RESUMEN
To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.
Asunto(s)
Cloroquina/análogos & derivados , Cloroquina/farmacología , Expresión Génica/efectos de los fármacos , Transgenes/efectos de los fármacos , Línea Celular Tumoral , Ciclodextrinas/química , ADN/administración & dosificación , ADN/genética , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Cambio de Movilidad Electroforética , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Luciferasas/genética , Relación Estructura-ActividadRESUMEN
Short nucleic acid sequences specific to oncogene targets such as bcl-2, bcr-abl, and c-myc have been shown to exhibit specific anti-cancer activity in vitro through antigene or antisense activity. Efficient in vivo delivery of oligonucleotides remains a major limitation for the therapeutic application of these molecules. We report herein on the preparation of transferrin-modified nanoparticles containing DNAzymes (short catalytic single-stranded DNA molecules) for tumor targeting as well as their biodistribution using various methods of administration in the mouse. Linear, beta-cyclodextrin-based polymers are complexed with DNAyzme molecules to form sub-50 nm particles termed "polyplexes". The surface properties of the cyclodextrin-containing polyplexes are modified by exploiting the ability of the beta-cyclodextrin substructure and adamantane to form inclusion complexes. Accordingly, conjugates of adamantane with poly(ethylene glycol) (PEG) are prepared and combined with the polyplexes. The adamantane form inclusion complexes with the surface cyclodextrins of the polyplexes to provide a sterically stabilizing layer of PEG. The stabilized polyplexes are also modified with transferrin for increasing targeting to tumor cells expressing transferrin receptors. The preparation, characterization, and in vitro application of these nanoparticles are discussed. The transferrin-polyplexes containing fluorescently-labeled DNAzyme molecules are administered to tumor-bearing nude mice and their biodistribution and clearance kinetics are monitored using a fluorescence imaging system. Four methods of administration are studied: intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. DNAzymes packaged in polyplex formulations are concentrated and retained in tumor tissue and other organs, whereas unformulated DNAzyme is eliminated from the body within 24 hours post-injection. Intravenous and intraperitoneal bolus injections result in the highest fluorescent signal (DNAzyme) at the tumor site. Tumor cell uptake is observed with intravenous bolus injection only, and intracellular delivery requires transferrin targeting.
Asunto(s)
Ciclodextrinas/química , ADN Catalítico/genética , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Neoplasias/terapia , Polietilenglicoles/farmacocinética , Transferrina/química , Adamantano/metabolismo , Animales , ADN Catalítico/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Infusiones Intravenosas , Infusiones Parenterales , Masculino , Ratones , Neoplasias/genética , Plásmidos/administración & dosificación , Plásmidos/química , Plásmidos/genética , Polietilenglicoles/química , Transferrina/metabolismoRESUMEN
Transferrin is a well-studied ligand for tumor targeting due to upregulation of transferrin receptors in numerous cancer cell types. Here, we report the development of a transferrin-modified, cyclodextrin polymer-based gene delivery system. The delivery system is comprised of a nanoparticle (formed by condensation of a cyclodextrin polycation with nucleic acid) that is surface-modified to display poly(ethylene glycol) (PEG) for increasing stability in biological fluids and transferrin for targeting of cancer cells that express transferrin receptor. A transferrin-PEG-adamantane conjugate is synthesized for nanoparticle modification. The transferrin conjugate retains high receptor binding and self-assembles with the nanoparticles by adamantane (host) and particle surface cyclodextrin (guest) inclusion complex formation. At low transferrin modification, the particles remain stable in physiologic salt concentrations and transfect K562 leukemia cells with increased efficiency over untargeted particles. The increase in transfection is eliminated when transfections are conducted in the presence of excess free transferrin. The transferrin-modified nanoparticles are appropriate for use in the systemic delivery of nucleic acid therapeutics for metastatic cancer applications.
Asunto(s)
Ciclodextrinas/química , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Transferrina/química , Adamantano/química , Secuencia de Carbohidratos , Carbohidratos/química , Línea Celular Tumoral , Portadores de Fármacos/metabolismo , Terapia Genética , Vectores Genéticos , Humanos , Células K562 , Lisina/química , Masculino , Datos de Secuencia Molecular , Estructura Molecular , Ácidos Nucleicos/metabolismo , Polietilenglicoles/química , Polietilenglicoles/farmacología , Neoplasias de la Próstata/metabolismo , Transfección , Transferrina/metabolismoRESUMEN
The localized, sustained delivery of growth factors for wound healing therapy is actively being explored by gene transfer to the wound site. Biocompatible matrices such as bovine collagen have demonstrated usefulness in sustaining gene therapy vectors that express growth factors in local sites for tissue repair. Here, new synthetic biocompatible materials are prepared and shown to deliver a protein to cultured cells via the use of an adenoviral delivery vector. The synthetic construct consists of a linear, beta-cyclodextrin-containing polymer and an adamantane-based cross-linking polymer. When the two polymers are combined, they create an extended network by the formation of inclusion complexes between the cyclodextrins and adamantanes. The properties of the network are altered by controlling the polymer molecular weights and the number of adamantanes on the cross-linking polymer, and these modifications and others such as replacement of the beta-cyclodextrin (host) and adamantane (guest) with other cyclodextrins (hosts such as alpha, gamma, and substituted members) and inclusion complex forming molecules (guests) provide the ability to rationally design network characteristics. Fibroblasts exposed to these synthetic constructs show proliferation rates and migration patterns similar to those obtained with collagen. Gene delivery (green fluorescent protein) to fibroblasts via the inclusion of adenoviral vectors in the synthetic construct is equivalent to levels observed with collagen. These in vitro results suggest that the synthetic constructs are suitable for in vivo tissue repair applications.
Asunto(s)
Materiales Biocompatibles/síntesis química , Ciclodextrinas/síntesis química , Marcación de Gen/métodos , Piel/efectos de los fármacos , Cicatrización de Heridas/genética , Materiales Biocompatibles/administración & dosificación , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclodextrinas/administración & dosificación , Ciclodextrinas/genética , Sistemas de Liberación de Medicamentos/métodos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Piel/citología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.