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1.
Colloids Surf B Biointerfaces ; 161: 519-527, 2018 Jan 01.
Article in English | MEDLINE | ID: mdl-29128838

ABSTRACT

This study performed a biophysical characterization (electrochemistry, structure and morphology) and assessment of the biological activity and cell biocompatibility of GCL/DOPE-pDNA lipoplexes comprised of plasmid DNA and a mixed lipid formed by a DOPE zwitterionic lipid and a gemini cationic lipid N-N'-(1,3-phenylene bis (methylene)) bis (N,N-dimethyl-N-(1-dodecyl) ammonium dibromide (12PH12) containing an aromatic spacer or its monomeric counterpart surfactant, N-benzyl-N,N-dimethyl-N-(1-dodecyl) ammonium bromide (12PH). Electrochemical results reveal that i) the gemini cationic lipid (12PH12) and the plasmid pDNA yield effective charges less than their nominal charges (+2 and -2/bp, respectively) and that ii) both vectors (12PH12/DOPE and 12PH/DOPE) could compact pDNA and protect it from DNase I degradation. SAXS and cryo-TEM experiments indicate the presence of a lamellar lyotropic liquid crystal phase represented as alternating layers of mixed lipid and plasmid. Transfection efficiency (by FACS and luminometry) and cell viability assay in COS-7 cells, performed with two plasmid DNAs (pEGFP-C3 and pCMV-Luc VR1216), confirm the goodness of the proposed formulations (12PH12/DOPE and 12PH/DOPE) to transport genetic material, with efficiencies and biocompatibilities comparable to or better than those exhibited by the control Lipofectamine 2000*. In conclusion, although major attention has been paid to gemini cationic lipids in the literature, due to the large variety of modifications that their structures may support to improve the biological activity of the resulting lipoplexes, it is remarkable that the monomeric counterpart surfactant with an aromatic group analyzed in the present work also exhibits good biological activity. The in vitro results reported here indicate that the optimum formulations of the gene vectors studied in this work efficiently transfect plasmid DNA with very low toxicity levels and, thus, may be used in forthcoming in vivo experiments.


Subject(s)
DNA/genetics , Drug Carriers/chemistry , Lipids/chemistry , Nanoparticles/chemistry , Transfection/methods , Animals , COS Cells , Cations/chemistry , Chlorocebus aethiops , Cryoelectron Microscopy , DNA/chemistry , Liposomes/chemistry , Microscopy, Electron, Transmission , Nanoparticles/ultrastructure , Phosphatidylethanolamines/chemistry , Plasmids/chemistry , Plasmids/genetics , Scattering, Small Angle , X-Ray Diffraction
2.
Nanomedicine (Lond) ; 11(5): 465-77, 2016.
Article in English | MEDLINE | ID: mdl-26892017

ABSTRACT

AIM: Development of EGF-liposomes (LP-EGF) for selective molecules delivery in tumors expressing EGFR. MATERIAL & METHODS: In vitro cellular interaction of EGF-LP and nontargeted liposomes (LP-N) was assayed at 37 and 4 °C in cells expressing different EGFR levels. Receptor-mediated uptake was investigated by competition with a monoclonal antibody anti-EGFR. Selective intracellular drug delivery and efficacy was tested by oxaliplatin encapsulation. In vivo biodistribution of LP-N and LP-EGF was done in xenograft model. RESULTS: LP-EGF was internalized by an active and selective mechanism through EGFR without receptor activation. Oxaliplatin LP-EGF decreased IC50 between 48 and 13% in cell EGFR+. LP-EGF was accumulated in tumor over 72 h postdosing, while LP-N in spleen. CONCLUSION: LP-EGF represents an attractive nanosystem for cancer therapy or diagnosis.


Subject(s)
Colorectal Neoplasms/drug therapy , Drug Delivery Systems , Epidermal Growth Factor/genetics , ErbB Receptors/genetics , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Epidermal Growth Factor/administration & dosage , Epidermal Growth Factor/chemistry , ErbB Receptors/antagonists & inhibitors , Humans , Liposomes/administration & dosage , Liposomes/chemistry , Mice , Molecular Targeted Therapy , Organoplatinum Compounds/administration & dosage , Organoplatinum Compounds/chemistry , Oxaliplatin , Xenograft Model Antitumor Assays
3.
Curr Pharm Des ; 21(29): 4193-200, 2015.
Article in English | MEDLINE | ID: mdl-26323430

ABSTRACT

Gene therapy, currently, represents one of the new therapies that have emerged for the treatment of different diseases. Between the different approaches that can be chosen concerning this therapy, gene delivery with non-viral systems has focused the attention during the past decades, because of the reduced toxicity compared to the viral systems. Non-viral vectors are formed by a group of different molecules with a wide variety of sources and features. Among them, polymeric systems have been extensively studied due to the ability to form nanoparticles in the presence of nucleic acids, protect the included nucleic acid and more importantly, improve the entrance of the desired nucleic acid fragment into the cell and hopefully achieve a therapeutic effect. In this review, some of the most used polymeric systems are commented with the main characteristics that can influence the activity of each of them, such as the molecular weight, N/P ratio (positive charges of cationic polymer/negative charges of pDNA), stability or the molecular structure.


Subject(s)
Gene Transfer Techniques , Nanoparticles/administration & dosage , Genetic Vectors/administration & dosage , Humans , Nanoparticles/therapeutic use , Polymers/administration & dosage , Polymers/pharmacokinetics
4.
Nanomedicine ; 10(2): 411-9, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23928214

ABSTRACT

The clinical application of gene silencing mediated by small interfering RNA (siRNA) has been limited by the lack of efficient and safe carriers. Phospholipid modification of low molecular weight polyethylenimine (PEI 1.8 kDa) dramatically increased its gene down-regulation capacity while keeping cytotoxicity levels low. The silencing efficacy was highly dependent on the nature of the lipid grafted to PEI and the polymer/siRNA ratio employed. Phosphoethanolamine (DOPE and DPPE) and phosphocholine (PC) conjugation did not change the physicochemical properties and siRNA binding capacity of PEI complexes but had a large impact on their transfection and ability to down-regulate Green Fluorescent Protein (GFP) expression (60%, 30% and 5% decrease of GFP expression respectively). We found that the micelle-forming structure of DOPE and DPPE-PEI dramatically changed PEI's interaction with cell membranes and played a key role in promoting PEI 1.8 kDa transfection, completely ineffective in the absence of the lipid modification. FROM THE CLINICAL EDITOR: While siRNA-based gene silencing methods could have numerous clinical applications, efficient delivery remains a major challenge. This team reports that DOPE-PEI and DPPE-PEI based micelle-forming nanostructures may be able to provide an efficient vector for siRNA transfection.


Subject(s)
Lipids/chemistry , Nanoparticles/chemistry , Phospholipids/chemistry , Polyethyleneimine/chemistry , RNA Interference , Animals , Cell Line , Cell Membrane/metabolism , Drug Carriers/chemistry , Ethanolamines/chemistry , Green Fluorescent Proteins/metabolism , Humans , Inhibitory Concentration 50 , Mice , Micelles , Molecular Weight , Nanomedicine , Phosphorylcholine/chemistry , RNA, Small Interfering/chemistry
5.
Eur J Pharm Biopharm ; 85(3 Pt A): 390-7, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23811437

ABSTRACT

AIM: Development and evaluation of a new targeted gene delivery system by first preforming self-assembled nanocomplexes from a polycationic amphiphilic cyclodextrin (paCD) and pDNA and then decorating the surface of the nanoparticles with folic acid (FA). EXPERIMENTAL SECTION: The cyclodextrin derivative (T2) is a tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups at the primary face of a cyclomaltoheptaose (ß-CD) core and 14 hexanoyl chains at the secondary face. RESULTS AND CONCLUSIONS: T2 complexed and protected pDNA (luciferase-encoding plasmid DNA, pCMVLuc) and efficiently mediated transfection in vitro and in vivo with no associated toxicity. The combination of folic acid with CDplexes afforded ternary nanocomplexes (Fol-CDplexes) that enhanced significantly the transfection activity of pCMVLuc in human cervix adenocarcinoma HeLa cells, especially when formulated with 1 µg FA/µg DNA. The observed transfection enhancement was associated to specific folate receptor (FR)-mediated internalization of Fol-CDplexes, as corroborated by employing a receptor-deficient cell line (HepG2) and an excess of free folic acid. The in vivo studies, including luciferase reporter gene expression and biodistribution, indicated that 24h after intravenous administration of the T2-pDNA nanocomplexes, transfection takes part mainly in the liver and partially in the lung. Interestingly, the corresponding Fol-CDplexes lead to an increase in the transfection activity in the lung and the liver compared to non-targeted CDplexes. Folate-CDplexes developed in this study have improved transfection efficiency and although various methods have been used for the preparation of ligand-DNA-complexes, covalent binding is usually needed and insoluble aggregates are formed unless the concentration of the components is minimized. However, the complexes developed by first time in this work were prepared by simple mixing. The synthetic nature of this formulation provides the potential of flexibility in terms of composition and the capability of inexpensive and large-scale production of the complexes. These nanovectors may be an adequate alternative to viral vectors for gene therapy in the future.


Subject(s)
DNA/administration & dosage , Folic Acid/chemistry , Gene Transfer Techniques , Genetic Therapy/methods , Cyclodextrins/chemistry , Female , Folate Receptors, GPI-Anchored/metabolism , Genes, Reporter/genetics , HeLa Cells , Hep G2 Cells , Humans , Liver/metabolism , Luciferases/genetics , Lung/metabolism , Nanoparticles , Plasmids , Polyamines/chemistry , Polyelectrolytes , Tissue Distribution , Transfection
6.
Methods Enzymol ; 509: 355-67, 2012.
Article in English | MEDLINE | ID: mdl-22568915

ABSTRACT

Cationic liposome-DNA complexes (lipoplexes) are used for the delivery of plasmid DNA to cultured cells and various tissues in vivo. In this chapter, we describe the preparation and evaluation of plain and targeted lipoplexes, using targeting ligands, including epidermal growth factor and transferrin. Ligand-associated lipoplexes may be used to target DNA or other nucleic acid drugs to specific cells, particularly cancer cells that overexpress the receptors for the ligands. We provide examples of the enhancement of gene expression mediated by epidermal growth factor in murine and human oral squamous cell carcinoma cells, and human hepatoblastoma and rat colon adenocarcinoma cells. We also summarize the studies on the use of transferrin-lipoplexes for enhancing gene delivery to cervical carcinoma, murine colon carcinoma, and African green monkey kidney cells. We outline two animal models in which transferrin-lipoplexes have been used for antitumor therapy by delivering either the gene encoding interleukin-12 or a suicide gene: a CT26 murine colon carcinoma, and a syngeneic, orthotopic murine oral squamous cell carcinoma.


Subject(s)
Liposomes/chemistry , Nanocapsules/chemistry , Transfection , Animals , Cell Line, Tumor , Cell Survival , Chlorocebus aethiops , Epidermal Growth Factor/genetics , Female , Genes, Reporter , Genes, Transgenic, Suicide , Genetic Therapy , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Interleukin-12/biosynthesis , Interleukin-12/genetics , Luciferases/biosynthesis , Luciferases/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Nanomedicine , Neoplasm Transplantation , Neoplasms/therapy , Plasmids/genetics , Rats , Transferrin/genetics
7.
Nanomedicine (Lond) ; 6(10): 1697-707, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22122582

ABSTRACT

AIM: In this study, a set of polycationic amphiphilic cyclodextrins featuring self-assembling capabilities in the presence of nucleic acids have been evaluated as therapeutic gene vectors for in vivo purposes. MATERIALS & METHODS: A tetradecacationic structure incorporating 14 primary amino groups and 7 thioureido groups in the primary face of the cyclooligosaccharide core and 14 hexanoyl chains in the secondary face was judged to be optimal for therapeutic gene delivery. RESULTS & CONCLUSION: This compound efficiently mediated serum-resistant transfection in HeLa and HepG2 cells, comparing favorably with branched poly(ethyleneimine), with a low associated toxicity. Further transfection experiments using an encoding therapeutic gene plasmid (pCMVIL-12) were effected to report expression levels of IL-12. Finally, in vivo gene delivery experiments by systemic injection in mice indicated relatively high transfection levels in the liver, overcoming trapping of the nanoparticles in lung cells, with low toxicity.


Subject(s)
Cyclodextrins/chemistry , DNA/metabolism , Gene Transfer Techniques/instrumentation , Nanoparticles/chemistry , Plasmids/metabolism , Polyamines/chemistry , Animals , Cell Line , Cell Survival/drug effects , Cyclodextrins/chemical synthesis , Cyclodextrins/metabolism , Cyclodextrins/toxicity , DNA/genetics , Humans , Mice , Plasmids/genetics , Polyamines/chemical synthesis , Polyamines/metabolism , Polyamines/toxicity , Polyelectrolytes , Transfection/methods
8.
Nanomedicine (Lond) ; 6(1): 89-98, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21182421

ABSTRACT

AIMS: In this work, we have evaluated the ability of targeted lipoplexes to enhance transgene expression in EGF receptor (EGFR) overexpressing tumor cells by using lipoplexes. MATERIALS & METHODS: We prepared DOTAP/cholesterol liposomes modified with EGF at 0.5/1, 1/1, 2/1 and 5/1 lipid/DNA (+/-) charge ratio by sequentially mixing the liposomes with the ligand and adding the reporter or the therapeutic plasmid gene, pCMVLuc (pVR1216) or pCMVIL12, respectively. HepG2, DHDK12proB and SW620 cells were used for in vitro experiments, which were performed in the presence of 60% serum. RESULTS: The characterization of EGF-lipoplexes indicated a size close to 300 nm and a variable net surface charge as a function of the amount of EGF associated to the cationic liposomes. EGF-lipoplexes, which showed an increased transfection activity, were positively charged, noncytotoxic and highly effective in protecting DNA from DNase I attack. Transfection activity in vitro resulted in an enhancement in the luciferase and IL-12 expression by EGF-lipoplexes compared with those without ligand (plain-lipoplexes) and to naked DNA. The results observed in SW620 cells, which are deficient in EGFR, confirmed that DNA uptake was predominantly via EGFR-mediated endocytosis. In vivo transfection activity was confirmed by luciferase imaging in living mice. Bioluminiscence could be detected mainly in the lung with a maximum signal 24 h after application. The resulting EGF-lipoplexes significantly increased the level of gene expression in mice compared with control or naked DNA. CONCLUSION: These findings indicate that these nanovectors may be an adequate alternative to viral vectors for gene therapy.


Subject(s)
DNA/administration & dosage , Epidermal Growth Factor/metabolism , Liposomes/chemistry , Transfection/methods , Animals , Cell Line, Tumor , DNA/metabolism , Deoxyribonucleases/metabolism , Epidermal Growth Factor/chemistry , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Genetic Therapy , Genetic Vectors , Humans , Interleukin-12/genetics , Mice , Mice, Inbred BALB C , Neoplasms/therapy , Up-Regulation
9.
J Control Release ; 146(1): 99-105, 2010 Aug 17.
Article in English | MEDLINE | ID: mdl-20438778

ABSTRACT

Polyplexes consisting of a standard CMV promoter driven luciferase plasmid condensed with PAMAM starburst dendrimers (generation 4 and 5) efficiently transfected tumor cells in vitro. Tail vein injection of PAMAM polyplexes into immune competent mice bearing subcutaneous, well vascularized murine neuroblastoma tumors (Neuro2A) led to predominant luciferase reporter gene expression in the tumor, and negligible transgene expression levels in other organs. Repeated PAMAM polyplex applications were well tolerated and prolonged transgene expression in the tumor. In vivo imaging studies using polyplexes fluorescently labeled with near infrared emitting semiconductor quantum dots (quantoplexes) revealed lung accumulation for both PAMAM and linear PEI (LPEI) based polyplexes, but only LPEI polyplexes induced high luciferase expression in lung, demonstrating that biodistribution and transgene expression of polyplexes does not necessarily correlate. With a luciferase plasmid devoid of immune modulatory CpG sequences and a combination of human CMV enhancer and human elongation factor 1 alpha promoter elements, Neuro2A tumor transgene expression after a single intravenous injection of generation 5 PAMAM polyplexes was observed for up to 1week as measured by luciferase bioluminescence imaging. Utilizing a human xenograft model (HUH7) in immune compromised nude mice, a low level of luciferase activity in the tumor area was observed after systemic PAMAM polyplex application.


Subject(s)
CpG Islands/genetics , Dendrimers/chemistry , Gene Expression Regulation, Neoplastic , Gene Transfer Techniques , Genetic Therapy/methods , Neuroblastoma/therapy , Plasmids/genetics , Transgenes , Animals , Cell Line, Tumor , Cell Survival , Fluorescent Dyes , Humans , Luciferases/genetics , Luminescent Measurements , Mice , Mice, Nude , Neuroblastoma/genetics , Neuroblastoma/pathology , Plasmids/administration & dosage , Plasmids/chemistry , Transfection , Xenograft Model Antitumor Assays
10.
J Gene Med ; 11(1): 38-45, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19021130

ABSTRACT

BACKGROUND: Transgene expression in vivo for therapeutic purposes will require methods that allow for efficient gene transfer into cells. Although current vector technologies are being improved, the development of novel vector systems with improved targeting specificity, higher transduction efficiencies and improved safety is necessary. METHODS: Asialoglycoprotein receptor-targeted cationic nanoparticles for interleukin (IL)-12 encapsulation (NP1) or adsorption (NP2) have been formulated by blending poly(D,L-lactic-co-glycolic) acid (PLGA) (50 : 50) with the cationic lipid 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and the ligand asialofetuin (AF), by using a modified solvent evaporation process. RESULTS: We present a novel targeted lipopolymeric vector, which improves significantly the levels of luciferase gene expression in the liver upon i.v. administration. Targeted-NP2 particles showed a five- and 12-fold higher transfection activity in the liver compared to non-targeted (plain) complexes or naked pCMV DNA, respectively. On the other hand, BNL tumor-bearing animals treated with AF-NP1 containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice, without signs of recurrence. High levels of IL-12 and interferon-gamma were detected in the sera of treated animals. Mice survival also improved considerably. Tumor treatment with AF-NP2 formulations lead only to a retardation in the tumor growth. CONCLUSIONS: In the present study, we have developed an efficient targeted non-viral vector for IL-12 gene transfer in hepatocellular carcinoma in vivo, by employing non-toxic cationic PLGA/DOTAP/AF nanoparticles. These results demonstrate for the first time that this cationic system could be used successfully and safely for delivery of therapeutic genes with antitumor activity into liver tumors with targeting specificity.


Subject(s)
Carcinoma, Hepatocellular/therapy , Interleukin-12/genetics , Liver Neoplasms/therapy , Nanoparticles/chemistry , Animals , Asialoglycoproteins/administration & dosage , Asialoglycoproteins/chemistry , Fatty Acids, Monounsaturated/administration & dosage , Fatty Acids, Monounsaturated/chemistry , Female , Fetuins , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Lactic Acid/administration & dosage , Lactic Acid/chemistry , Mice , Mice, Inbred BALB C , Nanoparticles/administration & dosage , Particle Size , Polyglycolic Acid/administration & dosage , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Quaternary Ammonium Compounds/administration & dosage , Quaternary Ammonium Compounds/chemistry , Transgenes , Tumor Cells, Cultured , alpha-Fetoproteins/administration & dosage , alpha-Fetoproteins/chemistry
11.
Eur J Pharm Biopharm ; 68(3): 503-12, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17881198

ABSTRACT

Biodegradable lactic acid-glycolic acid copolymer (PLGA) formulations incorporating cisplatin have been developed to evaluate the cytotoxicity of this agent in cultured cells. Two different W/O/W protocols were used to formulate micro- (MP) and nanoparticles (NP) under the solvent evaporation method. Although the amount of cisplatin encapsulated was higher in the MP, the efficiency of encapsulation was similar: 10.33% vs. 11.23%, for both MP and NP, respectively. The "in-vitro" release profiles displayed a significant difference in the initial burst effect, which had a significant impact in the antiproliferative effect of cisplatin. In addition, a duality in the cell cycle distribution was found for both formulations and low doses of free cisplatin (2.5, 10 microM) in comparison with the high doses of free cisplatin. The 50 microM caused a rapid inhibition of cells growth followed by a significant loss of cells in phases G0/G1 and G2/M which correlated with an increase in the number of cells in sub-G1. However, cisplatin released from controlled formulations induced an accumulation of cells in the phase G2/M. These results led to enhance the caspase-3 activity for MP and NP. These findings indicate that controlled release formulations of cisplatin are able to induce a more effective apoptosis than free cisplatin.


Subject(s)
Cisplatin/administration & dosage , Cisplatin/pharmacology , Lactic Acid/administration & dosage , Polyglycolic Acid/administration & dosage , Animals , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/chemistry , Delayed-Action Preparations , Nanoparticles , Polylactic Acid-Polyglycolic Acid Copolymer , Rats
12.
Curr Med Chem ; 10(14): 1213-20, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12678795

ABSTRACT

Cationic liposome-DNA complexes, also called "lipoplexes", constitute a potentially viable alternative to viral vectors for the delivery of therapeutic genes. Here we review the mechanisms of lipoplex-mediated gene delivery, the barriers to efficient gene expression, and novel cationic lipids used for transfection. We also describe methods for enhancing gene transfer via the use of proteins, including transferrin, albumin and asialofetuin, and synthetic peptides, including GALA and nuclear localization signal peptides. We underscore the importance of understanding the mechanisms of cytoplasmic and nuclear entry of DNA and its dissociation from lipoplexes. We emphasize that the in vitro transfection activity of new lipoplex constructs should be tested in the presence of high serum concentrations to emulate in vivo conditions.


Subject(s)
Drug Delivery Systems/methods , Genetic Therapy/methods , Liposomes/administration & dosage , Animals , Cations , Chemistry, Pharmaceutical , Humans , Lipids/administration & dosage , Lipids/genetics , Peptides/administration & dosage , Peptides/genetics , Proteins/administration & dosage , Proteins/genetics
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