A series of novel bioreducible poly(amido amine)s containing multiple disulfide linkages (SS-PAAs) were synthesized and evaluated as nonviral gene vectors. These linear SS-PAAs could be easily obtained by Michael-type polyaddition of various primary amines to the disulfide-containing cystamine bisacrylamide. The SS-PAA polymers are relatively stable in medium mimicking physiological conditions (pH 7.4, 150 mM PBS, 37 degrees C), but are rapidly degraded in the presence of 2.5 mM DTT, mimicking the intracellular reductive environment (pH 7.4, [R-SH] = 5 mM, 37 degrees C). The polymers efficiently condense DNA into nanoscaled (<200 nm) and positively charged (>+20 mV) polyplexes that are stable under neutral conditions but are rapidly destabilized in a reductive environment, as was revealed by both dynamic light scatting measurement and agarose gel assays. Moreover, most of the poly(amido amine)s possess buffer capacities in the pH range pH 7.4-5.1 that are even higher than polyethylenimine (pEI), a property that may favorably contribute to the endosomal escape of the polyplexes. Polyplexes of four of the seven SS-PAAs studied were able to transfect COS-7 cells in vitro with transfection efficiencies significantly higher than those of branched pEI, being one of the most effective polymeric gene carriers reported to date. Importantly, also in the presence of serum, a high level of gene expression could be observed when the incubation time was elongated from 1 h to 4 h. XTT assays showed that SS-PAAs and their polyplexes possess essentially no or only very low cytotoxicity at concentrations where the highest transfection activity is observed. The results indicate that bioreducible poly(amido amine)s have excellent properties for the development of highly potent and nontoxic polymeric gene carriers.

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Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by (1)H NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approximately 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.

A route was developed for the synthesis of three different cyclodextrin adsorbates:  heptakis{6-O-[3-(thiomethyl)propionyl)]-2,3-di-O-methyl}-β-cyclodextrin, heptakis{6-O-[12-(thiododecyl)dodecanoyl)]-2,3-di-O-methyl}-β-cyclodextrin (a short and long alkyl chain sulfide cyclodextrin adsorbate, respectively), and heptakis[6-deoxy-6-(3-mercaptopropionamidyl)-2,3-di-O-methyl]-β-cyclodextrin (a short alkyl chain thiol adsorbate). Self-assembled monolayers on gold of these three cyclodextrin adsorbates with seven sulfur moieties were fully characterized by electrochemistry, wettability studies, X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The electrochemical capacitance measurements show the differences between the thicknesses of the β-cyclodextrin monolayers, and the XPS-(S2p) measurements show the different effectivenesses of the sulfur moieties of the three monolayers in their binding to the gold surface. Sulfide-based β-cyclodextrin monolayers use on average 4.5 of the 7 attachment points whereas the thiol-based cyclodextrin monolayer only uses 3.2 of the 7 sulfurs. These experiments show that, for adsorbates with multiple attachment points, sulfides may be more effective than thiols. TOF-SIMS measurements confirm the robust attachment of these adsorbates on gold obtained by XPS.