Guanglong Ma

Reducing the Cytotoxity of Poly(amidoamine) Dendrimers by Modification of a Single Layer of Carboxybetaine

The surface primary amines of generation five poly(amido amine) (G5 PAMAM) dendrimer were modified by different amounts of carboxybetaine acrylamide (CBAA). As a result, the fully modified molecules (CBAA-PAMAM-20, obtained from the 20:1 molar ratio of CBAA molecules to amino groups in modification solution) show excellent compatibility with protein and cells. CBAA-PAMAM-20 and fibrinogen (Fg) could coexist in solution without forming aggregation, indicating very weak interaction force between CBAA-PAMAM-20 and fibrinogen. CBAA-PAMAM-20 exhibits almost undetectable hemolytic activity, while other partially modified ones cause severe hemolysis and fibrinogen aggregation. Furthermore, the membrane of human umbilical vascular endothelial cell (HUVEC) remains intact after 24 h incubation with CBAA-PAMAM-20. The cytotoxicity assay of HUVEC cells and KB cells also showed that the CBAA-PAMAM-20 was not cytotoxic up to a 2 mg/mL concentration (>90% cell viability). In short, a thin compact layer of zwitterionic carboxybetaine could reduce the cytotoxicity of PAMAM through minimizing the interaction with protein and cell membranes, which suggest that the carboxybetaine-coated PAMAM could be a useful platform for biocompatible carriers to load contrast agents and drugs.

Development of Zwitterionic Polymer-Based Doxorubicin Conjugates: Tuning the Surface Charge To Prolong the Circulation and Reduce Toxicity

Polymer-drug conjugates are commonly used as nano drug vehicles (NDVs) to delivery anticancer drugs. Zwitterionic polymers are ideal candidates to conjugate drugs because they show higher resistance to nonspecific protein adsorption in complex media than that of nonionic water-soluble polymers, such as poly(ethylene glycol). However, the charge balance characteristics of zwitterionic polymers used as NDVs will be broken from the inclusion of additional charged groups brought by conjugated drugs or functional groups, leading to the loss of resistance to protein adsorption. Consequently, the nonspecific protein adsorption on drug carriers will cause fast clearance from the blood system, an immune response, or even severe systemic toxicity. To overcome this drawback, a model zwitterionic polymer, poly(carboxybetaine methacrylate) (pCBMA), was modified by the introduction of a negatively charged component, to neutralize the positive charge provided by the model drug, doxorubicin (DOX). A DOX-conjugated NDV which possesses excellent resistance to nonspecific protein adsorption was achieved by the formation of a strongly hydrated pCBMA shell with a slightly negative surface charge. This kind of DOX-conjugated NDV exhibited reduced cytotoxicity and prolonged circulation time, and it accelerated DOX release under mild acid conditions. In tumor-bearing mouse studies a 55% tumor-inhibition rate was achieved without causing any body weight loss. These results indicate the importance of charge tuning in zwitterionic polymer-based NDVs.

Development of Nonstick and Drug-Loaded Wound Dressing Based on the Hydrolytic Hydrophobic Poly(carboxybetaine) Ester Analogue

A novel biocompatible polymer is developed for antimicrobial and nonstick coatings of wound dressing. The polymer is formed by copolymerization of carboxybetaine ester analogue methacrylate (CB-ester) and small partial poly(ethylene glycol) methacrylate (PEGMA) for cross-linking by hexamethylene diisocyanate (HDI), which is highly resistant to nonspecific protein adsorption and mammalian cell attachment after a quick hydrolysis. A small hydrophobic drug, aspirin, can be incorporated into the new polymer and slowly released to inhibit microorganism growth while the new polymer shows very low cytotoxicity. Moreover, the wound dressing, the new polymer coated medical gauze, shows good mechanic properties, such as flexibility and strength, for medical application. After all, this new nonfouling polymer offers great potential for an antimicrobial wound dressing and other applications.

Biocompatible long-circulating star carboxybetaine polymers

Polyethylene glycol (PEG) is considered to be the most effective material to prolong the circulation time of nanoparticles by reducing non-specific protein adsorption in blood. However, it is recognized that PEG decomposes in most physiological solutions, and an anti-PEG antibody has been detected in some normal blood donors as a response to injection with PEGylated polymer particles. Zwitterionic polymers are potential alternatives to PEG for biomedical applications because of their super resistance to non-specific protein adsorption. Thus, finding one polymer with a long circulation time and resistance to the immune response is of significant importance. Here, we prepared four star carboxybetaine polymers of different molecular weights via atom transfer radical polymerization (ATRP) from a β-cyclodextrin (β-CD) initiator for investigating the biocompatibility of carboxybetaine polymer, a typical zwitterionic polymer. The circulation half-life of the largest star polymer (123 kDa) in mice was prolonged to 40 h in vivo, with no appreciable damage or inflammation observed in the major organ tissues. Furthermore, the circulation time of repeat injections showed similar results to the first injection, with no obvious increase in the amount of antibody in blood. The internalization of the star carboxybetaine polymers by macrophage cells was a relatively slow process. The high cell viability in the presence of star carboxybetaine polymers up to 2 mg mL-1 was maintained. The hemolytic activity of the star carboxybetaine polymers at 5 mg mL-1 was almost undetectable. In vitro results prove a key prediction of excellent biocompatibility in vivo. All the results suggest that the carboxybetaine polymer, perhaps even most of the zwitterionic ones, might be a good alternative to PEG in the development of a drug delivery system.

Surface protonation/deprotonation controlled instant affinity switch of nano drug vehicle (NDV) for pH triggered tumor cell targeting

To realize a fast and selective capture of nano drug vehicles (NDVs) by malignant tumors, an instant affinity switchable NDV based on pH-sensitive surface protonation was prepared. This NDV is prepared from a zwitterionic polymer system with an ultra resistant poly(carboxybetaine) (pCB) shell and sulfo groups near a hydrophobic doxorubicin (DOX) loaded core. The results show that this new NDV system had an instant and sensitive affinity switch from strong resistance in physiological pH to a high affinity to tumor cell membranes in the slightly acidic extracellular pH of malignant tumors depending on if the net charge of the NDV is reversed or not. The pH of this affinity switch can be controlled by the amount of the strong acidic sulfo groups that act as the zeta potential adjustor of the NDV near the inner hydrophobic core. As a result, this CB-based NDV was captured by solid tumor tissue efficiently and thus inhibited tumor growth much more effectively than the PEGylated NDV system, while eliminating unwanted side effects in other healthy tissues. This indicates that the affinity switch caused by protein-like surface protonation of the NDV is much more efficient than the traditional charge switchable NDV approaches.

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 nonfouling polypeptides with uniform alternating charges by polycondensation of the covalently bonded dimer of glutamic acid and lysine

In this work, nonfouling polypeptides with homogenous alternating charges were synthesized by polycondensation of the covalently bonded dimer of glutamic acid (E) and lysine (K) (EK dimer) with benzyloxycarbonyl (Z)-protected side chains. This facile method successfully solved the uniformity problem of nonfouling peptides caused by the copolymerization of two different monomers and enabled the incorporation of various terminal functional groups for future applications. The molecular weights (MWs) of the nonfouling peptides can be easily controlled by the ratio of the terminal group, lipoic acid, to the EK dimer. The nonfouling peptides can form self-assembling monolayers (SAMs) on a gold surface through two terminal thiol groups, which were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and ellipsometry (ELL). The resistance to nonspecific protein adsorption, cell attachment and bacterial adhesion of these nonfouling peptide SAMs and the in vitro cytotoxicity and haemolytic activity of these peptides were also evaluated. The results show that the lowest relative protein adsorptions of antibody (anti-IgG) and fibrinogen (Fg) on the SAMs are 5.1 ± 1.6% and 7.3 ± 1.8%, respectively, determined by enzyme-linked immunosorbent assay (ELISA), where the protein adsorption on a tissue culture polystyrene (TCPS) surface was set to 100%. Almost no obvious cell attachment and bacterial adhesion were observed, and no cytotoxicity and no haemolytic activity in vitro were detected. With the advantages of biocompatibility, biodegradability and the abundance of moieties for ligand immobilization, these nonfouling peptides developed by the facile method can be used in a wide range of biomedical applications.

Different in vitro and in vivo behaviors between Poly(carboxybetaine methacrylate) and poly(sulfobetaine methacrylate)

Poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA) are two well-known zwitterionic polymers known for their excellent antifouling properties. In this work, these two zwitterionic polymers were compared both in vitro and in vivo. Both of them exhibited excellent antifouling properties and low macrophage uptake although there were negligible differences in resistance to nonspecific protein adsorption of their hydrogels and cell internalization of their star polymers. However, it is found that the β- Cyclodextrin-CBMA (CD-CBMA) showed a circulation time one order of magnitude longer than CD-SBMA, which implied that small differences in vitro may lead to a dramatic difference in vivo. This work demonstrated that pCBMA showed greater potential than pSBMA in biomedical applications.

Development of Long-Circulating Zwitterionic Cross-Linked Micelles for Active-Targeted Drug Delivery

Blood stability, active targeting, and controlled drug release are the most important features to design desirable drug carriers. Here, we demonstrate a zwitterionic biodegradable cross-linked micelle based on a penta-block copolymer, which utilizes poly(carboxybetaine methacrylate) as hydrophilic segment, poly(ε-caprolactone) as biodegradable hydrophobic segment, poly(S-2-hydroxyethyl-O-ethyl dithiocarbonate methacrylate) (PSODMA) block as thiol protecting segment for cross-linking, and cyclic Arg-Gly-Asp-d-Tyr-Lys [c(RGDyK)] as targeting ligand. As a result, this micelle possessed excellent colloidal stability at high dilution and in 50% fetal bovine serum. In vitro drug release experiment showed no burst release under physiological conditions but accelerated drug release in mimicking tumor tissue environment. In vivo tests showed that the drug-loaded micelles had prolonged half-life in bloodstream, enhanced therapeutic efficiency, and reduced cardiac toxicity and biotoxicity compared with free drug formulation. Taken together, the reported c(RGDyK)-modified zwitterionic interfacially cross-linked micelle has emerged as an appealing platform for cancer therapy.

Biodegradable copolypeptide hydrogel prodrug accelerates dermal wound regeneration by enhanced angiogenesis and epithelialization

Hydrogels are one of the most promising wound dressings. However, their effectiveness on wound healing is still largely limited due to either the non-degradability or the release of non-therapeutic degradable products. Herein, a biodegradable copolypeptide hydrogel based on the glutamic acid and lysine was synthesized and applied as both wound dressing and therapeutic prodrug. The hydrogel can degrade in the existence of elevated degradative enzymes in a wound environment, which will release therapeutic amino acids to enhance the wound healing. In vivo results found that the hydrogel could effectively promote wound regeneration in both macroscopic and microscopic scales. Further investigation revealed that the wound healing effect of the hydrogel was highly attributed to its enhanced impact on angiogenesis, cell proliferation and re-epithelialization of the wound. All in all, the present study proves that the degradable copolypeptide hydrogel can efficiently improve wound healing and indicates its potent clinical application for wound regeneration.