The assembly of gold nanoparticles (AuNPs) to AuNP assemblies is of interest for cancer therapy and imaging. Herein we introduce a new and general paradigm, thermally triggered AuNP assembly, for the development of novel intelligent platforms for cancer photothermal therapy (PTT) and multimodal imaging. Site-specific conjugation of a thermally sensitive elastin-like polypeptide (ELP) to AuNPs yields thermally sensitive ELP-AuNPs. Interestingly, ELP-AuNPs can in situ form AuNP assemblies composed of short necklace-like gold nanostructures at elevated temperatures and thus show strong near-infrared light absorption and high photothermal effect. These thermally responsive properties of ELP-AuNPs enable simultaneous photothermal/photoacoustic/X-ray computed tomographic imaging and PTT of melanoma after single intratumoral injection of ELP-AuNPs. The thermally triggered assembly of a variety of nanoparticles with optical, electronic, and magnetic properties into nanoparticle assemblies may open new ways for the establishment of intelligent platforms for various applications in biomedicine.Keywords: cancer therapy; elastin-like polypeptide; gold nanoparticle; hyperthermia; photothermal therapy;

Glucose oxidase (GOX) can convert glucose into gluconic acid and hydrogen peroxide (H2O2), which is potentially useful for synergistic cancer-starving and oxidation therapy. Herein we demonstrate a glucose-responsive nanomedicine made of GOX–polymer nanogels to regulate H2O2 production for synergistic melanoma starving and oxidation therapy. GOX–polymer nanogels showed glucose-responsive H2O2-generating activity in vitro, improved stability, and considerably enhanced tumor retention as compared to native GOX. More importantly, they exhibited high antimelanoma efficacy and no obvious systemic toxicity, whereas native GOX was ineffective and systemically toxic at the same dose. This work paves the way for establishing an endogenous and noninvasive cancer treatment paradigm that is based on intratumoral glucose-responsive, H2O2-generating chemical reactions.Keywords: cancer therapy; glucose oxidase; nanogel; protein−polymer conjugate; synergistic therapy;

The covalent attachment of protein-resistant polymers to therapeutic proteins is a widely used method for extending their in vivo half-lives; however, the effect of molecular weight of polymer on the in vitro and in vivo functions of protein-polymer conjugates has not been well elucidated. Herein we report the effect of molecular weight of poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) on the in vitro and in vivo properties of C-terminal interferon-alpha (IFN)-POEGMA conjugates. Increasing the molecular weight of POEGMA decreased the in vitro activity of IFN-α but increased its thermal stability and in vivo pharmacokinetics. Intriguingly, the in vivo antitumor efficacy of IFN-α was increased by increasing the POEGMA molecular weight from ca. 20 to 60 kDa, but was not further increased by increasing the molecular weight of POEGMA from ca. 60 to 100 kDa due to the neutralization of the improved pharmacokinetics and the reduced in vitro activity. This finding offers a new viewpoint on the molecular size rationale for designing next-generation protein-polymer conjugates, which may benefit patients by reducing administration frequency and adverse reactions, and improving therapeutic efficacy.治疗性蛋白质共价结合蛋白质抗性聚合物是被广泛使用的延长其体内半衰期的方法. 然而, 聚合物的分子量对蛋白质- 聚合物偶 联物体外和体内性能的影响尚未得到很好的阐明. 本文报道了聚(寡聚乙二醇甲基丙烯酸酯) (POEGMA)的分子量对C末端修饰干扰素-α (IFN)-POEGMA偶联物的体外和体内性能的影响. 增加POEGMA的分子量会降低IFN-α的体外活性, 但改善了其热稳定性和体内药代动力 学. IFN-α的体内抗肿瘤功效随着POEGMA分子量从20增加至60 kDa而增强, 但进一步提高POEGMA的分子量到100 kDa, 由于其生物活性 的降低中和了药代动力学的改善作用, 并不能进一步增强其体内抗肿瘤功效. 该发现为蛋白质- 聚合物偶联物分子尺寸效应提供了新的佐 证, 可为设计下一代蛋白质-聚合物偶联物提供参考, 通过减少给药频率和不良反应并改善治疗效果而使患者受益.

The challenge in photothermal therapy (PTT) is to develop biocompatible photothermal transducers that can absorb and convert near-infrared (NIR) light into heat with high efficiency. Herein, we report salt-induced aggregation of gold nanoparticles (GNPs) in biological media to form highly efficient and biocompatible NIR photothermal transducers for PTT and photothermal/photoacoustic (PT/PA) imaging of cancer. The GNP depots in situ formed by salt-induced aggregation of GNPs show strong NIR absorption induced by plasmonic coupling between adjacent GNPs and very high photothermal conversion efficiency (52%), enabling photothermal destruction of tumor cells. More interestingly, GNPs in situ aggregate in tumors to form GNP depots, enabling simultaneous PT/PA imaging and PTT of the tumors. These findings may provide a simple and effective way to develop a new class of intelligent and biocompatible NIR photothermal transducers with high efficiency for PT/PA imaging and PTT.

Conjugating therapeutic proteins and peptides to poly(ethylene glycol) (PEG) can improve their pharmacokinetics and therapeutic potential. However, PEGylation suffers from non-specific conjugation, low yield and immunogenicity. Herein we report a new and general methodology to synthesize a protein-polymer conjugate with site-specificity, high yield and activity, long circulation half-life and excellent therapeutic efficacy. A phospholipid polymer, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was grown solely from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) PMPC conjugate of interferon-alpha in high yield. Notably, the PMPC conjugate showed 194- and 158-fold increases in systemic exposure and tumor uptake as compared with interferon-alpha, respectively. The in vitro antiproliferative bioactivity of the PMPC conjugate was 8.7-fold higher than that of PEGylated interferon-alpha (PEGASYS). In a murine cancer model, the PMPC conjugate completely inhibited tumor growth and cured 75% mice, whereas at the same dose, no mice treated with interferon-alpha or PEGASYS survived. We believe that this new approach to synthesize C-terminal protein conjugates of PMPC may be applicable to a large subset of protein and peptide drugs, thereby providing a general platform for the development of next-generation protein therapeutics.

Tumor-homing and pH-responsive polypeptide–drug nanoparticles for targeted cancer therapy are precisely designed by site-specific drug conjugation to a bioactive and well-defined elastin-like polypeptide through an acid-labile linker. In a murine cancer model, these nanoparticles show significantly better anti-tumor efficacy and less systemic toxicity than not only free drugs, but also polypeptide–drug nanoparticles without the tumor-homing function.

•We introduce the concept of well-defined protein–polymer conjugates.•We describe how to synthesize well-defined protein–polymer conjugates.•We come up the concept of site-specific in situ polymerization (SIP).•We depict chemical and biological tools for site-specific protein modifications.Covalently conjugating proteins with synthetic polymers, particularly poly(ethylene glycol) (PEG) is widely used as a means to improve protein solubility and stability, prolong their circulating half-lives, and lower their immunogenicity. Conventionally, these polymers are attached to random locations on the protein surfaces through the modification of the reactive side chains of amino acid residues such as lysine and cysteine. The “grafting to” polymer conjugation usually leads to heterogeneous products with reduced activity and low yield, which may not be compatible with the intended applications. Therefore, it is highly desirable to synthesize well-defined protein–polymer conjugates by site-specific polymer conjugation. Recently, in situ growth of polymer conjugates from proteins (“grafting from”) has emerged as an alternative to the “grafting to” method. Particularly, site-specific in situ growth of polymer bioconjugates (SIP) is promising in overcoming the limitations of the “grafting to” method. In this review, we introduce the chemistry for synthesis of well-defined protein–polymer conjugates, and emphasize the SIP method as the next-generation platform for synthesis of well-defined protein–polymer conjugates. Furthermore, we exemplify biomedical applications of well-defined protein–polymer conjugates. In the end, we come up potential directions in this research field.Figure optionsDownload full-size imageDownload as PowerPoint slide

Conjugating poly(ethylene glycol) (PEG), PEGylation, to therapeutic proteins is widely used as a means to improve their pharmacokinetics and therapeutic potential. One prime example is PEGylated interferon-alpha (PEGASYS). However, PEGylation usually leads to a heterogeneous mixture of positional isomers with reduced bioactivity and low yield. Herein, we report site-specific in situ growth (SIG) of a PEG-like polymer, poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), from the C-terminus of interferon-alpha to form a site-specific (C-terminal) and stoichiometric (1:1) POEGMA conjugate of interferon-alpha in high yield. The POEGMA conjugate showed significantly improved pharmacokinetics, tumor accumulation and anticancer efficacy as compared to interferon-alpha. Notably, the POEGMA conjugate possessed a 7.2-fold higher in vitro antiproliferative bioactivity than PEGASYS. More importantly, in a murine cancer model, the POEGMA conjugate completely inhibited tumor growth and eradicated tumors of 75% mice without appreciable systemic toxicity, whereas at the same dose, no mice treated with PEGASYS survived for over 58 days. The outperformance of a site-specific POEGMA conjugate prepared by SIG over PEGASYS that is the current gold standard for interferon-alpha delivery suggests that SIG is of interest for the development of next-generation protein therapeutics.

Tumor-homing and pH-responsive polypeptide–drug nanoparticles for targeted cancer therapy are precisely designed by site-specific drug conjugation to a bioactive and well-defined elastin-like polypeptide through an acid-labile linker. In a murine cancer model, these nanoparticles show significantly better anti-tumor efficacy and less systemic toxicity than not only free drugs, but also polypeptide–drug nanoparticles without the tumor-homing function.