No abstract

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or “poly(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylate (AEMA) at three degrees of polymerization (n=34, 65, and 84). Two series of experiments were conducted to study these di block copolymers in detail and to compare their properties to two control polymer [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose)-coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nano-based technologies and macromolecules, in particular, those related to nanomedicine applications.

A series of diblock glycopolycations were created by polymerizing 2-deoxy-2-methacrylamido glucopyranose (MAG) with either a tertiary amine-containing monomer, N-[3-(N,N-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, N-(2-aminoethyl) methacrylamide (AEMA). Seven structures were synthesized via aqueous reversible addition–fragmentation chain transfer (RAFT) polymerization that varied in the block lengths of MAG, DMAPMA, and AEMA along with two homopolymer controls of DMAPMA and AEMA that lacked a MAG block. The polymers were all able to complex plasmid DNA into polyplex structures and to prevent colloidal aggregation of polyplexes in physiological salt conditions. In vitro transfection experiments were performed in both HeLa (human cervix adenocarcinoma) cells and HepG2 (human liver hepatocellular carcinoma) cells to examine the role of charge type, block length, and cell type on transfection efficiency and toxicity. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased in the glycopolycation-based polyplexes, toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Lastly, cell type played a major role in efficiency. These glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend, signifying that the MAG block could aid in hepatocyte transfection.

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of alpha-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multi-epitope nanofibers in a modular fashion.

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.