Magic Plastic Bullet:Active Targeting Of Tumors Through Conformational Epitope Imprinting

Molecular imprinting refers to a technology that embeds a template molecule in polymer matrices, creating cavities, after template dissociation, that not only retain the shape memory of the template but also specifically recognize it. This technology has been successfully used for constructing various polymer materials that bind specifically to peptide and protein templates, providing a promising tool for molecular design, drug discovery, and drug delivery. Active tumor/cancer-targeting drug delivery is a tumor-treatment strategy that improves tumor cell-specific uptake of drug molecules with enhanced therapeutic efficacy. This strategy entails specific recognition of the surface marker of a tumor cell by a ligand either directly conjugated to the drug molecule or fabricated on a vehicle containing it. The targeting ligands can be small molecules as well as macromolecules such as peptides and proteins. Unlike a small molecule ligand, a peptide or protein ligand enjoys superior specificity and high affinity for tumor markers; however, it often suffers poor in vivo stability, immunogenicity, and high costs of production. Novel chemistries are needed to design therapeutically viable ligands for anticancer drug delivery that share the best features of both small molecules and macromolecules. It has been challenging to develop imprinted polymer materials that recognize the conformational epitope of a protein- the mode of recognition that predominates in biology. Here, we report the design of a "plastic magic bullet" that specifically recognize the tumor cell membrane protein targets as a result of the combination of surface molecular imprinting and scaffold-based peptide design.In the first chapter, we explore that a novel strategy named conformational epitope imprinting. By inverse microemulsion polymerization, the surface imprinted nanoparticles was prepared by using acrylamide as functional monomer, N,N’-Methylenebisacrylamide as cross-linker, and apamin or a linear analog of apamin (in which four cysteines were changed to alanines) as the template. The obtained nanoparticles had a suitable sub-micrometer particle size with homogeneous distribution. Using transmission electron microscopy(TEM) to observe the morphological and structure of the nanoparticles, it showed that the epitope imprinting nanoparticles was prepared successfully. Notably, synthetic polymeric nanoparticles that were prepared using a linear analog of apamin as the imprinting template exhibited an extremely low response to apamin as compared with the nanoparticles prepared using apamin as the template. The specificity of this recognition of apamin by the nanoparticles in vivo was demonstrated by the neutralization of apamin by the nanoparticles. In spinal cord tissues, we observed a markedly reduced distribution of FITC-labeled apamin following either intravenous administration of a preincubated mixture of the nanoparticles and apamin or sequential injection of nanoparticles and apamin. Moreover, the imprinted nanoparticles functioned as a "defense system" and neutralized the "launched" apamin-modified micelles in vivo, regardless of whether or the micelles were administered before or after the nanoparticles.These results indicated that consistency in the secondary structure between the imprinted template and the target peptide is more critical than the consistency in the primary sequence is for achieving potent binding between a molecularly imprinted polymer and its corresponding target. In addition, we mastered the methods of preparing imprinted polymer nanoparticles and measuring the interactions of nanoparticles and template.Based on the results of the first chapter, in the second chapter, the dispersed residues located in the N-terminal a-helix of p32 were grafted into the corresponding site of apamin. The produced peptide, HAPPE (hybrid apamin-p32 polypeptide) almost maintained the exact featured structure stabilized by two disulfide bonds. Acrylamide and N,N’-methylenebisacrylamide were used as functional monomers and the molecularly imprinted polymeric nanoparticles (MIPNPs) were synthesized using inverse microemulsion polymerization. The nanoparticles obtained exhibited a uniform particle size of approximately 40 nm and a narrow size distribution. We used the fluorescence polarization (FP) technique to quantify the interaction of MIPNPs with recombinant p32. The binding affinity was calculated to be 11.9-40.1 nM. The results of other experiments showed that as compared with NIPNPs, MIPNPs exhibited no specific binding to phospholipase A2, which possesses an N-terminal a-helix, or to other proteins such as mouse nerve growth factor (NGF). The results of flow cytometry assays showed that 4T1 murine breast-cancer cells and BxpC-3 human pancreatic-cancer cells exhibited substantially three times higher uptake of MIPNPs than of NIPNPs. Our results showed that intravenous administration of nanoparticles encapsulating a near-infrared fluorophore (IR-783 dye) led to considerably higher accumulation in tumors of MIPNPs than of NIPNPs in the mouse xenograft 4T1 tumor model and BxPC-3 tumor model. Furthermore, the results of both cell-toxicity assays and pathological analysis of tissue sections demonstrated high biocompatibility of the prepared MIPNPs.In conclusion, we have presented a conformational epitope imprinting strategy to construct a novel tumor-targeted drug-delivery system; in this strategy, the functional peptide is used, for the first time, as an indirect targeting ligand of the targeted drug carrier. This work on the "plastic magic bullet" might provide a promising alternative for the peptide-modified nanocarriers that are currently used for tumor-targeted drug delivery and should have a broad impact on the development of polymeric nanoparticle-based targeted diagnoses and therapies used for a great variety of human diseases.