape capabilities, disulfide-crosslinked LMW-PEI has high transfection efficiency and low toxicity, and NLS peptides enhance nuclear delivery and improve p53 gene delivery efficiency; meanwhile, MnO2 nanosheets oxidize high intracellular concentration of glutathione (GSH), sensitizing p53 gene-mediated antitumor therapy. The results showed that the novel biofilm-camouflaged M@MPNs/p53 nanoparticles had a highly specific targeting effect on homologous cancer cells and could effectively inhibit tumor growth in vitro and in vivo. Besides, MnO2 loading improved p53-mediated tumor regression. This novel gene delivery platform is of great significance in improving gene delivery efficiency and enhancing anti-tumor therapy.

Intracellular delivery of p53 gene and drug using cationised pullulan thiomer lowers the effective therapeutic doses of chemotherapeutic drug in cancer cells

One of the limitations associated with chemotherapy is the multidrug resistance in cancer cells. Hence higher doses are required for a therapeutic effect which lead to toxic effects. In order to address this problem, 3,3 dithiodipropionate modified pullulan, a cationic thiomer, is developed for delivery of p53 gene along with the drug doxorubicin and efflux pump inhibition. The aim is to induce synergistic effect of drug retention (Doxorubicin) and gene expression (p53) on cancer cells so as to achieve high cell death at lower drug doses. Transfection efficacy of apoptotic gene p53, drug retention kinetics, IC50, dose dependent effect of DOX on viability, influence of glutathione on drug retention and polymer mediated efflux pump inhibition were studied in different cell lines. The cationic thiomer proves to be a good transfecting agent and efflux pump inhibitor. The p53 expression, Pgp inihitory potential and drug retention could synergistically kill the cells and demonstrate very high cell death even at lower drug doses compared with drug alone thus proving the hypothesis.

Inhibition of cancer cell proliferation by adenosine triphosphate-triggered codelivery system of p53 gene and doxorubicin

The codelivery of drugs and genes by stimuli-responsive nanocarriers is a promising strategy for achieving an effective cancer treatment. In this study, an adenosine triphosphate (ATP)-responsive nanosystem was constructed to codeliver doxorubicin (DOX) and p53 gene based on an ATP-triggered aptamer and polyethyleneimine (PEI). In this system, DOX interacts with the GC-rich motif of duplex formed by the aptamer and its cDNA sequence. Then, a ternary nanocomplex DOX-Duplex/PEI/p53 was constructed using cationic carrier PEI25K for facilitating the intracellular delivery and release of p53 gene and DOX. The DOX-Duplex/PEI/p53 nanocomplex was found to possess an efficient anticancer effect, which was attributed to the ability of the system to trigger cell apoptosis and meanwhile block the cell cycle at G2 phase. The favorable antiproliferative effect was found to be associated with the rapid DOX and p53 gene release in response to the intracellular ATP concentration and the synergistic effect of therapeutic drug and gene.

A chemoenzymatically synthesized cholesterol-g-poly(amine-co-ester)-mediated p53 gene delivery for achieving antitumor efficacy in prostate cancer.

BackgroundAn amphiphilic cationic copolymer cholesterol-g-poly(amine-co-ester), namely Chol-g-PMSC-PPDL synthesized in a chemoenzymatic route has been utilized as a carrier for p53 gene delivery to check its antitumor efficacy, using human prostate cancer cell line PC-3 (p53 null) as a model.Materials and methodsThe transfection efficiency was measured by quantitative PCR and Western blotting assay. The anti-proliferative effect was detected using MTT method, colony formation assay and Live/Dead staining. The anti-migration effect was evaluated through wound healing and Transwell migration assays.ResultsThe transfection efficiency assay indicated that the carrier-mediated p53 gene transfection could dramatically enhance the intracellular p53 expression level. Through p53 gene delivery, obvious anti-proliferative effect could be detected which was elucidated to be associated with the simultaneous activation of mitochondrial-dependent apoptosis pathway and cell cycle arrest at G1 phase. Meanwhile, the anti-migration effect could be obtained after p53 gene transfection.ConclusionChol-g-PMSC-PPDL-mediated p53 gene transfection could potentially be employed as a promising strategy for achieving effective anti-tumor response.

Inhibition of proliferation and migration of tumor cells through lipoic acid-modified oligoethylenimine-mediated p53 gene delivery

Inhibition of proliferation and migration of tumor cells through lipoic acid-modified oligoethylenimine-mediated p53 gene delivery.

Inhibition of cell proliferation and migration through nucleobase-modified polyamidoamine-mediated p53 delivery.

IntroductionThe nucleobase 2-amino-6-chloropurine-modified polyamidoamine (AP-PAMAM) was used as a carrier for p53 gene delivery to achieve the antitumor effects.Methods and materialsThe condensation of p53 plasmid was studied through gel retardation assay, and the transfection efficiency was evaluated through the transfection assay of pEGFP-N3 and pGL-3 plasmids. Using human cervical carcinoma cell line HeLa as a model, the inhibition of cell proliferation and migration was studied through flow cytometry, wound healing and Transwell migration assays, respectively. The p53 expression level was detected through quantitative polymerase chain reaction and Western blotting analyses.ResultsThe carrier could condense p53 plasmid into stable nanoparticles at N/P ratios of 2.0, and higher transfection efficiency than polyamidoamine (PAMAM) could be obtained at all the N/P ratios studied. AP-PAMAM-mediated p53 delivery could achieve stronger antiproliferative effect than PAMAM/p53. The antiproliferative effect was identified to be triggered by the induction of cell apoptosis (apoptotic ratio of 26.17%) and cell cycle arrest at S phase. Additionally, AP-PAMAM/p53 transfection has been found to suppress the cell migration and invasion of cancer cells. Finally, the enhanced p53 expression level could be detected after p53 transfection at mRNA and protein levels.ConclusionThe PAMAM derivative-mediated p53 delivery could be a promising strategy for achieving tumor gene therapy.

Chemoenzymatic synthesis of a cholesterol-g-poly(amine-co-ester) carrier for p53 gene delivery to inhibit the proliferation and migration of tumor cells

An amphiphilic cholesterol-g-poly(amine-co-ester) synthesizedviaa chemoenzymatic route has been successfully applied as a carrier in p53 gene delivery.

2-Amino-6-chloropurine-modified polyamidoamine-mediated p53 gene transfection to achieve anti-tumor efficacy

The modification of 2-amino-6-chloropurine on polyamidoamine was performed to synthesize a derivative, AP-PAMAM, which was then employed as a carrier for p53 gene delivery to achieve anti-tumor efficacy.

A Gain of Function p53 Gene Mutant Promotes Growth Suppression in Human Liver Cancer Cells

Primary liver cancer is one of the most common cancer in the world with highest cancer mortality rate. The most common type of primary liver cancer is hepatocellular carcinoma (HCC). There are many risk factors for liver cancer and currently available treatments for HCC are largely inadequate. Gene mutation and dysfunction of p53 are common and is recognized as an important molecular event in hepatocarcinogenesis. Therefore, replacement of the aberrant p53 gene is an attractive approach in the treatment of HCC providing an alternative treatment for primary HCC. In this study, we assessed whether the transfection with wild-type p53 gene is able to restore the pro-apoptotic effects and evaluate the feasibility of gene therapy in fixing a faulty p53 molecule. We established a non-viral cationic lipid-based p53 gene delivery into two human HCC cell lines namely HLF and PLC/PRF/5 cells. Both cell lines have mutations in the p53 gene. We compared the results with the normal liver cell line, WRL68, that constitutively expresses the wild-type p53 gene. In this study, the introduction of wild-type p53 gene into HLF and PLC/PRF/5 cells resulted in an increased of p53 gene expression, protein expression and cells growth inhibition shown in MTS reduction cell viability assay, FITC-Annexin V and PI apoptosis assay, western blot and caspase activity assay. In summary, the study provides a promising therapeutic approach for p53 gene delivery into HCC patients. The p53 gene delivery can be instituted together with chemotherapy as a combination treatment to induce apoptosis.

Novel luminescent silica nanoparticles (LSN): p53 gene delivery system in breast cancer in vitro and in vivo.

Mutations in the p53 tumor suppressor gene are one among the most common genetic abnormalities to be described in breast cancer. However, there are a few recant reports on non-viral vector-mediated p53 gene delivery in breast cancer. A new formulation of luminescent silica nanoparticles (LSNs) for gene delivery was produced by the two-step method with slight modification. The pp53 plasmid constructs (p53-EGFP)/LSNs complexes were transfected into human breast cancer cell (MCF-7) and transfection efficiency was determined by FACS analysis. The gene expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis respectively. Further the growth inhibition through induced apoptosis with pp53-EGFP/LSNs complex were assessed by trypan blue exclusion assay and annexin V staining, respectively. Interestingly the in vivo biodistribution of plasmid DNA study revealed the occurrence was investigated by PCR and RT-PCR. The transfection efficiency of LSNs showed the highest transfection efficiency among the LSN formulation was higher than that of commercially available Lipofectin®. The LSNs-mediated transfection of the p53 gene resulted in efficient high level of wild-type p53 mRNA and protein expression levels in MCF-7 cells. Selected tissues were analyzed for any potential toxicity by histological analysis the efficient reestablishment of wild-type p53 function in breast cancer cells restored the p53 dependent apoptotic pathway. Taken together, our results reveal that cationic LSN-mediated p53 gene delivery may have potential application as a non-viral vector-mediated breast cancer gene therapy due to its effective induction of apoptosis and tumor growth inhibition.

Biodegradable Poly(aminoester)-Mediated p53 Gene Delivery for Cancer Therapy.

Gene therapy is a promising strategy in cancer treatment. However, efficient gene translation still remains challenging. In the previous work, a hydrolytically degradable poly(aminoester) with good biocompatibility was synthesized. Herein, the poly(aminoester) was explored as a vector for gene delivery and cancer therapy. The experiments revealed that the poly(aminoester) condensed plasmid DNA into nanosized particles via electrostatic interaction. The pEGFP-N1 and pGL-3 were first used as two reporter genes to study intracellular transfection. The poly(aminoester) showed higher GFP expression (33%) than PEI 25 kDa (21%). Intracellular trafficking of Cy3-labelled pGL-3 also indicated that the poly(aminoester) showed superior DNA delivery ability to nucleus compared to PEI 25 kDa. Furthermore, the therapeutic gene (p53) was translated into the breast cancer cell line (MCF-7), and then induced cell apoptosis. These results suggested that the degradable poly(aminoester) is a promising and efficient gene delivery vector for gene therapeutic applications.

Non-viral Delivery Systems for the Application in p53 Cancer Gene Therapy.

A key barrier to the development of gene therapy remains the lack of safe, efficient and easily controllable vehicles for gene delivery. The fundamental problems associated with the viral vehicles, e.g. lack of specificity and immunogenic potential, have driven the development of non-viral systems of gene delivery. In the last decade, studies on p53 gene replacement therapy have dominated the literature. Although clinical trials of p53 gene therapy have achieved limited success, it remains the only tumor suppressor gene to be evaluated formally in clinical trials for cancer treatment, with increasing focus on delivery using non-viral systems. In this article, we particularly review current investigations on p53 gene delivery using non-viral methods, including both physical and chemical approaches, with an emphasis on the latter. The existing opportunities and challenges for successful p53 cancer gene therapy are also discussed.

Induction of apoptosis in cancer cells through N-acetyl-l-leucine-modified polyethylenimine-mediated p53 gene delivery

Herein, N-acetyl-l-leucine-modified polyethylenimine was successfully constructed through the EDC/NHS-mediated coupling reaction and employed as vectors to accomplish p53 gene delivery using HeLa (p53wt) and PC-3 cells (p53null) as models. Compared with PEI25K, the derivatives exhibited lower cytotoxicity, protein adsorption and hemolytic activity, together with satisfactory pDNA condensation capability and gene transfection efficiency. After p53 transfection, MTT analysis confirmed that the cell proliferation was inhibited. Flow cytometric analysis showed that the derivative-mediated p53 delivery could induce stronger early apoptosis than PEI25K and Lipofectamine2000. Further, PC-3 cells showed higher sensitivity to the exogenous p53 transfection than HeLa cells. The mechanism for inducing apoptosis was determined to be up-regulation of p53 expression at both mRNA and protein levels using RT-PCR and western blotting analysis. Expression level and activity analysis of caspase-3, -8 and -9, and mitochondrial membrane potential measurement revealed that p53 transfection mediated by these derivatives facilitated early apoptosis of tumor cells via a mitochondria-dependent apoptosis pathway. Thus, the derivatives showed potential as biocompatible carriers for realizing effective tumor gene therapy.

A polyethylenimine derivative-based nanocarrier for the highly efficient delivery of p53 gene to inhibit the proliferation of cancer cells

A polyethylenimine derivative-based nanocarrier for the highly efficient delivery of p53 gene to inhibit the proliferation of cancer cells

N-Isopropylacrylamide-modified polyethylenimine-mediated p53 gene delivery to prevent the proliferation of cancer cells

In this paper, N-isopropylacrylamide-modified polyethylenimine (PEN) was constructed through Michael addition and employed as a carrier to achieve the p53 gene delivery, using HeLa (p53wt) and PC-3 cells (p53null) as models. After PEN-mediated p53 transfection, expression level of p53 in HeLa and PC3 cells was up-regulated at both mRNA and protein levels. Due to the exogenous p53 expression, the inhibition of cell proliferation was observed through MTT analysis, attributing to the activation of apoptosis and cell cycle arrest. Using flow cytometric analysis, early apoptotic ratios of 54.95% and 27.06% after PEN-mediated p53 transfection were detected in PC-3 and HeLa cells, respectively, indicating that PC-3 cells were more sensitive to the exogenous p53 transfection than HeLa cells. Meanwhile, G1 phase arrest was detected in PC-3 cells while a unique G2 phase arrest was identified in HeLa cells after p53 transfection. Through Western blotting, activity analysis of caspase-3, caspase-8 and caspase-9 and mitochondrial membrane potential measurement, the apoptosis induced by PEN-mediated p53 transfection was conducted in a mitochondria-dependent apoptosis pathway. These results demonstrated that PEN could successfully mediate the p53 gene delivery and up-regulate the cellular p53 expression level, triggering a significant p53-dependent anti-proliferative effect on tumor cells.

Hyaluronic acid conjugated β-cyclodextrin-oligoethylenimine star polymer for CD44-targeted gene delivery

A new CD44-targeted gene delivery system, the star-shaped cationic polymer containing a β-cyclodextrin (β-CD) core and multiple branched oligoethylenimine (OEI) arms with conjugated oligomer of hyaluronic acid (HA), was synthesized by reductive amination between β-CD-OEI star polymer and HA, and was characterized for pDNA condensation and nanoparticle formation, followed by evaluation for targeted gene delivery of luciferase reporter gene and wild type p53 gene in CD44-positive and CD44-negative cell lines. The β-CD-OEI-HA polymer contained 6 arms of OEI (600Da) and a short HA segment. It could fully condense pDNA to form nanoparticles with sizes ranging from 100 to 200nm at N/P ratios of 8 or higher. The conjugation of HA reduced cytotoxicity of β-CD-OEI-HA/pDNA polyplexes. It was found that CD44 receptor was highly expressed and localized at the membrane of MDA-MB-231 breast cancer cell line, while no CD44 was found at the membrane of MCF-7 epithelial cell line. Compared with PEI (25kDa) and β-CD-OEI star polymers, β-CD-OEI-HA demonstrated significant increased gene transfection efficiency in MDA-MB-231 cells, while such effect was absent in MCF-7 cells. The targeted delivery of wild type p53 gene by β-CD-OEI-HA in MDA-MB-231 cells resulted in an increased cell cycle arrest at sub-G1 phase.

P0059 Induction of apoptosis in breast cancer cells through combined intracellular delivery of a tumour-suppressor gene and small interfering RNAs against growth factor receptor and anti-apoptotic genes

Background The mechanisms underlying the development of breast cancer are complex and vary among individual tumours. These mechanisms include genetic and epigenetic alterations and resulting changes in the activity of signalling pathways. Current treatment strategies for metastatic breast cancer (MBC) depend upon patient and tumour classification; menopausal status, hormone receptor status, and HER2 status can all be considered, as can site of metastatic disease (bone or soft tissue). The choice of treatment in metastatic disease usually involves systemic endocrine therapy or cytotoxic chemotherapy, with an anti-HER2 agent when appropriate. Novel agents are needed because many of the current therapies have limitations. These include drug resistance, lack of target receptor expression in tumours (e.g., only 25% of breast cancer tumours have HER2 expression), lack of specificity, and relatively small improvements in survival. Advances in the understanding of the aetiology and biology of breast cancer have identified key targets among the multiple signalling pathways involved in the development, growth, and survival of breast cancer cells. As such, targeted therapies are among the most promising new agents for the treatment of breast cancer. Methods The main objective of the project is the restoration of p53 activity and the silencing of HER2, EGFR, and IGF1 genes through the delivery of normal p53 gene and specific validated siRNA, respectively, using carbonate apatite nanoparticles both in vitro and in vivo. Concurrently, the sensitivity of breast cancer cells to the traditional anti-cancer drugs, such as doxorubicin, cisplatin, and paclitaxel will be tested following individually and combined delivery p53 gene and the siRNAs against both growth factor receptor genes and anti-apoptotic genes. Findings The in vitro analysis of siRNAs together and in combination with p53 and anticancer drugs helped to identify potential targets for the treatment of breast cancer. Interpretation Preclinical studies in animal models of breast cancer should be done through tumour-targeted delivery of siRNA in combination with passively diffusible anti-cancer drugs and p53.

Intracellular Delivery of ERBB2 siRNA and p53 Gene Synergistically Inhibits the Growth of Established Tumor in an Immunocompetent Mouse

Breast cancer is one of the leading causes of deaths worldwide in women with hormone therapy, chemotherapy, targeted therapies, or their combinations being the current options for treating the disease at the different stages (stages I-III) with associated side-effects or increasing life-span at the advanced stage (stage IV). Small interfering RNA (siRNA) as an effective tool to selectively knockdown of a particular gene could be harnessed in combination with plasmid DNA (carrying a gene of interest) and conventional anti-cancer drugs for precisely treating breast cancer with minimal side effects. However the limitation of the naked siRNA and DNA in penetrating the plasma membrane and their sensitiveness to nuclease-mediated cleavage render the technology rather complex in therapeutic intervention. Recently, we have developed pH-sensitive carbonate apatite as a potential nano-carrier to efficiently deliver siRNA or DNA across the cell membrane and facilitate them to escape endosomal acidic compartment resulting in specific cleavage of a particular mRNA transcript or expression of a desirable protein, respectively. Moreover, we demonstrated nanoparticle-assisted delivery of the siRNAs targeting cyclin B1, PLCgamma- 2/calmodulin1, NFκB1/NFκB2, ABCG2/ABCB1 and cROS1 mRNAs sensitizes cervical adenocarcinoma and breast cancer cells towards traditional anti-cancer drugs. Here, we report that co-delivery of the siRNA targeting HER2/ErbB2 gene transcript and p53 gene with the help of carbonate apatite nanoparticles synergistically induces inhibition of growth/proliferation of breast cancer cell lines as well as regression of the breast tumor induced in Balb/c mice. Additionally, combined delivery of nanoparticle-associated HER2/ErbB2 siRNA and p53 gene apparently slows down the growth of the established tumor in presence of doxorubicin or paclitaxel compared with the individual free drugs. Thus, the combination of HER2/ErbB2 knockdown and restoring of normal p53 function could be a highly promising approach necessitating further studies through pre-clinical trials with different models of breast cancer to establish the therapeutic role of this combination therapy prior to conducting clinical trials in breast cancer patients.

P53 mediated apoptosis by reduction sensitive shielding ternary complexes based on disulfide linked PEI ternary complexes

Reduction-sensitive hyaluronic acid derivatives (HA–SS–COOH) were shielded on the DNA/polyethylenimine (PEI) to construct ternary complexes (DNA/PEI/HA–SS–COOH, DPS ternary complexes) with efficient gene transfection. Details studied were conducted to investigation of factors influencing transfection efficiency, including the gene compression by fluorescence resonance energy transfer (FRET) spectrum and the intracellular fate of fluorescent labeled complexes by the confocal laser scanning microscope (CLSM). In the FRET study, DPS complexes were found to enhance condensation of DNA in preparation, while timely loosen gene under exposure to reductive reagent. Similar cellular uptake levels were observed for the designed reduction sensitive complexes and the stable one (DNA/PEI/HA, DPH ternary complexes), but the intracellular process was strikingly different for the two types of complexes. Only DPS showed obvious desired intracellular deshielding and endosomal escape, which contributed to highly efficient gene delivery. After loading with p53 plasmid, DPS complexes achieved significantly up-regulated p53 tumor suppressor gene expression at both mRNA and protein levels, as revealed by quantitative polymerase chain reaction (qPCR) and western blot investigations. Transgene induced apoptosis was evaluated by propidium iodide staining and flow cytometry analysis of cell cycle. Tumor cells transfected by DPS complexes containing p53 gene displayed almost 50% higher suppression in proliferation compared to those untreated cells, accompanied with a 46% elevation in the number of cells at sub-G1 phase and remarkable p53 dependent cell cycle perturbations prior to apoptosis. These results demonstrated that targeted delivery of p53 gene via reduction-sensitive DPS ternary complexes enabled up-regulated cellular p53 mRNA level through the exogenous p53 gene, inducing a significant p53-dependent anti-proliferative effect on tumor cells, which could be effective means of cancer treatment.

Supramolecular self-assembly forming a multifunctional synergistic system for targeted co-delivery of gene and drug

For developing a multifunctional bioreducible targeted and synergistic co-delivery system for anticancer drug paclitaxel (PTX) and p53 gene for potential cancer therapy, supramolecular self-assembled inclusion complex was prepared from PTX and star-shaped cationic polymer containing γ-cyclodextrin (γ-CD) and multiple oligoethylenimine (OEI) arms with folic acid (FA) conjugated via a disulfide linker. The inclusion complex, termed as γ-CD-OEI-SS-FA/PTX, was formed between PTX and the hydrophobic cavity of γ-CD core of the star polymer. The γ-CD-OEI-SS-FA/PTX complex further formed polyplexes with pDNA to give positively charged nanoparticles, becoming multifunctional supramolecular self-assembled co-delivery system for PTX and pDNA targeting to cancer cells that overexpress folate receptors (FRs). The results showed that the FA-targeted function induced higher gene transfection efficiency in the FR-positive KB cells. The redox-sensitive disulfide linker in the self-assembly system led to the detachment of the FA groups from the carrier after the FR-mediated endocytosis, which resulted in the release of the bound FRs followed by the recycling of the FRs from the cytosol onto the cell membrane surface, facilitating continuous FR-mediated endocytosis to achieve enhanced gene transfection. In addition, the complexed PTX was co-delivered to the cells with pDNA, which further enhanced the gene transfection even at low N/P ratios in the FR-positive KB cells. Further, the efficient delivery of wild-type p53 gene resulted in large cell population at sub G1 and G2/M phases, inducing significant cell apoptosis. Therefore, the multifunctional γ-CD-OEI-SS-FA/PTX self-assembly system with the synergistic effects of redox-sensitive FA-targeted and PTX-enhanced p53 gene delivery may be promising for cancer therapeutic application.