Modification of Î±-Cyclodextrin Polyrotaxanes by ATRP for Conjugating

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Modification of #-cyclodextrin polyrotaxanes by ATRP for conjugating drug and prolonging blood circulation Jialiang Zhang, Ling'e Zhang, Shun Li, Changfeng Yin, Cheng Li, Wei Wu, and Xiqun Jiang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00464 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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ACS Biomaterials Science & Engineering

Modification of α-cyclodextrin polyrotaxanes by ATRP for conjugating drug and prolonging blood circulation

Jialiang Zhang, Ling’e Zhang, Shun Li, Changfeng Yin, Cheng Li, Wei Wu*, Xiqun Jiang*

Department of Polymer Science & Engineering, College of Chemistry & Chemical Engineering, Nanjing University, Nanjing, 210023, People’s Republic of China

* To whom correspondence should be addressed Email: [email protected], [email protected]

ACS Paragon Plus Environment


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ABSTRACT: To enhance the water solubility of α-cyclodextrin (CD) polyrotaxanes (PRs) and achieve effective drug loading, we polymerized 2-hydroxyethyl methacrylate and 2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanaminium successively from a α-CD-PR-based macroinitiator by a two-step ATRP followed by cleaving the tert-butyl ester groups, providing a α-CD-PR-cored multiarm copolymer. The multiarm copolymer had reactive hydroxyl pendant groups in the inner block of the arms, which were used to incorporate antitumor agent paclitaxel (PTX). The outer block of the arms was zwitterionic poly(carboxybetaine) and had high hydrophilicity and zero net electric charge in neutral environment. This feature endowed the PTX-loaded multiarm copolymer with high water solubility and prolonged blood circulation. The blood circulation half-life of the PTX-loaded multiarm copolymer was determined to be about 7.7 h versus 18.8 ± 1.5 min of the reported blood circulation half-life of the PTX injected as commercial Taxol. The PTX-loaded multiarm copolymer was proved to be efficient in tumor accumulation and suppression.

KEYWORDS: α-cyclodextrin, polyrotaxane, multiarm copolymer, drug carrier, tumor treatment


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1. INTRODUCTION Cyclodextrin (CD) polyrotaxanes (PRs) are a type of stable supramolecular architectures consisting of multiple CD rings threaded by a linear polymer chain that is terminated by bulky capping group on each end to block the CD rings on it. When being used as drug carriers, CD PRs have many advantages such as good biocompatibility, abundant derivable hydroxyl groups, and tunable nanoscale size and chemical composition.1-4 Furthermore, due to the non-covalent interactions between the CD and polymer axle moieties, the CD rings can move freely on the polymer axle, which can effectively enhance the interactions between CD PRs and cells by adjusting the CD position to fit external changes.5-7 However, although CDs are highly water soluble, the as-prepared CD PRs are generally water insoluble because of the hydrogen bonds formed by the hydroxyl groups of adjacent CDs in PRs,8 which greatly hinders their biological applications. Therefore, hydrophilic modifications of CD PRs are prerequisite for their applications in drug delivery and other biologically related fields. So far, the hydrophilic modifications of CD PRs have been achieved by conjugating hydrophilic chains to PRs through hydroxyl groups, such as oligo(ethylene glycol) (OEG)9-11 and polyethylenimine (PEI),12,13 where the reactive groups in the hydrophilic chains could be used to incorporate functional molecules. However, due to the small length of the OEG chains and the positive charge of the PEI chains, which is easy to induce protein adsorption,14-18 these hydrophilic modifications are not suitable for prolonging the blood circulation time of PRs that is



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generally considered to be greatly favorable to the in vivo performance of therapeutic agents. Additionally, only one derivable group in an ethylene glycol chain can be used to link functional molecules to PRs, limiting greatly the loading of the functional molecules. So far, the hydrophilic modification of CD PRs that can provide both desirable antibiofouling ability and increased functional groups has not been reported yet. Herein, we report an effective strategy to the chemical modification and drug loading of α-CD PRs. Briefly, as shown in Scheme 1, α-CD PRs were synthesized by using poly(ethylene

glycol)

bisamine

(PEG-BA)

as

the

axle

and

N-carbobenzoxy-L-phenylalanine (Z-PHE-OH) as the end-capping reagent following reported procedures.19 A diblock copolymer chain was then introduced to the PRs by a grafting-from method via atom transfer radical polymerization (ATRP), affording a α-CD-PR-cored multiarm copolymer PR-PHEMA-PCB-tBu (Scheme 1). In each arm of the polymer, the block close to the PR core is poly(2-hydroxyethyl methacrylate) (PHEMA)

and

the

block

on

the

periphery

is

poly(2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanaminium) (PCB-tBu). The hydroxyl pendant groups in PHEMA block can be used to incorporate functional molecules, such as therapeutic agents or bioprobes. The PCB-tBu block can be readily converted to zwitterionic poly(carboxybetaine) (PCB) by cleaving the tert-butyl ester protective groups, which contains an anion charge and a cationic charge in one repeating unit, and thus has high hydrophilicity and zero net electric charge in neutral environment. PCB chains have been demonstrated to be very


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efficient in resisting the nonspecific protein adsorption and prolonging the blood circulation time of nanomaterials.20-22 To achieve the application of the water soluble α-CD-PR-cored multiarm copolymer in drug delivery, an antitumor agent, paclitaxel (PTX), was conjugated to the PHEMA block through a biodegradable ester linkage. The in vitro and in vivo properties of the drug-loaded polymer were studied, revealing the prolonged blood circulation time and enhanced antitumor effectiveness with respect to free PTX.

Scheme 1. Synthetic route of the PTX-loaded α-CD-PR-cored multiarm copolymer.

2. EXPERIMENTAL SECTION All reagents were purchased commercially without further purification. Experimental details are described in the experimental section in Supporting Information. 3. RESULTS AND DISCUSSION We synthesized α-CD PRs following published procedures by using PEG-BA as the



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axle and Z-PHE-OH as the end-capping reagent (Scheme 1).19 The chemical structure of the α-CD PRs was characterized by 1H NMR spectroscopy (Figure 1 and S1). As shown in Figure 1, because the rotaxanation reduces the conformational flexibility of the α-CD and polymer axle moieties, the proton signals of the α-CD PRs are broad and unresolved, indicating the formation of the supramolecular structure. In the 1H NMR spectrum, the signals from the end-capping groups, PEG axle and α-CD can be observed. Based on the integral intensity ratio of the C(1)H signal of α-CD with that of the aromatic protons in the end-capping groups, it can be calculated that about 18 α-CD molecules are locked on one polymer axle.

Figure 1. 1H NMR spectrum of the α-CD PRs.

To functionalize the α-CD PRs and increase their hydrophilicity, we conjugated a diblock copolymer chain PHEMA-b-PCB-tBu to the PRs by a grafting-from method through ATRP followed by cleaving the tert-butyl ester protective groups. The PR-based macroinitiator (PR-Br, Scheme 1) for ATRP was synthesized by the acylation of the hydroxyl groups in the α-CD PRs with 2-bromoisobutyryl bromide. The 1H NMR spectrum of PR-Br is shown in Figure S2. The degree of acylation per


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α-CD was calculated to be about 4 by comparing the integral intensity of the C(1)H signal of α-CD with that of the 2-bromoisobutyrate signal. 2-Hydroxyethyl methacrylate (HEMA) was then polymerized from the macroinitiator PR-Br by ATRP, providing PR-PHEMA. In the 1H NMR spectrum of PR-PHEMA (Figure S3), only the broad peaks from the PHEMA chains can be observed. The number-average polymerization degree of PHEMA chains in PR-PHEMA is then roughly evaluated to be about 6 according to the increase of the weight of the α-CD PRs after the polymerization. After obtaining PR-PHEMA, we performed the polymerization of 2-tert-butoxy-N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-2-oxoethanaminium

by

using PR-PHEMA as a macroinitiator and obtained the multiarm copolymer PR-PHEMA-PCB-tBu with a PR as the core (Scheme 1). Based on the weight increase after the polymerization, the number-average polymerization degree of the PCB-tBu blocks in PR-PHEMA-PCB-tBu is roughly evaluated to be about 32. Figure S4 shows the 1H NMR spectrum of PR-PHEMA-PCB-tBu. In this spectrum, only the proton signals from PCB-tBu segments are observed, because the signals of α-CD PR and PHEMA moieties are overlapped by those of the longer PCB-tBu block. To load PTX into PR-PHEMA-PCB-tBu covalently, a succinate-based PTX ester derivative (d-PTX, Scheme 1) was synthesized following reported procedures.23 Thereafter, the d-PTX activated previously with N,N'-diisopropylcarbodiimide (DIC) was conjugated to the PHEMA blocks in PR-PHEMA-PCB-tBu through esterification reaction. Subsequent cleavage of the tert-butyl ester groups in PCB-tBu block by trifluoroacetic acid (TFA) provides a water soluble PTX-loaded α-CD-PR-cored



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multiarm copolymer with zwitterionic PCB chains on periphery, PR-PTX-PCB (Scheme 1). The drug loading content of PR-PTX-PCB was determined to be about 6.6 % at the weight percent of PTX in PR-PTX-PCB by measuring the absorbance and using a pre-established calibration curve. High-resolution transmission electron microscopy (HR-TEM) was used to study the morphologic structure of PR-PTX-PCB. As shown in Figure 2, the molecules of PR-PTX-PCB exhibit a near-spherical shape with a diameter of about 3.5 nm, which is consistent with the previous work showing that the α-CD PRs behave as random coils in good solvents.24 The hydrodynamic diameter of PR-PTX-PCB in phosphate buffer solution (PBS, 0.1 M, pH 7.0) was determined to be about 6.0 nm by dynamic light scattering (DLS, the inset of Figure 2) with zeta potential of about 5.1 mV. This nearly neutrally charged surface of PR-PTX-PCB in neutral medium is favorable to its antibiofouling ability.

Figure 2. Typical TEM image of PR-PTX-PCB; the inset shows the hydrodynamic diameter distribution of PR-PTX-PCB in PBS (0.1 M, pH 7.0) determined by DLS.


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To assess the pharmacological activity of PR-PTX-PCB as well as the biosafety of PR-PHEMA-PCB, the in vitro cytotoxicities of PR-PTX-PCB and PR-PHEMA-PCB against

human

neuroblastoma

SH-SY5Y

cells

were

measured

by

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with commercial Taxol as a positive control after 48 h incubation with the samples. Figure 3 shows the cell viability after the incubation at different dosages. It can be seen that both PR-PTX-PCB and Taxol show dose-dependent cytotoxicity (Figure 3a). PR-PTX-PCB displays lower cytotoxicity than Taxol, which may be associated with the sustained drug release of PR-PTX-PCB. Furthermore, PR-PHEMA-PCB does not show significant cytotoxicity at all test concentrations, indicating its good cytocompatibility (Figure 3b).

a 120

PR-PTX-PCB Taxol

100

b 120 Relative cell viability (%)

Relative cell viability (%)

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80 60 40 20 0

100 80 60 40 20 0

1

2

4

8

16

PTX concentration (µg/ml)

15

30

60

121

242

PR-PHEMA-PCB concentration (µg/ml)

Figure 3. In vitro cytotoxicity of PR-PTX-PCB (a, black), Taxol (a, red) and PR-PHEMA-PCB (b) against SH-SY5Y cells.

Confocal laser scanning microscopy (CLSM) was used to trace the cellular uptake of PR-PTX-PCB after it was labeled with fluorescein isothiocyanate (FITC) through the



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reaction of the residue hydroxyl groups in the inner blocks of PR-PTX-PCB with the isothiocyanate group in the dye. Figure 4 shows the typical CLSM images of the SH-SY5Y cells incubated 4 h with the FITC-labeled PR-PTX-PCB at 37 °C. It can be seen that the FITC-labeled PR-PTX-PCB can enter the cells and stay mainly in cytoplasm with a punctate pattern, indicating that the cellular uptake of the labeled PR-PTX-PCB is most likely to be achieved through endocytosis.

Figure 4. CLSM images of SH-SY5Y cells incubated with FITC-labelled PR-PTX-PCB at 37 ºC for 4 h. The cell nuclei were stained by Hoechst 33258.

To study the in vivo behaviors of PR-PTX-PCB in tumor-bearing mice, PR-PTX-PCB was labeled with a near-infrared (NIR) dye, NIR-797, through the reaction of the residue hydroxyl groups in the inner blocks of PR-PTX-PCB with the isothiocyanate group in the dye, and imaged by a non-invasive real-time near-infrared fluorescence (NIRF) imaging systems after injected intravenously. All animal experiments were implemented according to the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Drum


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Tower Hospital (Nanjing, China). Figure 5 shows the NIRF images of a hepatic H22 tumor-bearing mouse at different time points after the tail-vein injection of NIR-797-labeled PR-PTX-PCB. From these images, it can be seen that the fluorescence signals from the labeled PR-PTX-PCB are mainly distributed in the regions of the liver and tumor over the monitoring period, and the signals in the tumor region continuously increase as time goes by, indicating the accumulation of the labeled PR-PTX-PCB in the tumor. The descent of the signals in the liver region may be caused by the hepatobiliary elimination of the labeled PR-PTX-PCB.

Figure 5. NIRF images of a hepatic H22 tumor-bearing mouse at different time points after the injection of the NIR-797-labeled PR-PTX-PCB via tail vein. The circled region is the tumor.

The biodistribution of the NIR-797-labeled PR-PTX-PCB was further studied by measuring the fluorescence intensities from the main organs dissected from the



hepatic H22 tumor-bearing mice at different times after tail-vein injection of the NIR-797-labeled PR-PTX-PCB (Figure 6). On the basis of the results shown in Figure 6, the half-life of the NIR-797-labeled PR-PTX-PCB in blood circulation can be calculated to be about 7.7 h, much longer than that of PTX injected as Taxol (18.8 ± 1.5 min) reported previously.25 The liver uptake of the labeled PR-PTX-PCB is highest among all the tested organs and tissues, and the maximum of the liver uptake among all the test time points is 44.5 % injected dose (ID) per gram of wet tissue at 4 h p.i.. In contrast, the uptake of the labeled PR-PTX-PCB in spleen, lung, kidney and heart is relatively lower, and the maximum uptakes are found to be 15.4 % ID/g at 1 h p.i. for spleen, 8.5 % ID/g at 1 h p.i. for lung, 7.9 % ID/g at 1 h p.i. for kidney and 4.1 % ID/g at 1 h p.i. for heart. The tumor uptake of the labeled PR-PTX-PCB increases continuously until 12 h p.i. and reaches 7.0 % ID/g at 8 h p.i., indicating the effective accumulation of the labeled PR-PTX-PCB in tumors. 60

1h 4h 12 h 24 h 48 h

50 40

% ID/g

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30 20 10 0

Heart Liver Spleen Lung Kiney Blood Tumor Figure 6. Biodistribution of NIR-797-labeled PR-PTX-PCB in different tissues of H22 tumor-bearing mice at different time points after the tail-vein injection of the


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labeled PR-PTX-PCB. The values were based on three mice per group.

The in vivo antitumor performance of PR-PTX-PCB was studied by using Taxol as a positive control at a dosage of 5 mg of PTX equivalent (equiv) per kilogram of body weight. PR-PHEMA-PCB (being equal to PR-PTX-PCB in mole) and saline were used as negative controls. Subcutaneous hepatic H22 tumor-bearing mice were used as model animals. After the treatments, the tumor size and survival rate of all the test groups were examined. Figure 7a shows the tumor volume evolutions after the treatments. It can be seen that both PR-PTX-PCB and Taxol exhibit significant antitumor activity. Among all the test groups, the group treated with PR-PTX-PCB shows the slowest tumor growth. From the 3th day p.i., the tumor volumes of the groups treated with PR-PTX-PCB and Taxol show a statistically significant difference (P < 0.05). On the 15th day p.i., the tumor growth inhibition (TGI) for the group treated with PR-PTX-PCB is calculated to be 67.0 %, in contrast, the TGI for the Taxol-treated group is 49.2 %, indicating that PR-PTX-PCB is much better than Taxol in tumor suppression (the calculation method for TGI can be found in Supporting Information). The groups treated with PR-PHEMA-PCB and saline do not show statistically significant difference in tumor volumes, indicating that PR-PHEMA-PCB does not have antitumor activity. Animal survival was recorded and shown in Figure 7b. It can be seen that all the mice died on the 22nd day after the treatment with saline, and only 1 and 3 of 8 mice survived in the PR-PHEMA-PCB and Taxol-treated groups on the same day, respectively. In contrast, in the PR-PTX-PCB-treated group,



5 mice survived on the 22nd day p.i., confirming the better antitumor effectiveness of PR-PTX-PCB. The body weights (Figure S5) and clinical situations of all the test groups were scrutinized and not significantly influenced by the treatments of either the two PTX formulations or PR-PHEMA-PCB when compared to the saline-treated group, indicating the well-tolerated dosage of drug and negligible toxicity of PR-PHEMA-PCB imposed on the test mice.

a

10000 PR-PTX-PCB Taxol PR-PHEMA-PCB Saline

8000

3

Tumor volume (mm )

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6000

4000

* *

2000

0 0

2

4

6

8

10

12

14

16

Days

Figure 7. (a) Tumor volume evolution of each treated group. *P < 0.05 (versus Taxol-treated group from the 3th day). Data are presented as mean ± SD (n = 8). (b) Kaplan-Meier curves showing survival of tumor-bearing mice in each treated groups.

4. CONCLUSION In conclusion, we synthesized a water soluble α-CD-PR-cored multiarm copolymer by a two-step ATRP. The multiarm copolymer had reactive hydroxyl pendant groups in the inner PHEMA blocks of the arms, which were used to incorporate antitumor PTX, affording a drug loading of ~6.6 % at the weight percent of PTX in the multiarm copolymer. The outer zwitterionic PCB block had high hydrophilicity and zero net


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electric charge in neutral environment. This feature endowed the PTX-loaded multiarm copolymer with high water solubility and prolonged blood circulation. The blood circulation half-life of the PTX-loaded multiarm copolymer was determined to be about 7.7 h versus 18.8 ± 1.5 min of the reported blood circulation half-life of the PTX injected as commercial Taxol. The PTX-loaded multiarm copolymer could enter tumor cells and were proved to be efficient in tumor accumulation and suppression.

ASSOCIATED CONTENT Supporting Information Experimental details, characterization data and additional results. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors [email protected]; [email protected].

ACKNOWLEDGMENTS This research was supported by the Natural Science Foundation of China (Nos. 51422303, 51690153 and 21474045).



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Modification of α-cyclodextrin polyrotaxanes by ATRP for conjugating drug and prolonging blood circulation Jialiang Zhang, Ling’e Zhang, Shun Li, Changfeng Yin, Cheng Li, Wei Wu*, Xiqun Jiang*

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Modification of Î±-Cyclodextrin Polyrotaxanes by ATRP for Conjugating

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