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.

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.

•A facile method to prepare biocompatible mimetic enzymes to maintain catalytic activity in complex medium was demonstrated.•The mimetic properties are ascribed to single compact zwitterionic layer composed by primary amine and carboxyl groups.•The single zwitterionic layer of mimetic enzymes significantly reduces the interaction between them and protein or cells.The dendrimer based synthetic mimetic enzyme has been drawing great attention. However, this mimetic enzyme is different from the natural enzymes, which are pH sensitive, biocompatible and keep their catalytic activity in biological complex medium. A single zwitterionic layer composed by primary amine and carboxyl groups may be a useful method to obtain these properties. Herein, we report a novel facile method to prepare a mimetic enzyme. The complexes of generation 5 poly(amido amine) dendrimers (G5 PAMAM) with free hemin (G5Hs) were modified by the maleic anhydride and cysteamine. Results showed that the mimetic enzymes (G5HMCs) had pH sensitivity and good stability by varying the pH from 4 to 9, while significant precipitation was observed for free hemin at pH 5 after two days. The G5HMC (3:1) showed optimal catalytic activity at its isoelectric point. Furthermore, G5HMCs displayed excellent biocompatibility. The G5HMCs incubated with fibrinogen were stable for 24 h, while G5Hs immediately formed large aggregates. G5HMC (3:1 2 mg/mL) displayed little cytotoxicity with HeLa cells or A549 cells for 24 h, while G5H (3:1) had serious cytotoxicity, which was also demonstrated by cell morphology observation. At last, G5HMCs fully preserved their catalytic activity in bovine serum albumin (BSA) solution compared with phosphate buffer saline (PBS) solution, while hemin decreased to 73.5–81.5% catalytic activity in BSA solution, which was caused by the less interaction with BSA for G5HMCs than free hemin. The surface functionalization schemes described in this report would represent a versatile method to prepare water-soluble, pH sensitive, biocompatible, and efficient artificial enzymes for biomedical related applications.Download high-res image (181KB)Download full-size image

The development of stable and biocompatible gold nanoparticles has been drawing great interest. Dendrimers could be used as templates to entrap gold nanoparticles. Herein, dendrimer templates were prepared by the modification of generation 5 polyamidoamine (G5 PAMAM) dendrimers with maleic anhydride and cysteamine. Then modified dendrimer-encapsulated gold nanoparticles (Au-G5MC NPs) were formed by incubation of the templates with chloroauric acid and reduction with sodium borohydride. The resulting Au-G5MC NPs showed high stability, biocompatibility, and catalytic efficiency in water, which is ascribed to the single zwitterionic layer composed of amine and carboxyl groups. The single zwitterionic layer was shown to provide the Au-G5MC NPs with excellent stability in both phosphate-buffered saline and fibrinogen solutions. The mixed solution of Au-G5MC NPs with fibrinogen remained stable within 24 h, while the mixed solution of G5 PAMAM dendrimer-entrapped gold nanoparticles (Au-G5 NPs) with fibrinogen formed obvious aggregates immediately. Au-G5MC NPs displayed little cytotoxicity (>90% cell viability) against HeLa cells up to 100 μg mL−1, while Au-G5 NPs showed obvious cytotoxicity (60% cell viability). In addition, Au-G5MC NPs displayed high catalytic efficiency for the reduction reaction of 4-nitrophenol (4-NP) in water. This method can be used to prepare a variety of highly stable, biocompatible, and efficient dendrimer-encapsulated catalysts.

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.

In this study, a versatile fabrication method for coating enzyme-based biosensors with ultrathin antifouling zwitterionic polymer films to meet the challenge of the long-time stability of sensors in vivo was developed. Electrochemically mediated atom transfer radical polymerization (eATRP) was applied to polymerize zwitterionic sulfobetaine methacrylate monomers on the rough enzyme-absorbed electrode surfaces; meanwhile, a refined overall bromination was developed to improve the coverage of polymers on the biosensor surfaces and to maintain the enzyme activity simultaneously for the first time. X-ray photoelectron spectroscopy and atomic force microscopy were used to characterize the properties of the polymer layers. The antifouling performance and long-time stability in 37 °C undiluted bovine serum in vitro were evaluated. The results showed that the polymer brush coatings diminished over 99% nonspecific protein adsorption and that the sensitivity of the evaluated sensor was maintained at 94% after 15 days. The overall sensitivity deviation of 7% was nearly 50% lower than that of the polyurethane-coated ones and also much smaller than the current commercially available glucose biosensors. The results suggested that this highly controllable electrodeposition procedure could be a promising method to develop implantable biosensors with long-time stability.

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.

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.

Polyurethane with zwitterionic side chains (PCB-ester-PU) based on a poly(carboxybetaine) ester analogue is developed for marine coatings and biomedical applications by introducing dihydroxy-terminated PCB-ester(OH)2 with different polymerization as the macrodiol, 4,4′-diphenylmethane diisocyanate (MDI) as the diisocyanate, and 1,4-butanediol (1,4-BD) as the chain extender. Robust coatings are obtained and exhibit long-term excellent resistance to nonspecific protein adsorption, bacterial adhesion, and human umbilical vein endothelial cell (HUVEC) attachment after hydrolysis. Tests of adhesion on different substrates and film hardness indicate that the material possesses far more stable mechanic properties than hydrogel coatings. Moreover, such a resistance can be generated not only by alkaline solution, but also by a physiological buffer (such as phosphate-buffered saline (0.15 M pH 7.4 PBS)) or by steam in an autoclave. Ultimately, its excellent long-term nonfouling property, its healing capability through self-regeneration and superior mechanic properties (such as hardness and elasticity), and its good adhesiveness as a paint on both polar and nonpolar substrates make this material an ideal candidate as a coating for marine and medical devices.Keywords: carboxybetaine; coatings; hydrolysis; nonfouling; polyurethane; zwitterionic polymers

Polymer–protein interactions are crucial for determining the activity of both polymer and protein for many bio-related applications. Poly(ethylene glycol) (PEG) as a well-known antifouling material is often coated on surfaces to form highly solvated brushes, which exhibit excellent protein-repellent properties. However, unlike surface-induced antifouling effects, little is known about the intrinsic PEG–protein interactions in aqueous solution, which is an important yet neglected problem. Here, we investigate the interactions between PEG and proteins in aqueous solution using fluorescence spectroscopy, atomic force microscopy (AFM), and nuclear magnetic resonance (NMR). Two important characteristics, molecular weight of PEG and mass ratio of PEG:protein, are examined to determine the effect of each on PEG–protein interactions as well as binding characteristics between PEG and proteins. In contrast to too long and too short PEG chains, collective results have shown that PEG with optimal molecular weight (MW) is more capable of interacting with proteins, which induces the conformational change of proteins through more stable binding sites and stronger interactions with long chain PEG. Enhanced PEG–protein interactions are likely due to the change of hydrophilicity to amphiphilicity of PEG with increasing MWPEG. In contrast to almost none or weak interactions of PEG surfaces with proteins, this work provides new evidence to demonstrate the existence of interactions between PEG and proteins in aqueous solution, which is important not only for better understanding of the structure–activity relationship of PEG both in solution and on surfaces, but also for the rational design of new PEG-based materials for specific applications.

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.

•Highly hemocompatible doxorubicin-loaded micelles with reversible cross-linkage were developed.•It is proved the effectiveness of zwitterionic carboxybetaine layer to resist nonspecific protein adsorption on nano drug vehicles (NDV).•The copolymers of two kind of monomers with large difference in hydrophobicity (carboxybetaine methacrylate vs 2-(methacryloyloxy)ethyl lipoate) were successfully prepared and used for drug encapsulation.•A simple method to pre-evaluate the hemocompatibility of NDV is developed.Both blood stability and intelligent-responsiveness after reaching the drug-targeting site are very important features to make desirable nano-drug vehicles (NDVs). Here, a highly nonfouling cross-linked micelle based on a copolymer composed of carboxybetaine methacrylate (CBMA) as hydrophilic segment and 2-(methacryloyloxy)ethyl lipoate (MAEL) as hydrophobic and cross-linked segment is reported. Furthermore, a simple method to evaluate the hemocompatibility of NDVs through examining the activation of a blood-clotting protein (fibrinogen) was introduced. The micelles can encapsulate anticancer drug doxorubicin (DOX) conveniently and release DOX quickly in response to an intracellular reductive environment. With the advantages of excellent stability in fibrinogen (1 mg/mL) PBS solution and 50% fetal bovine serum (FBS), and accelerated intracellular drug release, the biocompatible zwitterionic micelles stabilized by reversible cross-linkage might be a promising drug carrier for cancer chemotherapy.

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.

The dendrimer based synthetic enzyme mimic, so-called ‘dendrizyme’, has been of great interest since the early days of dendrimers. However, there is a lack of an effective way to obtain a natural enzyme mimic showing both good biocompatibility and high preservation of catalytic activity in biological complex medium simultaneously. Here we report a novel approach – the synthesis of a generation five dendrimer of poly(amido amine) (PAMAM) incorporating hemin through capping with carboxybetaine acrylamide (CBAA), which could function as peroxidase. Results showed that the nanocapsules of hemin using CBAA-modified PAMAM dendrimers (CBAA-H-PAMAM) exhibited excellent biocompatibility and full preservation of catalytic activity in bovine serum albumin (BSA) solution, compared with free hemin. Results indicated that the ultra-thin shell of zwitterionic CBAA groups reduced nonspecific interaction with proteins while it did not cause any obvious rise in hindrance to mass transfer. Furthermore, the synthetic peroxidase mimic (CBAA-H-PAMAM) exhibited remarkable temperature endurance as compared with natural proteins. Taken together, our results indicate that protein surface mimicking through CBAA attachment might open a new route for synthetic enzymes in biomedical related applications.

PEGylation of protein drugs has thus far been the most important method used in improving the stability and the circulation time, while lowering the immunogenicity of protein drugs. However, the loss of bioactivity of PEGylated protein drugs and the low recovery through the complicated separation process are the most challenging issues. To overcome these problems, a zwitterionic block copolymer poly(methyl acrylic acid-b-sulfobetaine methacrylate) (PMAA-b-PSBMA or abbreviated to PMS) has been chosen to modify a protein drug (uricase) since zwitterionic materials are superior to polyethylene glycol (PEG) in resistance to nonspecific protein adsorption, chemical stability and also low cytotoxicity. As indicated by the results, the conjugation between PMS and uricase could be achieved under 0.03 mg mL−1 of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.018 mg mL−1N-hydroxysuccinimide (NHS) through weak charge-induced adsorption of PMS on uricase, which is about 0.3% of the concentration for conventional protein–polymer conjugation. The conjugates, efficiently separated from the reaction solution and re-suspended in physiological solution through pH adjustment from pH 5.35 to 7.4, show about 133% of the original activity and better anti-trypsin digestion than the native uricase. These results demonstrate that the conjugation of a zwitterionic copolymer with a short poly(methyl acrylic acid) block could improve the stability of an enzyme without destructively affecting its bioactivity. This may open a new way for the delivery of therapeutic protein drugs.

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.Keywords: carboxybetaine; hydrolysis; nonfouling; nonstick; wound dressing;

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.

The strong surface hydration layer of nonfouling materials plays a key role in their resistance to nonspecific protein adsorption. Poly(sulfobetaine methacrylate) (polySBMA) is an effective material that can resist nonspecific protein adsorption and cell adhesion. About eight water molecules are tightly bound with one sulfobetaine (SB) unit, and additional water molecules over 8:1 ratio mainly swell the polySBMA matrix, which is obtained through the measurement of T2 relaxation time by low-field nuclear magnetic resonance (LF-NMR). This result was also supported by the endothermic behavior of water/polySBMA mixtures measured by differential scanning calorimetry (DSC). Furthermore, by comparing both results of polySBMA and poly(ethylene glycol) (PEG), it is found that (1) the hydrated water molecules on the SB unit are more tightly bound than on the ethylene glycol (EG) unit before saturation, and (2) the additional water molecules after forming the hydration layer in polySBMA solutions show higher freedom than those in PEG. These results might illustrate the reason for higher resistance of zwitterionic materials to nonspecific protein adsorptions compared to that of PEGs.

The strong surface hydration layer of nonfouling materials plays a key role in their resistance to nonspecific protein adsorption. Poly(ethylene glycol) (PEG) is an effective example of materials that can resist nonspecific protein adsorption and cell adhesion. Thus, the strong interaction between water molecules and PEG was investigated through each T2 component in water/PEG mixtures using multiexponential inversion of T2 relaxation time measured by the Carr–Purcell–Meiboom–Gill (CPMG) sequence of low-field nuclear magnetic resonance (LF-NMR). Results show that about one water molecule is tightly bound with one ethylene glycol (EG) unit, and additional water molecules over 1:1 ratio mainly swell the PEG matrix and are not tightly bound with PEG. This result was also supported by the endothermic behavior of water/PEG mixtures measured by differential scanning calorimetry (DSC). It is believed that the method developed could be also applied to investigate various interactions between macromolecules and other small molecules without using deuterium samples, which might open a novel route to quantitatively measure guest–host interactions in the future.

To overcome major challenges of non-specific protein adsorption on nanoparticles for nanosensing and nanodiagnosis, an efficient method for robust chemical modification was developed to achieve excellent specific biorecognition and long-term stability in complex biomedia. This method is demonstrated by a highly specific and sensitive immunoassay (IA), using superparamagnetic nanospheres (NSs) with high magnetite content. The non-specific protein adsorption on the NSs was suppressed dramatically when modified with dual functional poly(carboxybetaine methacrylate) (polyCBMA) via surface-initiated atom transfer radical polymerization (SI-ATRP) and chemically grafted with antibodies of the β subunit of human chorionic gonadotrop (anti-β-hCG). The response to hCG of IA NSs with polyCBMA coatings was highly consistent in either phosphate-buffered saline (PBS) or 50% fetal bovine serum (FBS), which is far less variable than the response of the IA NSs without polyCBMA coatings. After all, a very robust platform for IA NSs with excellent specific biorecognition was obtained. It is expected that this method for nanoparticle modification could be widely used in ultrasensitive nanosensing and nanodiagnosis in the future.

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.

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.

The dendrimer based synthetic enzyme mimic, so-called ‘dendrizyme’, has been of great interest since the early days of dendrimers. However, there is a lack of an effective way to obtain a natural enzyme mimic showing both good biocompatibility and high preservation of catalytic activity in biological complex medium simultaneously. Here we report a novel approach – the synthesis of a generation five dendrimer of poly(amido amine) (PAMAM) incorporating hemin through capping with carboxybetaine acrylamide (CBAA), which could function as peroxidase. Results showed that the nanocapsules of hemin using CBAA-modified PAMAM dendrimers (CBAA-H-PAMAM) exhibited excellent biocompatibility and full preservation of catalytic activity in bovine serum albumin (BSA) solution, compared with free hemin. Results indicated that the ultra-thin shell of zwitterionic CBAA groups reduced nonspecific interaction with proteins while it did not cause any obvious rise in hindrance to mass transfer. Furthermore, the synthetic peroxidase mimic (CBAA-H-PAMAM) exhibited remarkable temperature endurance as compared with natural proteins. Taken together, our results indicate that protein surface mimicking through CBAA attachment might open a new route for synthetic enzymes in biomedical related applications.

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.

PEGylation of protein drugs has thus far been the most important method used in improving the stability and the circulation time, while lowering the immunogenicity of protein drugs. However, the loss of bioactivity of PEGylated protein drugs and the low recovery through the complicated separation process are the most challenging issues. To overcome these problems, a zwitterionic block copolymer poly(methyl acrylic acid-b-sulfobetaine methacrylate) (PMAA-b-PSBMA or abbreviated to PMS) has been chosen to modify a protein drug (uricase) since zwitterionic materials are superior to polyethylene glycol (PEG) in resistance to nonspecific protein adsorption, chemical stability and also low cytotoxicity. As indicated by the results, the conjugation between PMS and uricase could be achieved under 0.03 mg mL−1 of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 0.018 mg mL−1N-hydroxysuccinimide (NHS) through weak charge-induced adsorption of PMS on uricase, which is about 0.3% of the concentration for conventional protein–polymer conjugation. The conjugates, efficiently separated from the reaction solution and re-suspended in physiological solution through pH adjustment from pH 5.35 to 7.4, show about 133% of the original activity and better anti-trypsin digestion than the native uricase. These results demonstrate that the conjugation of a zwitterionic copolymer with a short poly(methyl acrylic acid) block could improve the stability of an enzyme without destructively affecting its bioactivity. This may open a new way for the delivery of therapeutic protein drugs.

Polymer–protein interactions are crucial for determining the activity of both polymer and protein for many bio-related applications. Poly(ethylene glycol) (PEG) as a well-known antifouling material is often coated on surfaces to form highly solvated brushes, which exhibit excellent protein-repellent properties. However, unlike surface-induced antifouling effects, little is known about the intrinsic PEG–protein interactions in aqueous solution, which is an important yet neglected problem. Here, we investigate the interactions between PEG and proteins in aqueous solution using fluorescence spectroscopy, atomic force microscopy (AFM), and nuclear magnetic resonance (NMR). Two important characteristics, molecular weight of PEG and mass ratio of PEG:protein, are examined to determine the effect of each on PEG–protein interactions as well as binding characteristics between PEG and proteins. In contrast to too long and too short PEG chains, collective results have shown that PEG with optimal molecular weight (MW) is more capable of interacting with proteins, which induces the conformational change of proteins through more stable binding sites and stronger interactions with long chain PEG. Enhanced PEG–protein interactions are likely due to the change of hydrophilicity to amphiphilicity of PEG with increasing MWPEG. In contrast to almost none or weak interactions of PEG surfaces with proteins, this work provides new evidence to demonstrate the existence of interactions between PEG and proteins in aqueous solution, which is important not only for better understanding of the structure–activity relationship of PEG both in solution and on surfaces, but also for the rational design of new PEG-based materials for specific applications.

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.