Biomedical subjects

James W Yockman

Publications and source records attributed to James W Yockman.

11 recordsLinked to original sources

PubMed2007

Reducible poly(amido ethylenimine) directed to enhance RNA interference.

Designing synthetic macromolecular vehicles with high transfection efficiency and low cytotoxicity has been a major interest in the development of non-viral gene carriers. A reducible poly(amido ethylenimine) (SS-PAEI) synthesized by addition copolymerization of triethylenetetramine and cystamine bis-acrylamide (poly(TETA/CBA)) was used as a carrier for small interference RNA (siRNA). Poly(TETA/CBA) could efficiently condense siRNA to form stable complexes under physiological conditions and perform complete release of siRNA in a reductive environment. When formulated with VEGF-directed siRNA, poly(TETA/CBA) demonstrated significantly higher suppression of VEGF than linear-polyethylenimine (PEI) (L-PEI, 25kDa) in human prostate cancer cells (PC-3). After 5h of transfection, substantial dissociation and intracellular distribution of siRNA was observed in the poly(TETA/CBA) formulation, but not in the L-PEI formulation. The triggered release of siRNA by reductive degradation of poly(TETA/CBA) in the cytoplasm may affect the RNAi activity by increasing cytoplasmic availability of siRNA. These results suggest that the rational design of non-viral carriers should involve considerations for intracellular dissociation and trafficking of a nucleic acid drug to maximize its effect, in conjunction with formation of stable complexes under physiological conditions.

Aziridines↗

PubMed2006

Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma.

Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic growth factor that is a primary stimulant of the development and maintenance of a vascular network in the vascularization of solid tumors. It has been reported that a blockade of VEGF-mediated angiogenesis is a powerful method for tumor regression. RNA interference represents a naturally occurring biological strategy for inhibition of gene expression. In mammalian systems, however, the in vivo application of small interfering RNA (siRNA) is severely limited by the instability and poor bioavailability of unmodified siRNA molecules. In this study, we tested the hypothesis that a hydrophobically modified protein transduction domain, cholesteryl oligo-d-arginine (Chol-R9), may stabilize and enhance tumor regression efficacy of the VEGF-targeting siRNA. The noncovalent complexation of a synthetic siRNA with Chol-R9 efficiently delivered siRNA into cells in vitro. Moreover, in a mouse model bearing a subcutaneous tumor, the local administration of complexed VEGF-targeting siRNA, but not of scrambled siRNA, led to the regression of the tumor. Hence, we propose a novel and simple system for the local in vivo application of siRNA through Chol-R9 for cancer therapy.

Adenocarcinoma↗

PubMed2006

Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.

Angiogenesis Inhibitors↗

PubMed2005

Anti-GAD antibody targeted non-viral gene delivery to islet beta cells.

An islet cell targeting polymeric gene carrier was synthesized by conjugating anti-GAD Fab' fragment to PEI via PEG linker (PEI-PEG-Fab'). The Fab' fragment was prepared from a murine monoclonal antibody against glutamic acid decarboxylase (GAD), which has been identified as one of the major auto-antigens expressed in islet cells, and used as a targeting moiety for islet cell targeting. The electrophoretic migration of plasmid DNA (pCMVLuc)/PEI-PEG-Fab' complexes in agarose gel was completely retarded above the N/P ratio of 2. The complexes demonstrated a size of 100-275 nm with an almost neutral surface charge. Confocal microscopy revealed that the PEI-PEG-Fab' complexes showed much higher cellular binding and uptake efficiency compared to PEI-PEG complexes. The PEI-PEG-Fab' showed about 10-fold higher transfection efficiency (relative luciferase activity) than PEI-PEG in GAD-expressing mouse insulinoma cells (MIN6), however the transfection efficiency of PEI-PEG-Fab' reduced to that of PEI-PEG in GAD negative cells (293) and in the presence of competitive free Fab'. Considering the neutral surface charge of its complexes with DNA, and selectivity toward the islet cells expressing a specific antigen, the PEI-PEG-Fab' conjugate could be thought as a potential candidate of the systemic gene therapy for the treatment of type I diabetes.

Animals↗

PubMed2005

Soluble Flt-1 gene delivery using PEI-g-PEG-RGD conjugate for anti-angiogenesis.

Vascular endothelial growth factor (VEGF), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumors and closely associated with tumor growth and metastasis. It has been shown that a soluble fragment of VEGF receptor Flt-1 (sFlt-1) has anti-angiogenic properties by way of its antagonist activity against VEGF. In the present study, we demonstrated that the stable expression of sFlt-1 by endothelial cell targeted non-viral gene delivery inhibited the angiogenesis of endothelial cells. A targeted polymeric gene delivery system, PEI-g-PEG-RGD, was developed by incorporating the alphanubeta3/alphanubeta5 integrin-binding RGD peptide, ACDCRGDCFC (single-letter amino acid code), into the cationic polymer, polyethylenimine (PEI) via a hydrophilic polyethylene glycol (PEG) spacer. The functional analysis of therapeutic gene encoding sFlt-1/carrier complex was performed with an endothelial cell proliferation assay. The complex of sFlt-1 gene with PEI-g-PEG-RGD conjugate efficiently inhibited the proliferation of cultured endothelial cells, representing that expressed sFlt-1 predominantly bound to exogenous VEGF and blocked the binding of VEGF to the full-length Flt-1 receptor. These findings suggest that the combination of targeted gene carrier and sFlt-1 possesses the potential to be an efficient tool for the anti-angiogenic gene therapy to treat cancer.

Angiogenesis Inhibitors↗

PubMed2005

Local, non-viral IL-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment.

Development of improved gene transfer methods is needed for gene therapy to achieve its clinical potential. The use of biocompatible polymeric gene carriers has shown effectiveness in overcoming the current problems associated with viral vectors in safety, immunogenicity and mutagenesis. Previous work has demonstrated that repeated, local, non-viral interleukin-12 (IL-12) gene delivery successfully slows down tumor progression, while improving immunogenicity. Combining IL-12 gene delivery with systemic paclitaxel (PCT) chemotherapy as a treatment for various subcutaneous mouse mammary carcinomas, we used PCT with either a biodegradable polymeric solubilizer, HySolv or Cremophor EL for systemic treatment and injected water soluble lipopolymer (WSLP)/plasmid-encoding IL-12 gene (p2CMVmIL-12) complexes local once every week. The amount of lung metastases being essential for survival as well as subcutaneous tumor volume were compared against untreated controls. We showed inhibition of tumor growth and decreased lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the PCT only treated groups. Compared to Cremophor, HySolv performed better alone or in combination with IL-12. Using polymeric vectors as gene carrier systems in combination with improved systemic therapies provide evidence for the efficacy and feasibility of polymer-based drug delivery systems. Especially local cytokine gene delivery showed augmentation of systemic chemotherapy while reducing the hosts risk for further systemic toxicity.

Animals↗

PubMed2004

Tumor efficacy and biodistribution of linear polyethylenimine-cholesterol/DNA complexes.

Non-viral polymer/pDNA complexes were formed using linear polyethylenimine (LPEI) Mw 25 k conjugated to cholesterol in a T-shaped geometry (LPC-T) and pDNA encoding murine interleukin-12 (pmIL-12e). These complexes were subsequently injected weekly into BALB/c mice intravenously and locally for the treatment of murine renal cell adenocarcinoma (Renca) induced pulmonary metastases and subcutaneous (SC) Renca tumors, respectively. At the cessation of the pulmonary metastases study, the number of pulmonary metastases was significantly less (p < 0.001) with systemic injections of LPC-T/pmIL-12e formulations than with pmIL-12e alone or pmIL-12e complexed with LPEI, branched polyethylenimine (BPEI) Mw 25 k, or an LPEI/pEGFP control. In addition, biodistribution studies showed increased pulmonary levels of both the LPC-T carrier and pmIL-12e vector up to 3 hr after systemic injection of the LPC-T/pmIL-12e complexes into mice carrying pulmonary metastases. Furthermore, mice systemically treated with LPC-T/pmIL-12e showed a near linear profile in weight gain in the course of the pulmonary metastases study that suggests increased biocompatibility. Finally, due to favorable characteristics in vitro, LPC-T was also used for local (peritumoral) injection of SC Renca tumors. Tumor stasis and slight tumor regression were seen only with the LPC-T/pmIL-12e treated mice compared to BPEI/pmIL-12e, LPEI/pmIL-12e, and naked pmIL-12e controls. Thus, it was concluded that LPC-T is an effective carrier for passive targeting of the pulmonary tissue, treatment of Renca-induced pulmonary metastases, and local administration of Renca cell SC tumors.

Animals↗

PubMed2004

Combination of local, nonviral IL12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model.

Repeated, local, nonviral IL12 (interleukin-12) gene delivery decreased tumor progression and increased immunogenicity. We combined our IL12 gene delivery with systemic paclitaxel chemotherapy as a treatment for paclitaxel (PCT)-resistant 4T1 subcutaneous mouse mammary carcinomas and PCT-sensitive, immunogenic/nonimmunogenic tumors. We mixed PCT with either a biodegradable polymeric solubilizer, HySolv, or Cremophor EL for bimonthly systemic treatments and injected water-soluble lipopolymer (WSLP)/p2CMVmIL-12 (plasmid encoding IL12 gene) complexes locally every week. We compared treated subcutaneous tumor volume and lung metastasis with controls. HySolv alone performed better compared to Cremophor EL in combination with WSLP/p2CMVmIL-12. We showed inhibition of 4T1 tumor growth and lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the paclitaxel-only treated groups. In parallel experiments we also demonstrated additive responses for tumor growth and number of lung metastases within other PCT-sensitive mammary tumor models using this combination strategy. Our combination therapy provides evidence for the efficacy and feasibility of improved drug delivery systems. Local cytokine gene delivery can augment local and systemic chemotherapy without placing the host at risk for further systemic toxicity.

Animals↗

PubMed2003

Tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p2CMVmIL-12 complexes.

The recruitment of the body's own immune system is amongst the most potent defenses known against cancer. Recent attempts to harness this response have enlisted the use of the immune modulating cytokine, interleukin-12 (IL-12). The objective of this work is to investigate the organ distribution and anti-tumor response in vivo after intratumoral administration of IL-12 expression plasmid complexed with water soluble lipopolymer (WSLP). Formulations of WSLP/p2CMVmIL-12 at N/P mol ratio of 20:1 were prepared in the presence of 5% (w/v) glucose. Organ distribution data following intratumoral injection of CT-26 subcutaneous tumor-bearing BALB/c mice demonstrated enhanced retention of WSLP/p2CMVmIL-12 complexes within the tumor and limited accumulation in other organs for up to 96 h. Tumor-bearing BALB/c mice received either single or repeated intratumoral injections at 4- or 8-day intervals to examine the efficacy of single versus repeated injections on tumor regression and survival. Significant tumor growth inhibition during 4- and 8-day injection trials was observed with maximal survival in mice receiving 4-day injections of WSLP/p2CMVmIL-12 complexes. In conclusion, the water-soluble non-toxic lipopolymer complexed with p2CMVIL-12 showed enhanced transgene expression in vivo, inhibits the rate of tumor growth, and significantly increases survival.

Animals↗

PubMedDate not supplied

Modified linear polyethylenimine-cholesterol conjugates for DNA complexation.

Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M(w) 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of approximately 250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.

Animals↗

PubMedDate not supplied

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties.

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.

Animals↗