Between Dec 19, 2024 and Jan 2, 2025, datasets can be submitted to DRUM but will not be processed until after the break. Staff will not be available to answer email during this period, and will not be able to provide DOIs until after Jan 2. If you are in need of a DOI during this period, consider Dryad or OpenICPSR. Submission responses to the UDC may also be delayed during this time.

Browsing by Subject "Nucleic acid delivery"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Synthesis and Characterization of Polycations with Various Structural Features for Nucleic Acid Delivery
    (2014-07) Wu, Yaoying
    Cationic polymers have been widely explored as non-viral nucleic acid delivery vectors for gene therapy, as they are able to complex with nucleic acids, protect genetic materials from degradation, and facilitate the internalization of transgene expression process. To understand how various structural elements impact the nucleic acid delivery, several classes of polycations were synthesized and examined for their in vitro transfection performance. Reversible addition-fragmentation chain transfer (RAFT) polymerization was employed for the synthesis of several series of diblock glycopolycations with different carbohydrate containing blocks, including poly(2-deoxy-2-methacrylamido glucopyranose) (PMAG) and poly(methacrylamidotrehalose) (PMAT). Amine containing monomers were subsequently polymerized through chain extension to yield cationic blocks for nucleic acid binding, including N-[3-(N, N-dimethylamino) propyl] methacrylamide (DMAPMA), N-(2-aminoethyl) methacrylamide (AEMA), aminoethylmethacrylate (AEMT), N-methyl aminoethylmethacrylate (MAEMT), N,N-dimethyl aminoethylmethacrylate (DMAEMT), and N,N,N-trimethylammoniumethylmethacrylate (TMAEMT). Initially, it was demonstrated that these polymers were all able to complex plasmid DNA into polyplex structures and prevent colloidal aggregation of polyplexes in physiological salt conditions. More importantly, glycopolymers with PMAT block can prevent polyplexes from aggregation, and exhibit the ability to protect polyplexes through the cycle of lyophilization and reconstitution. The role of charge type, block length, and cell type on transfection efficiency and toxicity were studied for the in vitro transfection in both HeLa (human cervix adenocarcinoma) and HepG2 (human liver hepatocellular carcinoma) cells by comparing the polyplexes formulation created with PMAG-b-PAEMA and PMAG-b-PDMAPMA. 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, polyplex 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. 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. Lastly, a new class of lipophilic polycations were designed, poly(alkylamidoamine) (PAAA) was synthesized to understand the role of lyophilicity in pDNA transfection. PAAAs with various length of lipophilic linker (C3 to C6) were synthesized via step-growth polymerization, and examined for their in-vitro transfection efficiency and cytotoxicity in multiple cell types, including HDFa (human dermal fibroblasts, adult) cells, HeLa (human cervix adenocarcinoma) cells, HMEC (human mammary epithelial cells), and HUVEC (human umbilical vein endothelial cells). The PAAA vehicles exhibited comparable or even superior transfection efficiency to Lipofectamine 2000, a leading lipid-based transfection reagent. It was revealed that, for the PAAA polymers examined in this study, an increase in lipophilicity leads to higher cytotoxicity, but also raised the transfection efficiency in HeLa cells. Overall, we demonstrated the great potential of carbohydrate containing polymer block, which has potential to serve as a targeting moiety, stealthy coating, and even lyo-protectant for polyplex formulations. Different amine types were examined for their nucleic acid delivery ability, and we conclude that tertiary amine containing monomer, DMAPMA, exhibits high toxicity along with low transfection efficiency while primary amine block shows relatively lower toxicity and higher transfection efficiency. Finally, the lipophilic polycations, PAAAs, were shown to be important for improving transfection efficiency in multiple cell types.