Engineered Polymer–Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems

Abstract

Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems, which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface, and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatized sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anticancer drug (paclitaxel), and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the nonselectively derivatized conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tf-receptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel, when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems, which include engineering specific residues for conjugation that mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.

Precision Conjugation: An Emerging Tool for Generating Protein–Polymer Conjugates

“…Fore xample, Alexander and co-workers genetically engineered five transferrin variants whereby mutant cysteine was distributed at five distinct sites for the construction of five different sitespecific transferrin-polymer conjugates. [31] Secondly,t he dispersity of polymers synthesized by SIG is inevitable because of the intrinsic mechanisms of controlled polymerization techniques, [73] while the polypeptides synthesized by IPF have precisely defined molecular weights and sequences.T hirdly, multistep operations related to genetic engineering,p rotein chemistry,a nd polymer chemistry technologies are involved in SIG,w hich is unfavorable for technology transfer, but, on the other hand, makes SIG amodular and flexible method to generate multifunctional protein-polymer conjugates.I n contrast, only ao ne-step operation of genetic engineering is required in IPF,which makes IPF simpler and easier to realize technology transfer to the biopharmaceutical industry,a s evidenced by several ELP-and XTEN-fused proteins undergoing clinical trials. [63] Fourthly,the intrinsic biodegradability of the polypeptides synthesized by IPF is beneficial to in vivo applications,b ut leads to short circulation half-lives for their protein conjugates,whereas the polymers synthesized by SIG are typically non-biodegradable,but their protein conjugates have long circulation half-lives.Inthe cases of IFN-ELP and IFN-POEGMA, which have similar polymer molecular weights of about 40 kDa, their in vivo half-lives vary greatly, being 8.6 and 31 h, respectively.…”

“…Secondly, a high conjugation efficiency can be reached by SIG (typically >50 %) and IPF (100 %), which is particularly advantageous to the production of site‐specific protein–polymer conjugates with high molecular weights. For example, a conjugated synthetic polymer of over 600 kDa and an IDP of nearly 200 kDa were synthesized by SIG and IPF, respectively [16, 66] Thirdly, multifunctionality such as self‐assembly and responsibility can be introduced into site‐specific protein–polymer conjugates by SIG and IPF, [17, 20, 21, 23, 24, 26, 27, 29, 31, 36–39, 41, 60–62] which extremely expands the scopes of protein–polymer conjugates in terms of the structure, function, and application.…”

“…Firstly, the conjugation site of a protein in IPF is limited to the N‐/C‐terminus, while any site on a protein can, in principle, be the conjugation site in SIG through protein engineering technologies such as cysteine mutation and unnatural amino acid insertion. For example, Alexander and co‐workers genetically engineered five transferrin variants whereby mutant cysteine was distributed at five distinct sites for the construction of five different site‐specific transferrin–polymer conjugates [31] . Secondly, the dispersity of polymers synthesized by SIG is inevitable because of the intrinsic mechanisms of controlled polymerization techniques, [73] while the polypeptides synthesized by IPF have precisely defined molecular weights and sequences.…”

Insights on the Structure, Molecular Weight and Activity of an Antibacterial Protein–Polymer Hybrid

“…Protein–polymer hybrid molecules are inspiring biomaterials because they retain the functions/properties of both protein and polymeric materials . For example, upon attachment of neutral polymers, the host protein often shows enhanced stability in the biological environment .…”

“…Protein-polymer hybrid molecules are inspiring biomaterials because they retain the functions/properties of both protein and polymericm aterials. [1][2][3][4][5][6] For example, upon attachmento f neutralp olymers, the host protein often shows enhanced stability in the biological environment. [7][8][9][10] This is especially useful for protein delivery wherein the protein often experiences varied (and often harsh) biochemical environments.…”