RNA interference (RNAi) mediated through double stranded small interfering RNA (siRNA) is a promising therapeutic strategy for a variety of diseases.3
Effective siRNA delivery results in highly specific gene knockdown, providing a means to reduce expression of virtually any protein target with lower doses and less toxicity than other RNAi mechanisms.4,5
However, the successful in vivo delivery of siRNA is a formidable challenge. Although a wide variety of carriers for siRNA have been explored and consist of polymers, peptides, and lipids, a bottleneck for efficient siRNA therapy lies in the inability to specifically target active, non-degraded siRNA to tissues of interest.
Typically, cationic carriers have been employed for siRNA delivery.1,2,6–9
Through electrostatically complexation with negatively-charged phosphate groups of nucleic acids, cationic carriers imbibe siRNA with protection against enzymatic degradation and a means to enter cells through endocytosis. However, this approach renders a delivery system that lacks cellular specificity. Folic acid has shown great promise as a targeting ligand for a number of in vivo applications.10–13
While folic acid is internalized by the low affinity reduced folate carrier expressed by nearly all cells (Km
, it has greater affinity (Kd
towards the folate receptor, which is overexpressed in a number of tumors and cancer cell lines10–13
. Folic acid is internalized via the folate receptor in two steps. First, the folate binds to the receptor and then it is transferred into the cell by receptor-mediated endocytosis, allowing large drug carrier systems, including micelles, proteins, liposomes, and nanoparticles to enter the cell via endo-lysosomal trafficking17
. However, irrespective of specificity, once trafficked to the endo-lysosomal pathway, cargo typically becomes degraded in lysosomes. Thus an effective, efficient in vivo siRNA delivery system must be multifunctional and provide protection against enzymatic degradation, be targeted to and internalized by the desired cell type, and result in intracellular trafficking to the intracellular environment where the target is located.
We have previously reported the development of cationic and pH-responsive, endosomolytic diblock copolymers as potent siRNA delivery systems in vitro.1,2,9,18
These carriers were formed using reversible addition-fragmentation chain transfer polymerization (RAFT), a type of living radical polymerization (LRP). A distinct advantage of polymeric carrier systems, and particularly polymers synthesized via LRPs, is the ease with which modularity can be introduced to incorporate many functionalities.1,19–21
LRPs provide polymers with narrow molecular weight distributions and a wide range of functional polymers with defined architectures.19,20,22–25
RAFT, similar to other LRPs, uses a chain transfer agent (CTA), which typically consists of a dithioester or trithiocarbonate, and reactive Rand Z-groups, where the resultant polymer contains the same R and Z functionalities at its chain-ends. Reactive R- and Z-group modified CTAs have been employed to prepare alpha and omega-functional polymers, respectively, that are directly reactive toward biomolecules.26,27
More recently, this approach has been used to exploit pyridyl-disulfide groups to enable reversible conjugation of siRNA23
and proapoptotic peptides18 to polymers for enhanced delivery characteristics. In addition, chain extension strategies have been employed to specifically and reproducibly functionalize the omega-end of RAFT polymers with a multitude of functional groups, including folic acid.28
However, these synthetic schemes requires subsequent conjugation steps to introduce the targeting or drug functionality whereas the current approach achieves functionalization concurrently with RAFT polymer synthesis.
To our knowledge there have been no reports of targeting strategies introduced using the RAFT chain transfer agent directly. Herein, we report the straightforward de novo synthesis of a RAFT CTA with a folate-functionalized R-group. The strategy is different from reported LRP strategies to make targeted polymers29–33
and involves synthesis of a novel chain transfer agent (CTA) for RAFT polymerization. To achieve potent, pH-responsive siRNA carriers with precise presentation of folate and narrow polydispersities, RAFT polymerization was utilized in the synthesis of a diblock copolymer consisting of dimethylaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid (DMAEMA-b-DMAEMA-co-BMA-co-PAA), a terpolymer previously described for its potency for in vitro intracellular siRNA delivery.1,2
The presence of tertiary and partially protonated amines in the first block allows for complexation and protection of siRNA and the second block imbibes pH-responsive endosomolytic behavior. Moreover, each polymer presents targeting moieties meerly through use of the folate-functionalized CTA. As demonstrated through polymer characterization methods, competitive folate binding assays, and gene knockdown studies, this multifunctional polymer provides potent siRNA delivery to cancer cells that overexpress folate receptors.