Protein PEPylation: A New Paradigm of Protein-Polymer Conjugation.

Various polymers have been tested for protein conjugation with a goal of bridging the complementary advantages of both components. However, many of these polymers, including the most well-established PEG, are nondegradable, which raises potential concerns on their cumulative chronic toxicity. Moreover, the immunogenicity of PEG has recently evoked considerable controversy. Synthetic polypeptides, on the other hand, are biomimetic polymers with tunable degradability, versatile side chain functionalities, unique secondary structures, and fascinating self-assembly behaviors. These properties have made them promising materials in protein modification for various applications. In this Topical Review, we summarize recent advances and list a number of interesting future directions in protein-polypeptide conjugation, which we termed protein PEPylation.

Enzyme-Activatable Interferon-Poly(α-amino acid) Conjugates for Tumor Microenvironment Potentiation.

Protein-polymer conjugation is a clinically validated approach to enhanced pharmacokinetic properties. However, the permanent attachment of polymers often leads to irreversibly reduced protein bioactivity and poor tissue penetration. As such, the use of protein-polymer conjugates for solid tumors remains elusive. Herein, we report a simple strategy using enzyme-activatable and size-shrinkable protein-polypeptide conjugates to overcome this clinical challenge. Briefly, a matrix metalloproteinase (MMP)-responsive peptide sequence is introduced between a therapeutic protein interferon (IFN) and a synthetic polypeptide P(EG3Glu)20. The resulting site-specific MMP-responsive conjugate, denoted as PEP20-M-IFN, can, therefore, release the attached P(EG3Glu)20 to achieve both protein activation and deep penetration into the tumor microenvironment (TME). Compared to a similarly produced nonresponsive analogue conjugate PEP20-IFN, our results find PEP20-M-IFN to show higher bioactivity in vitro, improved tumor retention, and deeper penetration in a MMP2-dependent manner. Moreover, systemic administration of PEP20-M-IFN shows outstanding antitumor efficacy in both OVCAR3 and SKOV3 ovarian tumor models in mice. This work highlights the releasable PEPylation strategy for protein drug potentiation at the TME and opens up new opportunities in clinics for the treatment of malignant solid tumors.

Polysarcosine as an Alternative to PEG for Therapeutic Protein Conjugation.

The performance of many therapeutic proteins, including human interferon-α2b (IFN), is often impeded by their intrinsic instability to protease, poor pharmacokinetics, and strong immunity. Although PEGylation has been an effective approach to improve the pharmacokinetics of many proteins, a few noticeable limitations have aroused vast research efforts in seeking alternatives to PEG for bioconjugation. Herein, we report our investigation on the use of polysarcosine (PSar), a nonionic and hydrophilic polypeptoid, for IFN modification. The site-specific conjugate PSar-IFN, generated by native chemical ligation in high yield, is systematically compared with a similarly produced PEG-interferon conjugate (PEG-IFN) to evaluate the in vitro and in vivo behaviors. PSar is found to show comparable ability in stabilizing IFN from protease digestion in vitro and prolonging the circulation half-life in vivo. Interestingly, PSar-IFN retains more activity in vitro and accumulates more in the tumor sites upon systemic administration than PEG-IFN. Most importantly, PSar-IFN is significantly more potent in inhibiting tumor growth and elicits considerably less anti-IFN antibodies in mouse than PEG-IFN. Together, our results demonstrate for the first time that PSar is an outstanding candidate for therapeutic protein conjugation. Considering the low toxicity, biodegradability, and excellent stealth effect of PSar, this study suggests that such polypeptoids hold enormous potential for many biomedical applications including protein delivery, colloidal stabilization, and nanomedicine.

Phosphatase-triggered cell-selective release of a Pt(iv)-backboned prodrug-like polymer for an improved therapeutic index.

We describe here the synthesis and cell-selective delivery of a cationic Pt(iv)-backboned prodrug-like polymer P(DSP-DAEP). P(DSP-DAEP) features excellent aqueous solubility, unusually high (44.5%) drug loading, can be rapidly reduced to release the active cisplatin, and is more potent than its small molecular Pt(iv) precursor DSP. P(DSP-DAEP) can be formulated with an oppositely charged methoxyl poly(ethylene glycol)-block-poly(l-phosphotyrosine) (mPEG-b-PpY) to afford a polyion micelle (Pt-PIC) by taking advantage of polyelectrolyte coacervation. Preliminary in vitro cellular uptake and cytotoxicity assays indicate that Pt-PIC exhibits receptor (surface alkaline phosphatase)-dependent uptake and cytotoxicity. Overall, our results suggest a new approach to the improved therapeutic index of platinum-based anticancer drugs via cell-selective delivery.

Harnessing Phosphato-Platinum Bonding Induced Supramolecular Assembly for Systemic Cisplatin Delivery.

To improve the therapeutic index of cisplatin (CDDP), we present here a new paradigm of drug-induced self-assembly by harnessing phosphato-platinum complexation. Specifically, we show that a phosphato-platinum cross-linked micelle (PpY/Pt) can be generated by using a block copolymer methoxy-poly(ethylene glycol)-block-poly(l-phosphotyrosine) (mPEG-b-PpY). Coating of PpY/Pt with a R9-iRGD peptide by simple mixing affords a targeting micelle with near neutral-charged surface (iPpY/Pt). The micelles feature in well-controlled sizes below 50 nm and high stability under physiological conditions, and can withstand various environmental stresses. Importantly, the micelles demonstrate on-demand drug release profiles in response to pathological cues such as high ATP concentration and acidic pH. In vitro, the micelles are efficiently internalized and almost equally potent compared to CDDP. Moreover, iPpY/Pt induce greater cytotoxicity than PpY/Pt in a 3D tumor spheroid model likely due to its deeper tumor penetration. In vivo, the micelles exhibit prolonged circulation half-lives, enhanced tumor accumulation, excellent tumor growth inhibition in a xenograft HeLa model and an orthotropic mammary 4T1 model, and improved safety profiles evidenced by the reduced nephrotoxicity. Together, this work demonstrates for the first time that phosphato-platinum complexation can be exploited for effective delivery of CDDP, and suggests a paradigm shift of constructing nanosystems for other anticancer metallodrugs.