Spontaneously Restoring Specific Bioaffinity of RGD in Linear RGD-containing Peptides by Conjugation with Zwitterionic Dendrimers.

Peptides are more versatile than small molecule drugs, but their specific bioaffinities are usually lower than their original native proteins because of the loss of preferred conformations. To overcome this key obstacle, we demonstrated a hydrogen bond-induced conformational constraint method to enhance the specific bioaffinities of peptides to achieve a high success rate by using linear RGD-containing peptides as a model of bioactive peptides. By performing molecular simulation, we found that the chemically immobilized linear CRGDS via cysteine (C) at the N-terminus on zwitterionic PAMAM G-5 can not only spontaneously restore the natural conformation of the RGD segment through the assistance of the dynamic hydrogen bond from serine (S) at the C-terminus of the peptide, but it can also narrow the distribution of all possible conformations. Consequently, the conjugates showed comparable or even better high affinity than native proteins without the use of conventional, labor-intensive, synthesis-based structure search methods to construct a binding conformation. In addition, the conjugates showed globular protein-like characteristics chemically, physically, and physiologically. They exhibited not only high efficacy and biosafety both in vitro and in vivo, but they also showed extremely high thermostability even upon boiling in a solution. This approach offers great design flexibility for reviving functional peptides without impairing their high specific affinity for their targets. STATEMENT OF SIGNIFICANCE: In this work, we developed a swift approach to spontaneously restore the natural conformation of a linear peptide from a nature protein and thus enhance its specific bioaffinity instead of constructing a binding conformation by the labor-intensive, synthesis-based structure search method. In details, our new approach involves dynamically constraining the linear peptide on a zwitterionic PAMAM G-5 surface by a combination of chemical bonding at one terminus and dynamic hydrogen bonding at the other terminus of the linear peptide. The zwitterionic background offers abundant interaction sites for hydrogen bonding as well as resistance to nonspecific interactions. This approach fully restores the specific bioaffinity of RGD segments on a zwitterionic PAMAM G-5 through only one conjugation point at the C-terminus of the peptide. Moreover, the bioaffinity of all three types of RGD-containing peptides is successfully restored, which indicates the high rate of success of this approach in affinity restoring.

Development of Zwitterionic Polypeptide Nanoformulation with High Doxorubicin Loading Content for Targeted Drug Delivery.

Much attention has been drawn to targeted nanodrug delivery systems due to their high therapeutic efficacy in cancer treatment. In this work, doxorubicin (DOX) was incorporated into a zwitterionic arginyl-glycyl-aspartic acid (RGD)-conjugated polypeptide by an emulsion solvent evaporation technique with high drug loading content (45%) and high drug loading efficiency (95%). This zwitterionic nanoformulation showed excellent colloidal stability at high dilution and in serum. The pH-induced disintegration and enzyme-induced degradation of the nanoformulation were confirmed by dynamic light scattering and gel permeation chromatography. Efficient internalization of DOX in the cells and high antitumor activity in vitro was observed. Compared with the free drug, this nanoformulation showed higher accumulation in tumor and lower systemic toxicity in vivo. The DOX-loaded zwitterionic RGD-conjugated polypeptide vesicles show potential application for targeted drug delivery in the clinic.

Development of ionic strength/pH/enzyme triple-responsive zwitterionic hydrogel of the mixed l-glutamic acid and l-lysine polypeptide for site-specific drug delivery.

Environmentally responsive hydrogels show enormous potential in various applications, such as tissue engineering and drug delivery. The site-specific controlled drug delivery of hydrogels can improve the therapeutic outcome and minimize the negative side effects. In this work, enzymatically digestible hydrogels, which are composed of equally mixed l-glutamic acid (E) and l-lysine (K) polypeptides after being crosslinked by the coupling reaction between carboxyl groups and primary amines catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide·HCl (EDC·HCl), were prepared to improve the biocompatibility through reducing the nonspecific protein adsorption and cell attachment. Hydrogels loaded with two model drugs, doxorubicin hydrochloride (DOX·HCl) (positively charged anti-cancer drug) and diclofenac sodium (negatively charged anti-inflammatory drug), showed accelerated complete drug release and full enzymatic degradation in the presence of trypsin, which was reported to be expressed in various carcinomas and inflammations. The drug release also responds to the pH change through tuning charge-charge interaction. These indicated that the prepared hydrogels were promising candidates for drug delivery systems.

Development of polypeptide-based zwitterionic amphiphilic micelles for nanodrug delivery.

Protein molecules, which typically have a hydrophobic core and a zwitterionic shell with a polypeptide backbone, could be ideal materials for nanodrug vehicles (NDVs) with low side effects. Here, we synthesized poly(l-aspartic acid(lysine))-b-poly(l-lysine(Z)) (PAsp(Lys)-b-PLys(Z)) (PALLZ), a novel amphiphilic block polypeptide with key structures of protein to investigate the possibility for use as a NDV. This polypeptide can spontaneously self-assemble into micelles in aqueous solution with a zwitterionic brush (the PAsp(Lys) part) to provide the nonfouling shell and a hydrophobic core (the PLys(Z) part) for loading hydrophobic drugs. The doxorubicin (DOX) loaded PALLZ micelles showed excellent resistance to nonspecific protein adsorption in FBS, which leads to very low internalization. Moreover, PALLZ micelles showed no cytotoxicity to MCF7, HeLa and HepG-2 cells up to 500 µg mL-1. All these results indicated that zwitterionic amphiphilic block polypeptides could be promising materials for NDVs.