PEGylated J591 mAb loaded in PLGA-PEG-PLGA tri-block copolymer for targeted delivery: in vitro evaluation in human prostate cancer cells.

J591 monoclonal antibody (mAb) has high affinity for prostate specific membrane antigen (PSMA) on prostate cancer (PCA) cells. We coupled polyethylene glycol-J591 (PEGylated J591) to a salicyl hydroxamic acid (SHA)-derivatized polyethylenimine (PEI)/DNA-betagal vector to investigate the specificity and efficiency of targeting PSMA in PCA cells through encapsulation. Coupling was facilitated via the high affinity interaction between phenyl(di)boronic acid (PDBA) and SHA molecules yielding J591/PEG/PEI/DNA-betagal polyplex. After encapsulation with poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol-b-poly(d,l-lactic-co-glycolic acid) (PLGA-PEG-PLGA) tri-block copolymer, 8-10-fold increment of gene transfection levels were attained at the optimum concentration of 0.25% (w/v) using Pluronic F68 tri-block copolymer as a control. The enhanced transfection efficiency was attributed to increased internalization and uptake of the radiolabeled plasmid in the presence of PLGA-PEG-PLGA tri-block copolymer. The release of plasmid DNA (pDNA) from microparticles containing SHA-PEI-complexed pDNA showed little initial burst release followed by a 5% release over 48 h. The release accelerated thereafter and approximately 60% was released after 28 days. Deconvolution confocal microscopy showed polyplex/microparticle formulation localized in the cell nucleus as opposed to the polyplex without PLGA-PEG-PLGA indicating that an optimal concentration of PLGA-PEG-PLGA tri-block copolymer can be utilized to enhance endocytic process of J591-mediated targeting of PCA cells.

Uptake characteristics of NGR-coupled stealth PEI/pDNA nanoparticles loaded with PLGA-PEG-PLGA tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells.

We have investigated the in vitro uptake, toxicity, phenotypic consequences and transfection efficiency of a stealth NGR/PEG/PDBA-coupled-SHA-PEI/pDNA targeting polyplex loaded with PLGA-PEG-PLGA tri-block copolymer in human monocyte-derived dendritic cells (DCs). Modification with PEG effectively shielded and reduced non-specific phagocytosis by immature DCs to approximately 20%. Coupling the NGR cell-specific peptide to the PEGylated polyplex (NGR/PEG/PDBA-SHA-PEI/pDNA) however resulted in specific and enhanced phagocytosis in DCs without any observable toxicity at the optimum concentration of 0.25% of the copolymer. DNase treatment had no effect on DNA integrity in the encapsulated polyplex. Confocal microscopy confirmed intracellular localization of the targeting NGR/PEG/PDBA-SHA-PEI/pDNA microparticles, resulting in more enhanced uptake of the radiolabeled plasmid DNA and approximately 5- and 10-fold increase over the control tri-block Pluronic F68 copolymer and the non-targeting polyplex, respectively. More importantly, phagocytosis of the targeting microparticles neither altered the functionality of immature DCs nor the phenotypic expression of DC-specific cell surface molecules, CD80, CD86, CD40 and CD54 (ICAM-1), suggesting that uptake of the targeting microparticles by themselves did not induce DC maturation. Taken together, these results suggest that PLGA-PEG-PLGA encapsulation of this stealth targeting polyplex has no negative effects on key properties of immature DCs and should pave the way for targeting DCs for vaccination purposes.

Insulin-like growth factor binding protein-3 inhibits the growth of non-small cell lung cancer.

Insulin-like growth factors (IGFs) have mitogenic and antiapoptotic properties and have been implicated in the development of lung cancer. The effects of IGFs are modulated by insulin-like growth factor binding proteins (IGFBPs). This study explored the effects of IGFBP-3 on non-small cell lung cancer (NSCLC) cells after infection with an adenovirus constitutively expressing IGFBP-3 under the control of the cytomegalovirus promoter (Ad5CMV-BP3). We found that IGFs, especially IGF-I, stimulated the growth of NSCLC cells, and Ad5CMV-BP3 suppressed this IGF-I-induced NSCLC cell growth. We also found that the clonogenicity of H1299 cells in soft agar was markedly reduced by Ad5CMV-BP3. Furthermore, direct injection of Ad5CMV-BP3 into H1299 NSCLC xenografts s.c. established in athymic nude mice induced massive destruction of the tumors. Ad5CMV-BP3 did not induce detectable cytotoxicity on normal human bronchial epithelial cells, suggesting therapeutic efficacy of this virus. Ad5CMV-BP3 infection was accompanied by apoptotic cell death in vitro as detected by flow cytometry, DNA fragmentation analysis, and Western blot analysis on the expression of Bcl-2 and on the cleavage of poly(ADP-ribose) polymerase, a substrate of caspase 3. Immunofluorescence confocal microscopy was also used to show the apoptotic effect of Ad5CMV-BP3 in H1299 tumors established in nude mice. These findings indicated that IGFBP-3 was a potent inducer of apoptosis in NSCLC cells in vitro and in vivo. To delineate the underlying mechanism, we examined the effect of IGFBP-3 on Akt/protein kinase B and glycogen synthase kinase-3beta, downstream mediators of the phosphatidylinositol 3-kinase pathway, and on mitogen-activated protein kinase (MAPK), all three of which are activated by IGF-mediated signaling pathways and have important roles in cell survival. IGFBP-3 overexpression inhibited the phosphorylation of Akt and glycogen synthase kinase-3beta and the activity of MAPK. Furthermore, IGF-I rescued the NSCLC cells from serum depletion-induced apoptosis, and this rescue was blocked in Ad5CMV-BP-3-infected H1299 NSCLC cells. Transient transfection with activated Akt or constitutively active MAPK kinase-1, an upstream activator of MAPK, partially blocked IGFBP-3-induced apoptosis of NSCLC cells. These findings suggested that the growth-regulatory effect of IGFBP-3 on NSCLC cells was attributable in part to the inhibition of the IGF-induced survival pathway. These data demonstrate the importance of IGFBP-3 in the regulation of NSCLC cell proliferation, clonogenicity, and tumor growth, suggesting that IGFBP-3 is a target for the treatment of lung cancer and that Ad5CMV-BP3 is a potential therapeutic agent.

Efficient therapeutic gene delivery after systemic administration of a novel polyethylenimine/DNA vector in an orthotopic bladder cancer model.

Successful systemic gene therapy has been hindered by vector-related limitations, including toxicity and inefficient gene delivery to tumor cells after i.v. administration. To circumvent these problems, we developed a novel formulation between the polycation polyethyleneimine and DNA that mediates high-level tumor cell transduction in vitro and efficient i.v. gene delivery in that greater reporter gene expression occurred in tumor than in lung. Strikingly, administration of just 6 micro g of the polyethyleneimine/DNA-p53 vector every 3 days for 3 weeks indicated restoration of normal cell cycle regulation and apoptotic mechanisms as demonstrated by efficient p53 expression, increased apoptosis, and a 70% reduction in tumor size in an orthotopic bladder cancer model. This novel vector formulation represents a new method to increase i.v. delivery of genes to tumors.

Tumor-specific gene delivery mediated by a novel peptide-polyethylenimine-DNA polyplex targeting aminopeptidase N/CD13.

We have developed a novel polyethylenimine (PEI)-DNA vector formulation that is capable of efficient tumor-specific delivery after intravenous administration to nude mice. To further increase the specificity of delivery, we have attached the peptide CNGRC to the vector, which is specific for aminopeptidase N (CD13). The strategy for coupling this peptide to PEI was based on a novel method involving the strong affinity between phenyl(di)boronic acid (PDBA) and salicylhydroxamic acid (SHA) as well as a polyethylene glycol (PEG) linker to reduce steric hindrance between the vector and the peptide. In vitro assessment of targeting by the CNGRC/PEG/PEI/DNA vector carrying a beta-galactosidase (beta-Gal)-expressing plasmid showed as much as a 5-fold increase in transduction, relative to the untargeted PEG/PEI/DNA-betagal vector, of CD13-positive lung cancer, fibrosarcoma, bladder cancer, and human umbilical vein endothelial cells. Competition with free peptide resulted in up to a 90% reduction in delivery, indicating that gene delivery was specific for CD13-positive cells. Intravenous administration of the CNGRC/PEG/PEI/DNA-betagal vector to nude mice bearing subcutaneous tumors resulted in as much as a 12-fold increase in beta-Gal expression in tumors as compared with expression in either lungs or tumors from animals treated with the original PEI/DNA-betagal vector. In vivo transduction analysis using the CNGRC/PEG/PEI/DNA vector to target the intravenous delivery of a yellow fluorescence protein (YFP)-expressing plasmid to subcutaneous H1299 tumors confirmed delivery of YFP to both tumor cells and tumor endothelial cells. The use of this peptide to further increase tumor-specific delivery mediated by our novel PEI/DNA vector now provides a basis for developing tumor-targeted gene therapies for use in the clinical treatment of cancer.

Protein/DNA polyplexes for gene therapy.

The development of molecular conjugates as components of Protein/DNA polyplexes has resulted in the creation of a simple, non-virus vector for the targeted delivery of nucleic acids into specific cell types. This vector has many of the positive attributes of viruses, on without the limitations that continue to plague recombinant viruses. The simplicity of this vector allows for quick analysis of nucleic acids, expression vectors, and therapeutic genes in vitro and potentially in vivo, because the time that would be involved in the generation of recombinant viral vectors is not present. Essentially, the development of this delivery vector has resulted in the creation of a "synthetic virus" that has the capability of targeted delivery without the negative attributes of viruses.