Polysarcosine as an Alternative to PEG for ... - ACS Publications

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Polysarcosine as an Alternative of PEG for Therapeutic Protein Conjugation Yali Hu, Yingqin Hou, Hao Wang, and Hua Lu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00237 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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Bioconjugate Chemistry

Polysarcosine as an Alternative of PEG for Therapeutic Protein Conjugation Yali Hu,1,2 Yingqin Hou,1 Hao Wang, 1 and Hua Lu1,* 1

Beijing National Laboratory for Molecular Sciences, Center for Soft Matter Science

and Engineering, Key Laboratory of Polymer Chemistry and Physics of Ministry of Education, College of Chemistry and Molecular Engineering; 2Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies; Peking University, Beijing 100871, China.

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Abstract The performances of many therapeutic proteins, including human interferon-α2b (IFN), are often impeded by their intrinsic instability to protease, poor pharmacokinetics, and strong immunity. Although PEGylation has been an effective approach improving the pharmacokinetics of many proteins, a few noticeable limitations have aroused vast research efforts in seeking alternatives of PEG for bioconjugation. Herein, we report our investigation on the use of polysarcosine (PSar), a non-ionic and hydrophilic polypeptoids, 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.


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Introduction Protein-polymer conjugates are biohybrids in which the bioactive proteins are covalently modified with synthetic polymers. Presumably, the polymer-conjugation can stabilize the therapeutic proteins from protease digestion, improve their pharmacokinetics in vivo, and sometimes enhance their activities.1-3 Currently, all therapeutic protein-polymer conjugates on market are based on poly(ethylene glycol) (PEG), the so called PEGylation. Albeit effective, there is a growing awareness of a few intrinsic limitations of PEG including non-biodegradable backbone, reported patient hypersensitivity, and accelerated blood clearance (ABC effect).4 This has stimulated vast research efforts in seeking alternatives of PEG for protein conjugation including poly(2-oxazoline)s,5 polyglycerol,6 zwitterionic polymers,7 OEGylated poly(meth)acrylates,8,9 and poly(amino acid)s.10 Recently, polysarcosine (PSar), a polypeptoid based on the endogenous but non-proteinogenic amino acid sarcosine, has been considered an emerging “stealth” polymer for many biomedical applications.11-14 In one sense, the non-ionic and highly hydrophilic PSar can afford a large hydrodynamic volume and thus impart the desirable nonfouling character.15 More interestingly, there has been accumulated evidence suggesting that the biodegradable PSar is more biocompatible and less immunogenic than PEG. For instance, PSar has been used to coat the surface of inorganic particles such as quantum dots and gold nanorods, which incurred less nonspecific interactions and systemic toxicity, preserved higher colloidal stability, and gave longer circulation time in vivo than the PEGylated ones.16-18 PSar-conjugated Indocyanine Green (ICG), a small molecular photosensitizer for photoacoustic tumor imaging, has shown enhanced tumor accumulation and a higher signal/noise ratio in vivo as compared with a PEG-modified ICG analogue.19 Copolymers containing PSar designed for peptide or protein encapsulation have preserved more activity than PEG did.20,21 In light of these advances, we hypothesize that PSar is a promising material for therapeutic protein conjugation. Notably, although the conjugation of PSar to peptides



has been nicely done,22 the generation and systematic pharmacological evaluation of PSar-protein conjugate remains absent.

Results and discussion Synthesis and Characterization. To test the hypothesis, we synthesized PSar-IFN, a N-terminal specific PSar-modified human interferon-α2b (IFN). IFN was selected because it was a therapeutic cytokine requiring PEGylation for longer circulating half-life.23 A N-terminal cysteine functionalized IFN mutant (Cys-IFN) was produced for native chemical ligation (NCL) following a previously reported method.24 To generate PSar for Cys-IFN conjugation, we aimed to prepare a phenyl thioester-capped PSar via trimethylsilyl phenyl sulfide (PhS-TMS)-mediated ring-opening polymerization (ROP) of sarcosine N-carboxyanhydrides (SarNCA).25 However, our initial attempts failed because PhS-TMS was unable to directly initiate the ROP of SarNCA, possibly due to the steric hindrance of the N-methyl group. To circumvent the problem, as shown in Scheme 1, we charged a small amount (5 equiv) of γ-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)esteryl L-glutamate N-Carboxyanhydride (EG3GluNCA) to PhS-TMS (1 equiv) to extend a very short P(EG3Glu) linker before the addition of SarNCA (200 equiv). This allowed the smooth growth of PSar after P(EG3Glu) in one-pot and afforded the phenyl thioester-functionalized PSar (PhS-PSar). Gel permeation chromatography (GPC) and 1H NMR characterization of PhS-PSar collectively indicated that the polymer had a molecular weight ~12 kDa with a polydispersity index (PDI) of 1.06 (Figure 1A and S1). Next, the N-terminal specific protein conjugate PSar-IFN was generated in high yield via the chemoselective NCL under a mild condition. To include a positive control, a PEGylated IFN (PEG-IFN) was produced via the same method by coupling a thioester-functionalized PEG (1H NMR and MALDI available in Figure S2, PDI 1.04) and Cys-IFN (Scheme 1). We selected the 10 kDa PEG for conjugation because it has a comparable hydrodynamic radius with our PSar of 12 kDa.26 Size exclusion chromatography (SEC) analysis of the two conjugates gave the same elution time,


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suggesting the two conjugates shared a similar hydrodynamic volume (Figure 1B). This notion was also supported well by the dynamic light scattering (DLS) measurement, which gave 12.6 ± 0.7 and 12.8 ± 0.4 nm diameters for PSar-IFN and PEG-IFN, respectively (Figure 1C). SDS-PAGE gel analysis confirmed the successful generation and high purity of the two conjugates (Figure 1D). Interestingly, the apparent MW of both PEG-IFN and PSar-IFN, indicated by the protein ladder in the SDS-PAGE gel, did not agree well with their calculated sizes. Of note, such discrepancies were also frequently observed in the SDS-PAGE of other protein-polymer conjugates.27 Apart from the MW, many other parameters including the chemical structure, polarity and conformation of the polymers may alter their electrophoresis behaviors. Here, it was hypothesized that the rigidity difference of PEG and PSar (Kuhn length was 1.5 nm for PSar while 1.1 nm for PEG) 28 may affect the migration of the corresponding conjugates in the PAGE gel.

Scheme 1. Synthesis of PSar-IFN (A) and PEG-IFN (B).



Figure 1. Characterization: (A) gel permeation chromatography of the polymer PhS-PSar, (B) size exclusion chromatography, (C) dynamic light scattering, and (D) SDS-PAGE electrophoresis of the two conjugates PSar-IFN and PEG-IFN.

In vitro activity. To investigate the protease resistance and thermostability of the conjugates, we performed trypsin digestion and thermofluor assays. Compared with the wild type IFN (wt IFN) that was digested by trypsin rapidly within 2 h, both conjugates showed significantly enhanced stability under the same condition, with ~50% conjugates remain intact ~8 h after the treatment (Figure 2A and S3). Thermofluor assay revealed that the melting temperature (Tm) of the two conjugates were comparable with wt IFN, suggesting both PEG and PSar had very few influence to the thermostability of IFN (Figure S4). To test the antiproliferation activity of the two IFN conjugates, Daudi cells were incubated with the drugs at varied concentrations for 48 h. The half inhibition concentration (IC50) of wt IFN, PSar-IFN, and PEG-IFN was found to be 13, 80, and 136 pg/mL, respectively (Figure 2B). This result indicated that PSar-IFN was slightly more potent than PEG-IFN. Notably, both PSar and PEG polymers were essentially nontoxic to Daudi cells (Figure 2C),


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implying the cell killing ability of the conjugates was fully derived from the protein IFN. We further tested the binding affinity of the conjugates to the receptor of interferon-α2b (IFNAR2) by surface plasmon resonance (SPR) analysis (Figure 2D-F). The dissociation constant (KD) was measured to be 1.07, 1.37, and 1.70 nM for wt IFN29,30, PSar-IFN and PEG-IFN, respectively (Table 1). Moreover, it was found that the kon was 5.04 × 106 and 2.04 × 106 M−1 s−1 for PSar-IFN and PEG-IFN, respectively. This result indicated that whereas the binding affinity of the two conjugates were comparable, PSar-IFN could associate with the receptor molecule more rapidly than PEG-IFN, which might explain the toxicity difference shown in Figure 2B.

Figure 2. In vitro assessment of the conjugates. (A) trypsin digestion assay; (B and C)



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relative viabilities of Daudi cells treated with wt IFN, PSar-IFN, PEG-IFN (B), PSar, and PEG (C) for 48 h. Data are expressed as means ± SD. (D-F) Surface plasmon resonance analysis of the binding of wt IFN (D), PSar-IFN (E) and PEG-IFN (F) to IFNAR2.

Table 1. Surface plasmon resonance analysis of the binding of wt IFN, PSar-IFN and PEG-IFN to IFNAR2.

kon (M-1s-1)

koff (s-1)

KD (M)

wt IFN

1.57 × 107

0.01672

1.07 × 10-9

PSar-IFN

5.04 × 106

0.00692

1.37 × 10-9

PEG-IFN

2.04 × 106

0.003474

1.70 × 10-9

Pharmacokinetics, biodistribution, and immunogenicity. Next, we examined the performance of the conjugates in live animals. To analyze the pharmacokinetics (PK), wt IFN and the two conjugates were intravenously injected into Sprague−Dawley (SD) rats and the plasma IFN levels at different time points were measured by enzyme-linked immunosorbent assay (ELISA). The measured elimination half-life of wt IFN was almost equally extended from ~0.8 h to ~4.8 and 4.6 h for PSar-IFN and PEG-IFN, respectively (Figure 3A). To explore the biodistribution of different conjugates, all IFN variants were labeled with Cy5 (Figure S5) and injected intravenously into BALB/c nude mice bearing OVCAR3 tumors. The major organs and tumors were extracted 24 h after administration and analyzed with an in vivo imaging equipment. It was found that kidney was the brightest organ for all IFN variants, suggesting the renal clearance was the major elimination route (Figure 3B). This was not surprising considering the size of the conjugates shown in DLS (Figure 1C). Apart from the kidney, liver and tumor also displayed considerably higher fluorescent intensities than other organs including the heart, spleen and lung. Compared with PEG-IFN, interestingly, PSar-IFN was shown to accumulate more in the tumor (p value < 0.05) and have less exposure in the liver (p value < 0.01) (Figure


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3 B and C). To evaluate the ability of the polymers in shielding IFN from immune recognition, PSar-IFN and PEG-IFN were intravenously administrated to immune competent SD rats weekly (n = 4). ELISA analysis indicated that, upon repetitive administration, PSar-IFN elicited significantly less amount of anti-IFN IgG in week 3 than those produced by PEG-IFN (Figure 3D). Overall, the results suggested that PSar was an excellent stealth polymer for protein modification.

Figure 3. In vivo pharmacological evolution of the conjugates: (A) plasma IFN concentration at varied time points; (B) biodistribution at 24 h estimated by the fluorescence intensity of the extracted tissues; (C) fluorescent images of tumors at 24 h; (D) the development of anti-IFN IgG at different time after weekly administration of the conjugates. Data are expressed as means ± SD. *p

Polysarcosine as an Alternative to PEG for ... - ACS Publications

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