Functional properties and biodistribution of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) mixtures formed with nucleic acid.

The clinical success of non-viral gene delivery reagents is hampered by their inefficient cellular transgene delivery, which is largely influenced by carrier properties that are currently undefined and misunderstood. In an attempt to further define and understand the requirements for a safe and efficient non-viral gene delivery reagent, research labs often engineer and evaluate many putative products with subtle physiochemical differences in order to delineate requirements for improved in vitro and in vivo success. The synthesis of many putative reagents is often time-intensive, laborious and costly. In a previous manuscript published by our lab, different amounts of poly(triethylenetetramine/cystamine bisacrylamide) (p(TETA/CBA) and its pegylated counterpart, poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) (p(TETA/CBA)-g-PEG) were mixed together to easily identify optimal reagent properties and candidates in vitro, while avoiding the synthesis of many putative candidates for study. This report uses the aforementioned facile approach to evaluate reagent properties of products that were obtained via one-pot synthesis, which improved synthetic ease. As such, synthesis time was reduced from 6days to 3days and had comparable or improved transfection and viability compared to previous works. Moreover, this synthesis resulted in higher molecular weight products than were used in the previous study and allow for lower polymer doses to be used for complexation, which is useful for systemic delivery that is used herein. The physiochemical properties of the formulations derived using these novel reagents was studied prior to investigating their in vivo biodistribution profiles in a murine colon adenocarcinoma model. Interestingly, negatively charged complexes exhibited greater passive tumor accumulation compared to positively charged complexes following their systemic administration. These studies warrant further investigation for the use of negatively charged drug and gene delivery reagents for passive tumor targeting, and they substantiate the use of polycation/PEG-polycation mixtures for facile product evaluation in order to elucidate design and formulation mandates for the clinical success of non-viral gene delivery formulations.

EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide-diamino hexane) for the delivery of therapeutic pCMV-RAE-1γ to pancreatic islets.

The pathogenesis of type-1 diabetes is complicated, and a clear, single mechanism has yet to be identified. Reports have indicated that the activating receptor NKG2D plays an important role in the development of disease. Exploiting a natural phenomenon observed in tumors, plasmid DNA encoding for a soluble ligand to NKG2D (sRAE-1γ) was isolated and engineered into a plasmid expression system. A polymeric gene delivery system was developed to deliver the soluble RAE-1 plasmid locally to the pancreatic islets for the prevention of type-1 diabetes. The bioreducible cationic polymer poly(cystamine bisacrylamide-diamino hexane) (p(CBA-DAH)) was modified with poly(ethylene glycol) (PEG) and the targeting peptide CHVLWSTRC, known to target the EphA2 and EphA4 receptors. The PEG serves to improve stability and tissue selectivity, while the peptide will target EphA2 and A4, overexpressed in the pancreatic microvasculature. The targeting polymer Eph-PEG-p(CBA-DAH) shows selective uptake by the target cell line, indicative of the targeting properties that will be seen in systemic administration. Using the delivery system, the therapeutic plasmid can be delivered to the pancreas, reduce interactions between the beta-cells and infiltrating NKG2D positive lymphocytes, and effectively protect beta-cells from autoimmune destruction and prevent type 1 diabetes.

Mechanism of sodium uptake in PNA negative MR cells from rainbow trout, Oncorhynchus mykiss as revealed by silver and copper inhibition.

The rate of acid-stimulated and phenamil-sensitive sodium (Na(+)) uptake was measured in three different cell lineages: pavement cells (PVC), total mitochondrion-rich (MR) cell populations, and peanut lectin agglutinin-negative mitochondrion-rich cells (PNA(-) MR) isolated from the rainbow trout gill epithelium. Despite the presence of basal levels of Na(+) uptake in PVC, this transport was not enhanced by acidification, nor was it inhibited by independent treatment with bafilomycin (i.e., a V-type H(+)-ATPase inhibitor), phenamil (i.e., a specific inhibitor of ENaC), or Ag (a specific inhibitor of active Na(+) transport in fish). In contrast, Na(+) uptake in PNA(-) MR cells was increased by ~220% above basal levels following acidification of near 0.4 pH units in the presence of 1.0 mM external Na(+). Acid-stimulated Na(+) transport was entirely inhibited by both phenamil and bafilomycin. Silver (Ag) and copper (Cu), which are known to interfere with active Na(+) transport in fish, were also responsible for inhibiting acid stimulated Na(+) uptake in PNA(-) MR cells, but by themselves had no effect on basal Na(+) transport. Thus, we demonstrate that Ag specifically prevented acid-stimulated Na(+) uptake in PNA(-) MR cells in a dose-dependent manner. We also demonstrate rapid (<1 min) and significant inhibition of carbonic anhydrase (CA) by Ag in PNA(-) MR cells, but not in PVC. These data lend further support to the idea of a PNA(-) MR cell type as the primary site for Na(+) uptake in the freshwater (FW) gill phenotype of rainbow trout. Moreover, these findings provide support for the importance of intracellular protons in regulating the movement of Na(+) across the apical surface of the fish gill.

Mixtures of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)-g-poly(ethylene glycol) for improved gene delivery.

Branched disulfide-containing poly(amido ethyleneimines) (SS-PAEIs) are biodegradable polymeric gene carrier analogues of the well-studied, nondegradable, and often toxic branched polyethylenimines (bPEIs), but with distinct advantages for cellular transgene delivery. Clinical success of polycationic gene carriers is hampered by obscure design and formulation requirements. This present work reports synthetic and formulation properties for a graft copolymer of poly(ethylene glycol) (PEG) and a branched SS-PAEI, poly(triethylentetramine/cystaminebisacrylamide) (p(TETA/CBA)). Several laboratories have previously demonstrated the advantages of PEG conjugation to gene carriers, but have also shown that PEG conjugation may perturb plasmid DNA (pDNA) condensation, thereby interfering with nanoparticle formation. With this foundation, our studies sought to mix various amounts of p(TETA/CBA) and p(TETA/CBA)-g-PEG2k to alter the relative amount of PEG in each formulation used for polyplex formation. The influence of different PEG/polycation amounts in the formulations on polymer/nucleic acid nanoparticle (polyplex) size, surface charge, morphology, serum stability and transgene delivery was studied. Polyplex formulations were prepared using p(TETA/CBA)-g-PEG2k, p(TETA/CBA), and mixtures of the two species at 10/90 and 50/50 volumetric mixture ratios (wt/wt %), respectively. As expected, increasing the amount of PEG in the formulation adversely affects polyplex formation. However, optimal polymer mixtures could be identified using this facile approach to further clarify design and formulation requirements necessary to understand and optimize carrier stability and biological activity. This work demonstrates the feasibility to easily overcome typical problems observed when polycations are modified and thus avoids the need to synthesize multiple copolymers to identify optimal gene carrier candidates. This approach may be applied to other polycation-PEG preparations to alter polyplex characteristics for optimal stability and biological activity.

Dual bio-responsive gene delivery via reducible poly(amido amine) and survivin-inducible plasmid DNA.

A bioreducible poly(amido amine) (SS-PAA) gene carrier, known as poly (amido-butanol) (pABOL), was used to transfect a variety of cancer and non-cancer cell lines. To obtain cancer-specific transgene expression for therapeutic efficiency in cancer treatment, we constructed survivin-inducible plasmid DNA expressing the soluble VEGF receptor, sFlt-1, downstream of the survivin promoter (pSUR-sFlt-1). Cancer-specific expression of sFlt-1 was observed in the mouse renal carcinoma (RENCA) cell line. pABOL enhanced the efficiency of gene delivery compared to traditional carriers used in the past. Thus, a dual bio-responsive gene delivery system was developed by using bioreducible p(ABOL) for enhanced intracellular gene delivery and survivin-inducible gene expression system (pSUR-sFlt-1 or pSUR-Luc reporter gene) that demonstrates increased gene expression in cancer that has advantages over current gene delivery systems.