Polycarboxybetaine Esters for Gene Delivery: Molecular Tuning, Controlled Degradation and Vaccine Applications

The safe and controlled delivery of genes to targeted cells promises exceptional advancements in clinical disease treatment, next-generation vaccines, and tissue engineering. [1-3] An ideal polymeric vector would condense nucleic acids into stable polyplexes that can successfully navigate the extracellular environment and pass through the cell membrane, as well as protect genes from degradation inside and outside the target cells and assist with escape from the endosome or trafficking vesicle. We have developed a gene delivery platform based on polycarboxybetaines (PCBs) modified with degradable esters, with the goal of optimizing each function without detriment to the others.

When the anionic carboxylate groups in zwitterionic PCB side chains are esterified, the polymer is rendered cationic and thus able to bind and condense nucleic acids. [4,5] A tertiary amine can be introduced in place of the quaternary ammonium to make the polymer pH-responsive, buffering pH changes during polyplex trafficking and providing a mechanism via the "proton sponge" for endosomal or phagosomal escape. Another key function of this platform is the hydrolytic conversion of cationic PCB esters to zwitterionic PCB. The rate of this charge shifting can be easily modified by the incorporation of different ester leaving groups and different distances between the ester and the tertiary amine groups. This appends a "smart" characteristic to this polymer; DNA is first strongly complexed by cationic PCB-esters, but is efficiently released when ester degradation reveals the anionic carboxylate groups, rendering PCB zwitterionic and nontoxic.

In our recent study [6], the side chain length was varied to include either one or two alkyl spacers denoted between the amine and the carboxylate/ester groups. Additionally, each chain length was synthesized with both an ethyl ester and a tert-butyl ester hydrolytic group. These molecular modifications result in monomers and resulting polymers with varying proton buffering capacities and hydrolytic profiles. An alkyl spacer length of one brings the pKa of each amine into the optimal range for endosomal buffering, and the hydrolytic ester can be easily modified to speed or slow charge switching. An optimized polymer is able to deliver plasmid DNA an order of magnitude more effectively than polyethyleneimine, without demonstrating any cytotoxicity.

We have also developed a novel photolabile o-nitrobenzyl ester of polycarboxybetaine (PCB-NBE) to give this platform a UV- sensitive "switch" for active degradation control, to study how the charge neutralization caused by ester degradation directly catalyzes DNA release from a polyplex. Photoinitiated ester degradation precipitates the rapid release of over 70% of complexed DNA from PCB-NBE polyplexes. This molecule is promising for triggered release of nucleic acid therapies in vitro.

Extending the functionality of this polymer class, we have applied a similar platform to the passively targeted delivery of DNA vaccines with polycarboxybetaine micro-"gels". [7] These hydrogel microparticles target the phagocytosis pathway of immunostimulating cell types to delivery nucleic acid vaccines adsorbed to their surface. Tertiary amines and hydrolytic esters confer similar benefits of attraction, buffering, and release as they do in nanosized polyplexes. A model vaccine delivered to macrophage cells has resulted in over 10x higher expression than that delivered from a cationic CTAB microparticle control. This platform is also nontoxic and easily tunable.

References:

[1]        Sheridan, C. Nat. Biotechnol. 2011, 29, 121-128.

[2]        Ferraro, B.; Morrow, M. P.; Hutnick, N. A.; Shin, T. H.; Lucke, C. E.; Weiner, D. B. Clin. Infect. Dis. 2011, 53, 296-302.

[3]        Vallet-Regi, M.; Ruiz-Hernandez, E.; Gonzalez, B.; Baeza, A. J. Biomater. Tissue Eng. 2011, 1, 6-29.

[4]        Zhang, Z.; Cheng, G.; Carr, L. R.; Vaisocherova,́ H.; Chen, S.; Jiang, S. Biomaterials 2008, 29, 4719-4725.

[5]        Carr, L. R.; Jiang, S. Biomaterials 2010, 31, 4186-4193.

[6]        Sinclair, A.; Bai, T.; Carr, L. R.; Ella-Menye, J-R.; Zhang, L.; Jiang, S. Biomacromolecules 2013 (DOI: 10.1021/bm400221r)

[7]        Zhang, L.; Sinclair, A.; Cao, Z.; Ella-Menye, J-R.; Xu, X.; Carr, L. R.; Pun, S. H.; Jiang, S. Small 2013 (DOI: 10.1002/smll.201202727)