A series of nine poly(2-deoxy-2-methacrylamido glucopyranose)-b-poly(methacrylate amine) diblock copolycations The cationic block was varied in length and in the degree of methyl group substitution (secondary, tertiary, quaternary) on the pendant amine in an effort to optimize the structure and activity for plasmid DNA delivery. Upon a thorough kinetic study of polymerization for each polymer, the glycopolymers were prepared with well-controlled Mn and Ð. The binding and colloidal stability of the polymer-pDNA nanocomplexes at different N/P ratios and in biological media has been investigated using gel electrophoresis and light scattering techniques. The toxicity and transfection efficiency of the polyplexes has been evaluated with Hep G2 (human liver hepatocellular carcinoma) cells; several polymers displayed excellent delivery and toxicity profiles justifying their further development for in vivo gene therapy.
The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.
Natural product feedstocks such as carbohydrates and vegetable oils offer tremendous potential for creating sustainable cross-linked epoxy resin thermosets for numerous applications. Herein, we designed and synthesized trehalose- and β-cyclodextrin-based carboxylic acid hardeners to cure with epoxidized soybean oil forming predominantly sustainable epoxy resins. Trehalose (Tr) and β-cyclodextrin (Cd) were functionalized with heptanoyl chloride (H) and succinic anhydride (S). The resulting carboxylic acid hardeners were homogeneously formulated and cross-linked with epoxidized soy bean oil (ESO) at three different COOH/epoxide ratios. The cured resins were thermally stable up to 300 °C and stable in neutral and acidic aqueous conditions. Yet, degradation into water-soluble components could be triggered upon exposure to basic aqueous media. The physical properties of these materials are tunable based on feedstock composition and identity of the carbohydrate hardener. The glass transition temperatures ( T g) of the Tr-based epoxy polymers ranged from −3 to 3 °C, whereas the Cd-based polymers exhibited T g values of 28–36 °C. The mechanical properties including tensile strength and Young’s moduli also varied where the Cd-thermosets offered higher performance due to the structural rigidity of the cup-like structure. Homogeneous epoxy resin films of these materials were examined for their ability to promote cell adhesion and proliferation using neonatal human dermal fibroblast (HDFn) cells. The results indicated that films composed of the Cd-based epoxy resin with a 50/50 ratio of −COOH/epoxide promoted cell adhesion and proliferation with density similar to that of the well-studied control polymer poly(d l-lactide-co-glycolide) (PLG). Interestingly, the Tr-based epoxy films completely prevented cell adhesion and growth. The starkly different cell adhesion results and favorable physical characteristics of these predominantly sustainable epoxy resins support their promise as benign surfaces and scaffolds for a variety of applications ranging from adhesives and antifouling coatings to wound healing and tissue engineering.
Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose-block-2-methylaminoethyl methacrylate) [P(MAG-b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG-b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG-b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG-b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG-b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG-b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG-b-MAEMt) N/P = 5 and P(MAG-b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG-b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG-b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.
It was discovered that the original version ofFigure 2 contained errors, where incorrect image files were used in panels a, c, and f. Panels b, d, and e were correct and thus not in need of change. The correct image files were recovered out of existing raw data for Figure 2a (pMAT-b-AEMA-1 before lyophilization; the original micrograph in 2d was correct), whereas new electron micrographs for Figure 2c and 2f (pMAT-b-AEMA-3
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