Polymerization Induced Self-Assembly of a Site-Specific Interferon Î±

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Polymerization induced self-assembly of a site-specific interferon alpha-block copolymer conjugate into micelles with remarkably enhanced pharmacology Xinyu Liu, Mengmeng Sun, Jiawei Sun, Jin Hu, Zhuoran Wang, Jianwen Guo, and Weiping Gao J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06013 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018

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Journal of the American Chemical Society

Polymerization induced self-assembly of a site-specific interferon alpha-block copolymer conjugate into micelles with remarkably enhanced pharmacology Xinyu Liu, Mengmeng Sun, Jiawei Sun, Jin Hu, Zhuoran Wang, Jianwen Guo, Weiping Gao* Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China Supporting Information Placeholder ABSTRACT: Conjugating a hydrophilic and protein-

resistant polymer to a protein is a widely used strategy to extend the in vivo half-life of the protein; however, the benefit of the half-life extension is usually limited by the bioactivity decrease. Herein we report a supramolecular self-assembly strategy of site-specific in situ polymerization induced self-assembly (SI-PISA) to address the dilemma. An amphiphilic block copolymer (POEGMAPHPMA) was directly grown from the C-terminus of an important therapeutic protein interferon alpha (IFN) to in situ form IFN-POEGMA-PHPMA conjugate micelles. Notably, the in vitro bioactivity of the micelles was 21.5fold higher than that of the FDA-approved PEGylated interferon alpha PEGASYS. Particularly, the in vivo half-life of the micelles (83.8 h) was 1.7- and 100-fold longer than those of PEGASYS (49.5 h) and IFN (0.8 h), respectively. In a tumor-bearing mouse model, the micelles completely suppressed tumor growth with 100% animal survival, whereas at the same dose, PEGASYS and IFN were much less effective. These findings suggest that SI-PISA is promising as a next-generation technology to remarkably enhance the pharmacological performance of therapeutic proteins with short circulation half-lives.

alpha (IFN) therapeutics, PEGASYS and PEGINTRON, are widely used in clinic for chronic hepatitis and cancer therapies7-10. However, PEGylation typically produces a product composed of positional isomers with significantly decreased bioactivities. For instance, PEGASYS was composed of 9 positional isomers with distinct reduced bioactivities11. The in vivo half-life of PEGASYS (77 h) in humans was 8.6-fold longer than that of native IFN (9 h), but the in vitro bioactivity of PEGASYS was just 17% of native IFN12. Most recently, we have found that increasing the molecular weight of polymer of sitespecific IFN-polymer conjugates prolonged the in vivo half-life of IFN, but decreased the in vitro bioactivity of IFN and could not always increase the in vivo therapeutic efficacy of IFN13. Therefore, it is highly valuable to address the dilemma that the benefit of the half-life extension is usually limited by the bioactivity decrease. Scheme 1. Site-specific in situ polymerization induced self-assembly (SI-PISA) for the synthesis of an IFN-POEGMA-PHPMA micelle (IFNmicelle) with greatly enhanced pharmacology for tumor therapy. Br

Initiator

ATRP

Sortase A IFN-Br

IFN

Therapeutic proteins are increasingly becoming popular in drug market owing to their intrinsic high activity and specificity1. However, most of therapeutic proteins except antibodies have short circulation half-lives due to their small size, poor stability, and potential immunogenicity, which limits their widespread applications in clinic2. Conjugating hydrophilic and protein-resistant polymers to proteins is a widely used strategy to extend the circulation half-lives of the proteins3,4. Poly(ethylene glycol) (PEG) is the most widely used polymer to modify proteins. Indeed, twelve PEGylated proteins have been approved by the USA Food and Drug Administration, and many more PEGylated proteins are under development5,6. As a prime instance, PEGylated interferon

OEGMA IFN-POEGMA O

ATRP O

HPMA

NH O N H

IFN-POEGMA-PHPMA (IFN-micelle)

Br

O

N H NH 2

Initiator

n

n

O

O

O

O 9O

POEGMA

O HO

PHPMA

The structure image of IFN was generated from PDB entry: 1lTF.

Herein we present a supramolecular self-assembly strategy of site-specific in situ polymerization induced self-assembly (SI-PISA), by combining site-specific in situ growth (SIG)14-16 with polymerization induced selfassembly (PISA)17-20, to address this challenge (Scheme 1). In this proof-of-concept study, a controlled radical

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polymerization technique, atom transfer radical polymerization (ATRP)21-26, was applied to directly grow an amphiphilic block copolymer, poly(oligo(ethylene glycol) methyl ether methacrylate)poly(2-hydroxypropyl methacrylate) (POEGMAPHPMA), from the C-terminus of IFN to in situ form an IFN-POEGMA-PHPMA conjugate micelle (IFNmicelle). POEGMA is a PEG-like polymer, which has been demonstrated to be useful as an alternative to PEG in prolonging the circulation half-life of proteins14-16. PHPMA is a biocompatible and water-insoluble polymer, but its monomer is water-soluble. It has been widely employed in polymerization induced self-assembly (PISA) processes17-20. We reason that IFN-micelle would not only greatly extend the circulation half-life of IFN but also well retain the bioactivity of IFN as compared to PEGASYS, owing to their supramolecular selfassembly behavior. IFN-micelle was synthesized by SI-PISA, and the reactions and purifications were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (Figure S1). First, a functionalized ATRP initiator was solely attached to the C-terminal end of IFN through sortase A mediated protein ligation to produce a C-terminal macroinitiator IFN-Br16. Second, ATRP was carried out to directly grow POEGMA from IFN-Br to yield a C-terminal IFN-POEGMA conjugate. Third, PHPMA was directly grown from IFN-POEGMA by ATRP to in situ form an IFN-POEGMA-PHPMA conjugate micelle (IFN-micelle). To determine the numberaverage molecular weight (Mn) and dispersity (Ð) values of POEGMA and POEGMA-PHPMA, proteinase K was used to treat IFN-POEGMA and IFN-POEGMAPHPMA to remove IFN (Figure S2), and then gel permeation chromatography (GPC) was used to analyze the POEGMA and POEGMA-PHPMA residues (Figure S3). The Mn, Ð and protein content are shown in Table S1. The IFN-POEGMA and IFN-POEGMA-PHPMA conjugates were further analyzed by proton nuclear magnetic resonance (1H-NMR), which confirmed the successful in situ growth of POEGMA and POEGMA-PHPMA from the protein (Figure S4). The hydrodynamic radius (Rh) of IFN-micelle (64.9 nm) measured by dynamic light scattering (DLS) was 28- and 9.4-fold bigger than those of IFN (2.3 nm) and IFN-POEGMA (6.9 nm), respectively (Figure 1a), suggesting self-assembly of the IFNPOEGMA-PHPMA conjugate into micelles. This result was confirmed by transmission electron microscopy (TEM) (Figure 1b), Cryo-TEM and atomic force microscopy (AFM) (Figures S5 and S6), which revealed sphere-like nanoparticles with an average diameter of 112 ± 23 nm. The protein binding and serum stability of IFN-micelle were characterized by DLS (Figures S7 and S8). The critical micelle concentration (CMC) of IFNmicelle was measured to be ~ 0.5 µg/mL (Figure S9), indicating that IFN-micelle could dissociate into unimers at low concentrations. The secondary structure of IFN

was not disturbed by SI-PISA, as indicated by the coincidence of the circular dichroism (CD) spectra of IFNmicelle, IFN-POEGMA and IFN (Figure 1c).

Figure 1. In vitro characterization of IFN-micelle. a) DLS analyses of IFN, IFN-POEGMA and IFN-micelle, where Rh is hydrodynamic radius. b) TEM image of representative IFN-micelle. c) CD spectra of IFN-micelle, PEGASYS, IFN-POEGMA, and IFN. d) Antiproliferative activities of IFN, IFN-POEGMA, PEGASYS and IFNmicelle. Data are shown as mean ± standard deviation (n = 3).

IFN is a versatile cytokine with multiple biological functions such as antivirus, antiproliferation, and immunoregulation27,28. The antiproliferative activity of IFNmicelle was evaluated by using human lymphoma Daudi B cells (Figure 1d)29,30. Half maximal inhibitory concentrations (IC50’s) and activity retentivities relative to IFN of IFN-micelle, PEGASYS, IFN-POEGMA, and IFN were summarized in Table S2. The activity retentivities of IFN-micelle (41.9%) and IFN-POEGMA (70.3%) were 21.5- and 36.1-fold higher than that of PEGASYS (1.95%), respectively. Notably, the IC50 of IFN-micelle (33.0 pg/mL) was lower than the CMC (0.5 µg/mL), suggesting that IFN-micelle dissociated into unimers at the IC50. This was further confirmed by chemically cross-linking IFN-micelle to yield a cross-linked IFNmicelle without a CMC (Supplementary information and Figure S10), where the activity retentivity of the crosslinked IFN-micelle (10.0 %) was much lower than that of IFN-micelle (41.8 %), but was higher than that of PEGASYS (Figure S11). Collectively, these results indicated that SI-PISA did not significantly decrease the biological activity of IFN as compared to non-specific PEGylation of IFN. The pharmacokinetics of IFN-micelle was studied in a mouse model (Figure 2a). The plasma level of IFN quickly dropped upon intravenous injection. In contrast, the plasma IFN levels of IFN-micelle, IFN-POEGMA, and PEGASYS slowly decreased. Furthermore, the pharmacokinetic parameters of these samples were cal-


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c

Plasma IFN evel (ng/mL)

1000

b

100 IFN IFN-POEGMA *** PEGASYS ** * IFN-micelle

10 1 0.1

0

20

40 60 Time (h)

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**

100

**

80 60 40 20 0

*** *** **

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IFN IFN-POEGMA PEGASYS IFN-micelle

200 150 100 50

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ns ** *

*** * ns

** * *

** ** *

* ns ns

a

1500 1200 900 600 300 0

c

b PBS IFN IFN-POEGMA PEGASYS IFN-micelle

****

100 * **

PBS IFN IFN-POEGMA PEGASYS IFN-micelle

75 50 25

**

0 10 20 30 40 50 60 70 80 Time (day) PBS

IFN

0

0

IFN-POEGMA

20

40 60 80 Time (day)

PEGASYS

100 120

IFN-micelle

100 µm d

Figure 3. In vivo antitumor activity of IFN-micelle. a) Suppression of tumor growth. The arrows indicate the time points of dosing. P value was determined by Student's t test, **P < 0.01, *P < 0.05. b) Cumulative survival of mice. Data are shown as mean ± standard deviation (n = 6 ~ 8, P value was determined by Log-rank (Mantel-Cox) test, ****P < 0.0001). c) H&E staining of tumors at 49 days post the first administration.

** IFN level (ng/g tissue)

a

tion, the IFN level of IFN-micelle (87.69 ng/g tissue) in tumors was 3.23-, 2.68- and 32.5-fold higher than those of PEGASYS (27.17 ng/g tissue), IFN-POEGMA (32.66 ng/g tissue), and IFN (2.70 ng/g tissue), respectively (Figure 2c). These data showed that SI-PISA could considerably enhance the tumor accumulation of IFN as compared with PEGylation and POEGMA conjugation of IFN. Percent survival

culated by fitting the data with a two-compartment model (Table S3). The clearance (CL) of IFN-micelle (0.212 mL/h) was 1.74-, 3.05- and 376-fold lower than those of PEGASYS (0.368 mL/h), IFN-POEGMA (0.647 mL/h), and IFN (79.66 mL/h), respectively. Notably, the terminal half-life (t1/2) of IFN-micelle (83.8 h) was 1.69-, 2.68- and 100-fold longer than those of PEGASYS (49.5 h), IFN-POEGMA (31.3 h), and IFN (0.834 h), respectively. These data indicated that SI-PISA could substantially prolong the circulation half-life of IFN as compared to PEGylation and POEGMA conjugation of IFN. The area under the curve (AUC) of IFN-micelle (94.0 mg/L•h) was 1.73-, 3.04- and 375-fold larger than those of PEGASYS (54.4 mg/L•h), IFN-POEGMA (30.9 mg/L•h), and IFN (0.251 mg/L•h), respectively, which indicated that SI-PISA could remarkably increase the plasma exposure of IFN as compared to PEGylation and POEGMA conjugation of IFN. Collectively, all the results indicated that SI-PISA could significantly improve the pharmacokinetics of IFN as compared to PEGylation and POEGMA conjugation of IFN.

IFN level (ng/g tissue)

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Tumor size (mm3 )

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** ** ns

0

Figure 2. In vivo pharmacokinetics and biodistribution of IFN-micelle. a) Plasma concentrations of IFNmicelle, PEGASYS, IFN-POEGMA, and IFN versus time post intravenous administration (n = 3). b) Biodistributions of IFN-micelle, PEGASYS, IFN-POEGMA, and IFN at 24 h post intravenous administration (n = 3). c) Tumor concentrations of IFN-micelle, PEGASYS, IFNPOEGMA, and IFN at 24 h after intravenous administration (n = 3). Data are shown as mean ± standard deviation. P value was determined by Student's t test, ***P < 0.001, **P < 0.01, *P < 0.05.

We next studied the biodistribution of IFN-micelle in an ovarian tumor-bearing mouse model (Figure 2b). The IFN levels of IFN-micelle in major organs and tissues were higher than those of PEGASYS, IFN-POEGMA and IFN. Notably, at 24 h post intravenous administra-

Based on these in vitro and in vivo results, we further studied the antitumor activity of IFN-micelle in a mouse model of ovarian cancer (Figure 3). Mice with an average tumor size of 24 mm3 were intravenously injected with IFN-micelle at a dosage of 1 mg IFN-equivalent/kg body weight once weekly until all the mice treated with PBS were executed. IFN-micelle completely inhibited tumor growth, while at the same dose, PEGASYS, IFNPOEGMA and IFN could not do so (Figure 3a and Figure S12). At 51 d post the first injection, the tumor size of the mice treated with IFN-micelle (21.0 mm3) was 6.7-, 11.7-, 28.1- and 51.9-fold smaller than those of the mice injected with PEGASYS (141 mm3), IFNPOEGMA (245 mm3), IFN (590 mm3), and PBS (1090 mm3), respectively. The remarkably increased inhibition of tumor growth was correlated to the prolonged animal survival (Figure 3b). The percent survival of the mice treated with IFN-micelle was 100%, in which 62.5% of the mice were tumor-free and the left mice bore small tumors of about 50 ~ 100 mm3 in size that did not grow any more. In contrast, the percent survival of the mice in all the control groups was 0%. The median survival times of PEGASYS, IFN-POEGMA, IFN, and PBS were 67, 48, 32, and 23 d, respectively. Furthermore, hematoxylin-eosin (H&E) staining of tumor tissues confirmed the enhanced in vivo antitumor activity of IFNmicelle (Figure 3c). Notably, a severe cell destruction



was observed for the treatment with IFN-micelle as compared to other treatments. Moreover, no systemic toxicity was observed for all the treatments (Figures S13-16). Collectively, these results demonstrated that SIPISA could greatly enhance the antitumor activity of IFN in a mouse model of ovarian cancer as compared with PEGylation and POEGMA conjugation of IFN. In summary, we have reported a supramolecular selfassembly strategy of SI-PISA to design IFN-micelle that is distinct from hydrophilic IFN-polymer conjugates in structures and functions. IFN-micelle is a self-assembled nanostructure of an amphiphilic IFN-POEGMAPHPMA conjugate, whose size is much larger than those of PEGASYS and IFN-POEGMA that are hydrophilic conjugates. As a result, the in vivo half-life of IFNmicelle is as long as 83.8 h, which, to our knowledge, is much longer than those of protein-polymer conjugates previously reported in the literature. Notably, based on the allometric scaling principle31,32, we estimate that IFN-micelle might have a longer half-life of over 250 h in humans, suggesting that this IFN-micelle system might be applicable for a twice-monthly dosing in humans. On the other hand, when the concentration of IFN-micelle is below its CMC, IFN-micelle can dissociate into much smaller unimers of IFN-POEGMAPHPMA conjugates, which results in a well-retained in vitro bioactivity as compared to cross-linked IFNmicelle and PEGASYS. Furthermore, the tumor accumulation of IFN-micelle is much greater than those of PEGASYS and IFN-POEGMA owing to the substantially extended half-life. More importantly, IFN-micelle can completely suppress tumor growth, whereas at the same dose, PEGASYS and IFN-POEGMA are not so effective. Additionally, the remarkably enhanced therapeutic efficacy does not induce systemic toxicity. These characteristics suggest that IFN-micelles might be widely useful as novel, long-acting, highly potent, and safe therapeutics for the treatment of viral diseases and cancers. Furthermore, we believe that SI-PISA is of great promise as a new, general and effective platform technology to remarkably enhance the pharmacology of many therapeutic proteins with short circulation half-lives.

Corresponding Author

*[email protected] ORCID

0000-0002-2916-3044 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank Dengli Qiu of Bruker Corp (Beijing) for carrying out AFM imaging, and we also thank Danyang Li for the technical assistance in Cryo-TEM. This study was financially supported by grants from the National Natural Science Foundation of China (Grants 21534006 and 21274043).

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ASSOCIATED CONTENT Supporting Information. Materials; full experimental details including synthesis, purification, and in vitro and in vivo characterization of the IFN-micelle; and supplementary results includ1 ing SDS-PAGE, H-NMR, GPC, Cryo-TEM, AFM, protein binding, serum stability, CMC, cross-linked IFNmicelle properties, representative images of mice, biosafety data and pharmacokinetic parameters (PDF)

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The Supporting Information is available free of charge on the ACS Publications website at DOI:

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Polymerization Induced Self-Assembly of a Site-Specific Interferon Î±

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