RESUMEN
The membrane-bound folate receptor (FR) is overexpressed on a wide range of human cancers, such as those originating in ovary, lung, breast, endometrium, kidney and brain. The vitamin folic acid is a high affinity ligand of the FR which retains its receptor binding properties when conjugated to other molecules. Consequently, "folate targeting" technology has successfully been applied for the delivery of protein toxins, chemotherapeutic agents, radio-imaging and therapeutic agents, MRI contrast agents, liposomes, gene transfer vectors, antisense oligonucleotides, ribozymes and immunotherapeutic agents to FR-positive cancers. These folate-bearing delivery systems have produced major enhancements in cancer cell specificity and selectivity over their non-targeted formulation counterparts. Hence, it is hopeful that this targeting strategy will lead to improvements in the safety and efficacy of clinically-relevant anti-cancer agents. Therefore, the focus of this review will be to highlight the current status of folate-targeted technology with particular emphasis on the recent advances in this field as well as possible directions for future development.
Asunto(s)
Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Inmunoterapia/métodos , Neoplasias/diagnóstico por imagen , Neoplasias/terapia , Receptores de Superficie Celular/metabolismo , Animales , Medios de Contraste , Diseño de Fármacos , Receptores de Folato Anclados a GPI , Humanos , Neoplasias/metabolismo , CintigrafíaRESUMEN
A folate-targeted, cationic lipid based transfection complex was developed and found to specifically transfect folate receptor-expressing cells and tumors. These liposomal vectors were comprised of protamine-condensed plasmid DNA, a mixture of cationic and neutral lipids, and a folic acid-cysteine-polyethyleneglycol-phosphatidylethanolamine (FA-Cys-PEG-PE) conjugate. Pre-optimization studies revealed that inclusion of low amounts (0.01 to 0.03%) of FA-Cys-PEG-PE yielded the highest binding activity of dioleoylphosphatidylcholine/cholesterol liposomes to folate receptor-bearing cells. In contrast, higher amounts (>0.5%) of FA-Cys-PEG-PE progressively decreased cellular binding of the liposomes. In vitro studies with cationic lipid/dioleoylphosphatidylethanolamine formulations indicated that as little as 0.01 to 0.3% of FA-Cys-PEG-PE was needed to produce optimal targeted expression of plasmid DNA. Similarly, using a disseminated intraperitoneal L1210A tumor model, maximum in vivo transfection activity occurred with intraperitoneally administered formulations that contained low amounts (0.01 mol%) of the FA-Cys-PEG-PE targeting lipid. Overall, folate-labeled formulations produced an eight- to 10-fold increase in tumor-associated luciferase expression, as compared with the corresponding non-targeted cationic lipid/DNA formulations. These results collectively indicate that transfection of widespread intraperitoneal cancers can be significantly enhanced using folate-targeted techniques.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Fólico/genética , Marcación de Gen/métodos , Terapia Genética/métodos , Neoplasias Peritoneales/terapia , Receptores de Superficie Celular , Animales , Línea Celular , Femenino , Receptores de Folato Anclados a GPI , Ácido Fólico/metabolismo , Liposomas , Luciferasas/genética , Ratones , Ratones Endogámicos DBA , Neoplasias Peritoneales/metabolismo , Unión Proteica , Transfección/métodosRESUMEN
Viral vectors with high transfection efficiencies are not always those with optimal target cell binding specificities. As a consequence, virus pseudotyping has been developed to endow transfection competent viruses with improved cell binding specificities and affinities. We have hypothesized that chemical conjugation of a virus to a cell specific ligand might also alter its target cell specificity and produce a virus that would transfect only the desired cell type. To test this concept, an ecotropic replication-defective myeloproliferative sarcoma retrovirus and an amphotropic murine adenovirus containing the gene for beta-galactosidase were chemically derivatized with folic acid. As expected from its strong ecotropism, the unmodified retrovirus did not induce beta-galactosidase expression in nonhost KB cells, while the amphotropic adenovirus yielded high levels of gene expression in the same cell line. Surprisingly, although folate derivatization enabled avid binding of both viruses to folate receptor expressing KB cells, the folate conjugation did not promote retroviral gene expression and actually prevented the normal beta-galactosidase expression seen with the adenoviral vector. The fact that co-administration of excess free folic acid to block uptake by folate receptor-mediated endocytosis restored adenoviral gene expression to the level obtained with unmodified virus suggests that folate derivatization per se does not hamper viral activity. We, therefore, conclude that neither retroviral nor adenoviral delivery via the folate endocytosis pathway is compatible with viral gene expression in KB cells.
Asunto(s)
Adenoviridae/genética , Proteínas Portadoras/efectos de los fármacos , Endocitosis/efectos de los fármacos , Vectores Genéticos , Receptores de Superficie Celular , Retroviridae/genética , Conjugación Genética , Receptores de Folato Anclados a GPI , Humanos , Radioisótopos de Yodo , Operón Lac/genética , Virus de la Leucemia Murina de Moloney/genética , Transducción Genética , Transfección , Células Tumorales Cultivadas , beta-Galactosidasa/metabolismoRESUMEN
Liposomal gene therapy vectors that penetrate cells by endocytosis must escape an endosomal compartment in order to enter the target cell's nucleus. Because such endosomal compartments are generally acidic in nature, pH-sensitive liposomes have been designed that are stable at extracellular pH ( approximately pH 7.4) but fusogenic at endosomal pH values ( approximately pH 5). We report here the use of a novel folate-targeted, pH-sensitive, anionic liposomal vector that mediates the efficient delivery of DNA into folate receptor-bearing cells and discharges the DNA into the cytoplasm. N-Citraconyl-dioleoylphosphatidylethanolamine (C-DOPE), a derivative of dioleylphosphatidylethanolamine (DOPE) that hydrolyzes rapidly at pH 5 to yield DOPE, was synthesized and incorporated with DOPE and folate-polyethyleneglycol-DOPE into liposomes. The resulting liposomes were stable at neutral pH but fusogenic at pH 5. Folate-labeled gene transfer vectors were prepared by compacting plasmid DNA with polylysine at a 1:0.75 (w/w) ratio and complexing the condensed cationic plasmid with the above anionic liposomes. Association of the polylysine-DNA with the liposomes was confirmed by sucrose gradient centrifugation, where migration of the folate-labeled vectors was midway between that of the free liposomes and condensed polylysine-DNA. Transfection of cultured cancer cells with the pH-sensitive liposomal vectors was found to be significantly more efficient than transfection with DOPE-cholesterol hemisuccinate-based vectors, the more commonly used pH-dependent, liposomal transfection formulation. Optimization studies revealed that inclusion of only 3% C-DOPE and 0.1% folate-derivatized DOPE yielded the highest transfection activity. Nearly quantitative competition with free folic acid as well as direct correlation of transfection efficiency with folate receptor density for several different cell lines further documented that vector uptake was mediated by folate receptor endocytosis. Taken together, these data argue that C-DOPE warrants further consideration as a pH sensitive component of lipid-based gene delivery formulations.
Asunto(s)
Etanolaminas/química , Ácido Fólico/metabolismo , Técnicas de Transferencia de Gen , Liposomas/farmacocinética , Células Cultivadas , ADN/metabolismo , Endocitosis , Etanolaminas/síntesis química , Vectores Genéticos/fisiología , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Plásmidos , Polilisina/química , Transfección , beta-Galactosidasa/metabolismoRESUMEN
A folate-targeted transfection complex that is internalized by certain cancer cells and displays several properties reminiscent of enveloped viruses has been developed. These liposomal vectors are comprised of a polycation-condensed DNA plasmid associated with a mixture of neutral and anionic lipids supplemented with folate-poly(ethylene glycol)-dioleylphosphatidylethanolamine for tumor cell-specific targeting. N-Citraconyl-dioleylphosphatidylethanolamine is also included for pH-dependent release of endosome-entrapped DNA into the cytoplasm, and a novel plasmid containing a 366-bp segment from SV40 DNA has been employed to facilitate transport of the plasmid into the nucleus. Because formation of the DNA core is an important step in the assembly of liposomal vectors, considerable effort was devoted to comparing the transfection efficiencies of various DNA condensing agents. It was found that complexation of plasmid DNA with high molecular weight polymers such as acylated-polylysine and cationic dendrimers leads to higher folate-mediated transfection efficiency than DNA complexed with unmodified polylysine. In contrast, compaction of plasmid DNA with small cationic molecules such as spermine, spermidine, or gramicidin S yields only weakly active folate-targeted liposomal vectors. Compared to analogous liposomal vector preparations lacking an optimally compacted DNA core, a cell-specific targeting ligand, a caged fusogenic lipid, and a nucleotide sequence that facilitates nuclear uptake, these modified liposomal vectors display greatly improved transfection efficiencies and target cell specificity.
Asunto(s)
Proteínas Portadoras/fisiología , ADN/administración & dosificación , Ácido Fólico/química , Terapia Genética/métodos , Liposomas/química , Receptores de Superficie Celular , Aniones/química , ADN/química , ADN/genética , Receptores de Folato Anclados a GPI , Ácido Fólico/administración & dosificación , Ácido Fólico/metabolismo , Vectores Genéticos/administración & dosificación , Vectores Genéticos/síntesis química , Vectores Genéticos/química , Humanos , Células KB , Liposomas/síntesis química , Fosfatidiletanolaminas/química , Plásmidos/administración & dosificación , Plásmidos/química , Plásmidos/genética , Poliaminas/química , Polielectrolitos , Polilisina/química , Transfección/métodosRESUMEN
We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34(+) cells express both the beta and gamma isoforms of FR. The alpha isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-alpha), the abundance of FR-beta on CD34(+) cell surfaces was relatively low (approximately 8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34(+) cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34(+) cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function.
Asunto(s)
Proteínas Portadoras/metabolismo , Células Madre Hematopoyéticas/metabolismo , Antígenos CD34 , Línea Celular , Citometría de Flujo , Receptores de Folato Anclados a GPI , Humanos , Isoformas de Proteínas/metabolismo , Receptores de Superficie Celular/metabolismoRESUMEN
The vitamin folic acid (FA) enters cells either through a carrier protein, termed the reduced folate carrier, or via receptor-mediated endocytosis facilitated by the folate receptor (FR). Because folate-drug conjugates are not substrates of the former, they penetrate cells exclusively via FR-mediated endocytosis. When FA is covalently linked via its gamma-carboxyl to a drug or imaging agent, FR binding affinity (KD approximately 10(-10)M) is not measurably compromised, and endocytosis proceeds relatively unhindered, promoting uptake of the attached drug/imaging agent by the FR-expressing cell. Because FRs are significantly overexpressed on a large fraction of human cancer cells (e.g., ovarian, lung, breast, endometrial, renal, colon, and cancers of myeloid hematopoietic cells), this methodology may allow for the selective delivery of a wide range of imaging and therapeutic agents to tumor tissue. Folate-mediated tumor targeting has been exploited to date for delivery of the following molecules and molecular complexes: (i) protein toxins, (ii) low-molecular-weight chemotherapeutic agents, (iii) radioimaging agents, (iv) MRI contrast agents, (v) radiotherapeutic agents, (vi) liposomes with entrapped drugs, (vii) genes, (viii) antisense oligonucleotides, (ix) ribozymes, and (x) immunotherapeutic agents. In virtually all cases, in vitro studies demonstrate a significant improvement in potency and/or cancer-cell specificity over the nontargeted form of the same pharmaceutical agent. Where live animal studies have been conducted, they also reveal significant promise.