Efficient Synthesis of Polymer Prodrug by Thiol-Acrylate Michael

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Efficient Synthesis of Polymer Prodrug by Thiol-Acrylate Michael Addition Reaction and Fabrication of pH-Responsive Prodrug Nanoparticles Chao-Ran Xu, Liang Qiu, Cai-Yuan Pan, Chun-Yan Hong, and Zong-Yao Hao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00531 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 5, 2018

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Bioconjugate Chemistry

Efficient Synthesis of Polymer Prodrug by ThiolAcrylate Michael Addition Reaction and Fabrication of pH-Responsive Prodrug Nanoparticles Chao-Ran Xu†, Liang Qiu‡, Cai-Yuan Pan†, Chun-Yan Hong*,†, Zong-Yao Hao*,§ †

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and

Engineering, University of Science and Technology of China, Hefei, Anhui, 230026, China. ‡

Institute of Biophysics, Hebei University of Technology, Tianjin, 300401, China.

§

Institute of Urology, Anhui Medical University, Hefei, Anhui, 230026, China.

E-mail: [email protected], [email protected].

ABSTRACT: In this study, an efficient method is proposed for the synthesis of polymer prodrug with acid-liable linkage via thiol-acrylate Michael addition reaction of the camptothecin with tethering acrylate group and polymer scaffold containing multiple thiol groups. The polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) is prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization of the methacrylate of lipoic acid (HEMA-LA) using poly(2-(2-hydroethoxy) ethyl methacrylate) (PHEO2MA) as macro-RAFT agent, followed by reduction of the disulfides in lipoic acid (LA) groups to give polymer scaffold with dihydrolipoic acid (DHLA) pendent groups. Acrylate-tethering camptothecin (ACPT) is connected to P(HEO2MA)-b-P(HEMA-DHLA) via Michael addition reaction between thiol and acrylate with

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a high coupling efficiency (95%). Amphiphilic polymer prodrug P(HEO2MA)-b-P(HEMADHLA-CPT) spontaneously self-assembles into nanoparticles in an aqueous solution, and exhibits a CPT loading content as high as 40.1%. The prodrug nanoparticles with the acid-liable β-thiopropionate linkages can release CPT under acidic condition, and the prodrug nanoparticles show similar cytotoxicity to HeLa cells as free CPT. Overall, the prodrug nanoparticles with high drug loading contents and acid-liable linkages are promising for pH-responsive anticancer therapy.

Introduction Chemotherapy is often used for cancer treatment, however, most anticancer drugs have some drawbacks, such as poor water solubility, rapid blood clearance, low tumor selectivity, and side effects for healthy tissues.1 Nanosized vehicles2,3 including liposomes,4,5 vesicles,6,7 polymer nanoparticles8-11 and some inorganic materials12,13 are used as carriers of anticancer drugs to greatly enhance their performances in clinical. Among these nanocarriers, polymer nanoparticles have attracted considerable attention to perform drug delivery in cancer therapy. Significant advances on polymer nanoparticles encapsulating anticancer drugs in their cores have been reported, but some obstacles including burst release, poor drug loading content and low miscibility of drugs with polymer matrix need to be overcome.14,15 A promising approach to addressing these problems is to conjugate free drug with polymer scaffold to form polymer prodrug, which has been widely investigated in recent years.16-18 Polymer prodrugs can improve the therapeutic efficacy of conventional small molecule drugs by increasing their loading capacity and stability in the blood circulation, decreasing their exposure to normal tissues, and controlling their release in response to extracellular or intracellular stimuli.19-22


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The conjugation of drugs to pre-synthesized polymers is one of the main strategies for the synthesis of polymer prodrugs.23 The “conjugation to” strategy is to graft drugs on the side chains of polymers via organic reactions.24-27 Organic and platinum anticancer drugs have been linked to the side chains of preformed polymers via coupling strategies mainly including imine formation,28 carbodiimide reaction,29,30 amidation reaction,31,32 Diels-Alder coupling reaction33 and photochemical [2 + 2] addition reaction.34 However, these reactions have lower reaction efficiency compared with copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Drugs can be coupled to polymer scaffold efficiently via CuAAC reaction,35 but the residual Cu species in the reaction products are hard to be fully removed, which is not suitable for biomedical applications. The Michael addition reaction between thiol and acrylate doesn’t need heavy metallic salts, which is known as a highly efficient and “green” reaction in bioconjugation chemistry.36 Additionally, the thiol-acrylate Michael addition reaction can generate pHresponsive β-thiopropionate linkers.37 It is reported that the β-thiopropionate linkage can hydrolyze in acidic solution at a sustained rate,38,39 thus, it can be used for controlled drug release. So far literatures on drug release via the hydrolysis of β-thiopropionate linkage are few. Zou et al. synthesized paclitaxel-conjugated linear block copolymer with β-thiopropionate linkages by the post-functionalization of paclitaxel molecule onto the polymer backbone, and the conjugation efficiency was 50% when the feed molar ratio of the paclitaxel to polymer scaffold was 20 to 1.40 Recently, our group fabricated camptothecin-bonded hyperbranched star copolymer by atom transfer radical polymerization (ATRP) of prodrug monomer with βthiopropionate linkage in the presence of copper salt.41 In this paper, Michael addition reaction between thiol and acrylate is used for synthesizing polymer prodrug with β-thiopropionate linkages via the “conjugation to” approach. This


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synthetic method can control the drug loading capacity, improve the drug-loading efficiency and enable the conjugated drugs to be controllably released in response to acidic milieu. As shown in Scheme 1A, the polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) was synthesized by RAFT polymerization of HEMA-LA using PHEO2MA as macro-RAFT agent, and successive reduction reaction of lipoic acid groups in P(HEO2MA)-b-P(HEMA-LA). ACPT was then conjugated to the polymer scaffold via Michael addition reaction with a high efficiency, forming polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT) with β-thiopropionate linkages. Moreover, the drug loading capacity of polymer prodrug could be controlled via adjusting the feed molar ratio of ACPT and polymer scaffold. The polymer prodrug spontaneously self-assembled into prodrug nanoparticles in water (Scheme 1B). The prodrug nanoparticles were easily internalized by cancer cells via cell endocytosis, and CPT was released in a controlled manner owing to the hydrolysis of β-thiopropionate linkage in the subacid condition. Such prodrug nanoparticles have potential applications in anticancer chemotherapy.

Scheme 1. Schematic illustration of A) the synthesis of polymer prodrug P(HEO2MA)-bP(HEMA-DHLA-CPT) with acid-liable linkage, and B) fabrication of pH-responsive prodrug nanoparticles for drug delivery.


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Results and Discussion Synthesis and Characterization of Polymer Prodrug with Acid-liable β-Thiopropionate Linkages In this study, the polymer prodrug was synthesized by conjugating acrylate-tethering camptothecin (ACPT) to polymer scaffold containing multiple thiol groups, and the detailed synthetic route was shown in Scheme 1. PHEO2MA was synthesized via the RAFT polymerization of HEO2MA, which was further used as macro-RAFT agent in the RAFT polymerization of HEMA-LA. The block copolymer P(HEO2MA)-b-P(HEMA-LA) was successively reduced into polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) with thiol groups. ACPT was then connected to the polymer scaffold via Michael addition reaction between acrylate and thiol, forming polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT). The P(HEO2MA) component in the polymer prodrug is hydrophilic, which is used for protecting the covalently conjugated drugs from undesirable external influence. The RAFT polymerization of HEO2MA was carried out in tetrahydrofuran at 70 oC for 6 h, using 4-cyanopentanoic acid dithiobenzoate (CPADB) as chain transfer agent and azobisisobutyronitrile (AIBN) as initiator. The feed molar ratio of [HEO2MA]/[CPADB]/[AIBN] was fixed at 50/1/0.1. The GPC curve of PHEO2MA (Figure S1A in the supporting information) was unimodal, and the number-average molecular weight (Mn,GPC) and the molecular weight distribution (Mw/Mn) were 13500 g/mol and 1.09, respectively. In the 1H NMR spectrum of PHEO2MA (Figure 1A), the characteristic peaks of PHEO2MA emerged at δ 4.15 ppm (b), 3.75 ppm (c) and 3.58 ppm (d), corresponding to the CH2 groups on the side chain of PHEO2MA. Meanwhile, the representative peaks at δ 7.88 ppm, 7.54 ppm and 7.38 ppm appeared in the spectrum, which were attributed to the aromatic protons from CPADB end group. The Mn,NMR of


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PHEO2MA was calculated based on the integral values of the signals at 4.15 ppm (b) and 7.2~8.0 ppm (a), and PHEO2MA with degree of polymerization (DP) = 44 and Mn,NMR = 7700 g/mol was used in the following study. The

block

copolymer

P(HEO2MA)-b-P(HEMA-LA)

was

synthesized

by

RAFT

polymerization of HEMA-LA using PHEO2MA as macro-RAFT agent with a feed molar ratio of [HEMA-LA]/[PHEO2MA]/[AIBN] = 40/1/0.1 in THF at 70 oC for 6 h. Compared with Figure 1A, new peaks at δ 4.27 ppm (a), 2.37 ppm (i), 1.2~1.8 ppm (j, k, l), 3.53 ppm (m), 3.08 ppm (n), 2.47 ppm (o) and 1.92 ppm (p) emerged in the 1H NMR spectrum of P(HEO2MA)-b-P(HEMALA) (Figure 1B), corresponding to the CH and CH2 groups on the side chain of P(HEMA-LA) block. The DP of P(HEMA-LA) block was calculated based on integral values of the signals at δ 2.47 ppm (o) and 4.0~4.5 ppm (a, b). The polydispersity index (PDI) value of the copolymer measured by GPC (Figure S1A) was 1.21, and block copolymer P(HEO2MA)44-b-P(HEMALA)12 was used in the following experiments.

Figure 1. 1H NMR spectra of PHEO2MA (A) and P(HEO2MA)-b-P(HEMA-LA) (B) recorded in CDCl3.


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Under mild condition, the LA groups in the P(HEO2MA)-b-P(HEMA-LA) could be reduced to DHLA pendent groups by NaBH4, giving P(HEO2MA)-b-P(HEMA-DHLA). After the reduction reaction, the signals of CH2 and CH protons (n and m) of P(HEO2MA)-b-P(HEMA-DHLA) near both sulfur atoms at δ 3.08 ppm and 3.53 ppm in Figure 1B shifted to δ 2.71 ppm and 2.95 ppm, respectively; and the CH2 resonances (o, p) of P(HEO2MA)-b-P(HEMA-LA) at δ 2.47 and 1.96 ppm in Figure 1A merged together at δ 1.90 ppm (Figure 2A), suggesting the disappearance of cis-trans isomerism in the DHLA pendent groups. GPC curves (Figure S1B) showed almost no change in the molecular weight or PDI of polymer after reduction, and Fourier transform infrared spectrum (Figure S2) confirmed the successful synthesis of P(HEO2MA)-b-P(HEMA-DHLA) from P(HEO2MA)-b-P(HEMA-LA), as the thiol peak emerged at 2560 cm-1. The above analysis results showed that the disulfides in lipoic acid groups have almost converted to thiol groups, which were available for the following Michael addition reaction. Then ACPT was coupled to P(HEO2MA)-b-P(HEMA-DHLA) by Michael addition reaction between thiol and acrylate, forming polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT). In the 1H NMR spectrum of polymer prodrug (Figure 2B), the CPT resonances (s, t, u, v, w, x, y) at δ 7.4~8.5 ppm and 5.1~5.8 ppm appeared. Meanwhile, representative new peaks emerged at δ 2.10 and 2.70 ppm (i and z), which were attributed to CH2 protons on the newly formed β-thiopropionate linkages. Such results suggested that the polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT) was synthesized successfully via the “conjugation to” approach.


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Figure 2. 1H NMR spectra of P(HEO2MA)-b-P(HEMA-DHLA) (A) and P(HEO2MA)-bP(HEMA-DHLA-CPT) (B) recorded in CDCl3. As is well known, the Michael addition reaction of thiol and acrylate usually has almost quantitative conversion under mild reaction condition.36 In this manuscript, the conjugation of ACPT to the polymer scaffold was carried out using triethylamine (TEA) as catalyst, and the reaction was performed at room temperature in THF for 24 h. When the feed molar ratio of [ACPT]/[DHLA] was 2:1, the drug loading content (DLC) of the polymer prodrug (Prodrug-1) was 40.1% and the drug loading efficiency (DLE) was as high as 88%. Upon decreasing of [ACPT]/[DHLA] ratios to 1:1 and 1:2, the DLCs of the polymer prodrugs (Prodrug-2 and Prodrug-3) decreased to 26.6% and 16.6%, and the DLEs increased to 90% and 95%, respectively. The DLCs of polymer prodrugs could be controlled via adjusting the feed ratio of ACPT-to-DHLA in the Michael addition reaction, and detailed results were listed in Table 1. GPC traces of the polymer prodrugs shown in Figure S3 revealed that the molecular weight of polymer prodrug increased with the increase of the feed molar ratio of [ACPT]/[DHLA]. All the


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above results verified that this synthetic method endowed the polymer prodrug controllable drug loading contents, high drug-loading efficiency and acid-liable β-thiopropionate linkages. Table 1. Characterizations of prodrug nanoparticles with different drug loading content Feed molar ratio of [ACPT]/[DHLA]a

Diameter(TEM) b (nm)

Diameter(DLS) c (nm)

PDI(DLS)d

DLC(UV)e (%)

Prodrug-1

2:1

38

42

0.168

40.1

45.7

88

Prodrug-2

1:1

38

43

0.152

26.6

29.6

90

Prodrug-3

1:2

40

45

0.110

16.6

17.4

95

Sample

DLC(Theory)f (%)

DLEg (%)

a [ACPT] and [DHLA] refer to the acrylate of CPT and dihydrolipoic acid pendent group in the block copolymer P(HEO2MA)44b-P(HEMA-DHLA)12, respectively; b

The average diameter of prodrug nanoparticles determined by TEM;

c

The number-average diameter of prodrug nanoparticles measured by DLS;

d

The polydispersity index (PDI) of prodrug nanoparticles measured by DLS.

e

DLC refers to drug loading content, which was determined by ultraviolet quantitative analysis;

f The theoretical DLC is calculated according to the feed weight ratio of CPT/(CPT + polymer scaffold) in the Michael addition reaction; g The drug loading efficiency was calculated according to the equation: Drug loading efficiency (DLE) (%) = DLC measured/theoretical DLC × 100%.

Fabrication of Prodrug Nanoparticles Polymeric nanoparticles have been proven to be promising vehicles for targeted delivery of drugs, because their nanoscopic sizes can protect them from being taken up by the phagocytic cells of the reticuloendothelial system (RES), and they can passively accumulate in the solid tumor through the enhanced permeability and retention (EPR) mechanism.43,44 Herein, the polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) is hydrophilic, but the covalently coupled CPT in polymer prodrug is almost water-insoluble. The polymer prodrug P(HEO2MA)-bP(HEMA-DHLA-CPT) could spontaneously self-assemble into nanoparticles in water. Polymer prodrug (12 mg) was dissolved in 1.0 mL of dimethyl sulfoxide (DMSO), and deionized water


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(7.0 mL) was then added into the solution slowly through a syringe pump. The mixture was dialyzed against deionized water, forming prodrug nanoparticles with a concentration of 1.5 mg/mL. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to characterize the formed assemblies. As shown in Figure 3A, prodrug nanoparticles fabricated from prodrug-1, prodrug-2 and prodrug-3 had similar diameters, which were characterized by TEM. The DLS curves were shown in Figure S4, and the sizes of prodrug nanoparticles detected by DLS were listed in Table 1. The sizes obtained from DLS were slightly larger than the sizes from TEM, because prodrug nanoparticles were swollen in DLS measurement and collapsed in TEM measurement. The critical micelle concentration (CMC) of prodrug nanoparticles was determined using pyrene as a fluorescence probe. As shown in Figure 3B, the CMCs of prodrug nanoparticles increased from 5.75 to 7.33 µg/mL with the decrease of CPT contents in polymer prodrugs, because camptothecin occupied the hydrophobic region of prodrug nanoparticles. These low CMC values indicated that prodrug nanoparticles were stable in water. Overall, the CPT-incorporated nanoparticles were successfully prepared, and prodrug nanoparticles fabricated from prodrug-1 were used in the following measurements.


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Figure 3. (A) TEM images of prodrug nanoparticles (a: prodrug-1, b: prodrug-2, c: prodrug3); (B) Plot of the I339/I332 ratio from pyrene excitation spectra versus the concentration of polymer prodrugs on a log scale. Release of CPT from Prodrug Nanoparticles As has been reported previously, the hydrolysis of β-thiopropionate in acidic solution at a relatively slow rate is driven by the generation of a partial positive charge on the ester carbonyl carbon because of the inductive effect of sulfur atom.45 The prodrug nanoparticles with βthiopropionate linkages were thus expected to be pH-sensitive. In vitro drug release experiments of the prodrug nanoparticles were performed in the phosphate buttered saline (PBS) solution at


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pH 5.0, 6.0 and 7.4, respectively, and typical release profiles were shown in Figure 4. When prodrug nanoparticles were incubated at pH 7.4, almost no CPT was released, indicating that the prodrug nanoparticles were stable in the neutral solution, because β-thiopropionate linkage between CPT molecule and the polymer scaffold was unable to hydrolyze at pH 7.4.38,39 Upon treatment of prodrug nanoparticles at pH 6.0, 23.4% (for 24 h) and 50.4% (for 96 h) of CPT in the nanoparticles were released. When the drug release was performed at pH 5.0, the release rate of CPT from prodrug nanoparticles increased, and 38.4% (for 24 h) and 79.7% (for 96 h) of CPT were released, this is due to that the hydrolysis rate of β-thiopropionate linkages increased with the decrease of pH values.41 It was clearly shown that the release of CPT from prodrug nanoparticles in a subacid condition needed a couple of days to accomplish, which was appropriate for controlled drug release in a sustained manner. It is speculated that, in the acidic condition, such as in the cancer cells, CPT can be controllably released from the prodrug nanoparticles.

Figure 4. In vitro CPT release profiles of prodrug nanoparticles fabricated from prodrug-1 after incubation in PBS buffer at pH values of 5.0, 6.0 and 7.4, respectively.


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In Vitro Cytotoxicity Assay The cytotoxicity of CPT, polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) and prodrug nanoparticles with acid-liable linkages against HeLa cells was investigated. Before evaluating the cytotoxicity of prodrug nanoparticles, it was necessary to study the cytotoxicity of the nanocarriers, because biocompatible nanocarriers could reduce the side effect in drug delivery.46 The cells were incubated with P(HEO2MA)-b-P(HEMA-DHLA) at various concentrations in the Dulbecco’s modified Eagle’s medium (DMEM) at 37 oC and 5% CO2 for 24 hours, and the cells cultured with free DMEM were used as control. As shown in Figure 5A, P(HEO2MA)-bP(HEMA-DHLA) was almost nontoxic to HeLa cells even at a concentration of 500 µg/mL, which was in accordance with the well-recognized biocompatibility of PHEO2MA.47 The cytotoxicity of prodrug nanoparticles was compared with that of CPT, and the result was shown in Figure 5B. Prodrug nanoparticles showed similar cytotoxicity to free CPT, and the IC50 (inhibitory concentration for 50% cell viability) of prodrug nanoparticles was 3.05 µg/mL, which was slightly higher than that of free CPT (IC50 = 2.01 µg/mL). Moreover, in vitro experiment of cell apoptosis was conducted to quantitatively detect and differentiate live, apoptotic and necrotic cells. Figure 5C showed that cells treated with prodrug nanoparticles at CPT concentration of 5 µg/mL demonstrated 16.0% early apoptotic (AV+/PI-) populations and 83.0% necrotic/late apoptotic cells (AV+/PI+), which was comparable to free CPT (10.1% early apoptotic populations and 89.6% necrotic/late apoptotic cells). The above results revealed that prodrug nanoparticles exhibited anticancer activity via releasing covalently conjugated CPT molecules in response to the acidic microenvironment of cancer cells.


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Figure 5. Relative cell viability values of HeLa cells evaluated by MTT assay after incubation with different concentrations of samples: (A) the polymer scaffold P(HEO2MA)-b-P(HEMADHLA), (B) the prodrug nanoparticles (black line) compared with free CPT (red line); (C) Annexin V/PI assay of free CPT and prodrug nanoparticles at the CPT concentration of 5 µg/mL after incubation for 48 h. Cellular Uptake of the Prodrug Nanoparticles To further investigate the pH-responsive intracellular release of CPT molecules from prodrug nanoparticles, confocal laser scanning microscope (CLSM) was used to study the internalization of the prodrug nanoparticles by cancer cells. HeLa cells were incubated with free CPT or


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prodrug nanoparticles at CPT equivalent doses of 3.0 µg/mL for 4 h, and then the cells were stained with calcein, which could label survived cells with green fluorescence. CLSM images in Figure 6A revealed that blue fluorescence of CPT mainly appeared at the perinuclear region of HeLa cells, and green fluorescence of calcein appeared in the overall cells. This observation indicated that prodrug nanoparticles were internalized by HeLa Cells. Moreover, HeLa cells cultured with free CPT displayed relatively weaker blue fluorescence compared with HeLa cells cultured with prodrug nanoparticles. The quantitative results by flow cytometry analysis (Figure 6B) further demonstrated that prodrug nanoparticles exhibited approximately 1.5-fold cellular internalization amount compared with free CPT. The probable reason is that the poor water solubility of free CPT makes it difficult for CPT to be endocytosed by cells.48,49 Prodrug nanoparticles overcame this obstacle successfully, and it was easier for prodrug nanoparticles to realize cell endocytosis compared with free CPT, because small size of nanoparticle was available for cellular uptake and PHEO2MA in prodrug nanoparticles could improve the cells’ absorption of nanopartilces.50,51


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Figure 6. (A) Confocal laser microscopic images of HeLa cells incubated with fresh DMEM medium containing free CPT (a) or prodrug nanoparticles (b) for 4 h at a CPT (blue) equivalent dose of 3.0 µg/mL. The cells were then counterstained with calcein (green); (B) Cellular uptake by flow cytometry for free CPT and prodrug nanoparticles at a CPT equivalent dose of 3.0 µg/mL. Antitumor Efficacy Therapeutic potentials of the prodrug nanoparticles were further evaluated using subcutaneous tumor model of Male 7-week-old CD-1 (ICR) mice. ICR mice bearing subcutaneous tumors (~ 50 mm3) were treated with PBS, free CPT and prodrug nanoparticles at a dose of 5 mg equivalent CPT per kilogram mouse, respectively. As shown in Figure 7, the tumor of ICR mice treated with PBS continued to grow for 12 days, and the tumor volume increased from 50 mm3 to 326 mm3. Prodrug nanoparticles showed better tumor growth suppression than CPT, and the tumor volume of mice treated with prodrug nanoparticles was only 73 mm3 after 12 d. The high


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anticancer efficacy was presumably attributed to the synergistic effect of tumors’ (the enhanced permeability and retention) effect43 and promoted cellular internalization of prodrug nanoparticles. In contrast, CPT is difficult for iv administration because its water solubility is poor. Such prodrug nanoparticles have potential applications in anticancer chemotherapy.

Figure 7. Tumor growth curves of Male 7-week-old CD-1 (ICR) mice after intravenous injection of PBS, free CPT and prodrug nanoparticles. Data are expressed as means ± SEM, n = 3. **P < 0.005, ***P < 0.0001. Conclusions In summary, a method for the synthesis of polymer prodrugs with high drug-loading efficiency, acid-liable linkages and controllable drug loading capacity was proposed. ACPT was efficiently coupled to the polymer scaffold P(HEO2MA)-b-P(HEMA-DHLA) via acrylate-thiol Michael addition reaction, forming polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT)


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with pH-responsive β-thiopropionate linkage. The drug loading contents of polymer prodrugs could be controlled via adjusting the feed molar ratio of [ACPT]/[DHLA] in the Michael addition reaction. Then the polymer prodrug self-assembled into nanoparticles in water with a CPT loading content as high as 40.1%. Owing to the hydrolysis of β-thiopropionate linkage in acidic solution, most of CPT was released from the prodrug nanoparticles at pH 5.0 in 4 days. Cell viability studies revealed that the polymeric carrier P(HEO2MA)-b-P(HEMA-DHLA) was of low cytotoxicity, whereas prodrug nanoparticles displayed similar cytotoxicity to free CPT, showing high anticancer activity. Moreover, prodrug nanoparticles were more easily internalized by HeLa cells compared with free CPT. In vivo anticancer experiments revealed that the prodrug nanoparticles showed better tumor growth suppression than free CPT. Therefore, this study provides an approach for fabrication of prodrug nanoparticles in anticancer chemotherapy. Experimental Section Materials. Camptothecin (CPT, TCI, >97%), triethylamine (TEA, Aladdin, 99.5%), diethylene glycol (TCI, 99.5%), 2-hydroxyethyl methacrylate (HEMA, TCI, >95%), lipoic acid (LA, Energy Chemical, 98%), methacryloyl chloride (Energy Chemical, 97%), acryloyl chloride (Energy Chemical,

98%),

N,N’-dicyclohexylcarbodiimide

(DCC,

Aladdin,

99.0%),

4-

dimethylaminopyridine (DMAP, Aladdin, 99%) and calcein (TCI) were used as received. Azobisisobutyronitrile (sigma, 98%) was recrystallized from ethanol. Tetrahydrofuran (THF) was refluxed over sodium for 24 hours and distilled prior to use. 4-Cyanopentanoic acid dithiobenzoate (CPADB) was synthesized according to the previous report.42 All other reagents with analytical grade were purchased from Shanghai Chemical Reagent Co and used as received. Instrumentations.


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All NMR spectra were recorded on a Bruker AV300 NMR spectrometer using CDCl3 as solvent. Mass spectrum analysis was performed by using a LC-MS instrument (Thermo Scientific, LTQ Orbitrap XL), and the system was equipped with an ESI source. Molecular weight and molecular weight distribution (Mw/Mn) were determined on a Waters 150C gel permeation chromatography (GPC) instrument equipped with microstyragel columns (500, 103 and 104 Å) and a RI 2414 detector set at 30 oC; monodispersed polystyrene standards were used in the calibration of the number-average molecular weight Mn and Mw/Mn, and DMF was used as an eluent at a flow rate of 1.0 mL/min. Ultraviolet (UV)/visible (Vis) light measurement was performed on a Unico (United Products & Instruments, Inc., NJ, USA) UV/Vis 2802PCS spectrophotometer. Transmission electron microscopy (TEM) observations were conducted on a JEM-100SX electron microscope at an acceleration voltage of 100 kV. Dynamic light scattering (DLS) measurements were carried out on a Dynapro light scattering instrument (model Dynapro99E) at 25 oC with an 824.3 nm laser, and the data were analyzed with DYNAMICS V6 software. Confocal laser scanning microscopy (CLSM) observations were conducted on a Leica TCS SP5 microscope. Fourier transform infrared spectra were acquired on Vector-22 fourier infrared spectrometer. Synthesis of 2-(2-hydroethoxy) ethyl methacrylate (HEO2MA). Diethylene glycol (1.06 g, 10 mmol) and TEA (1.51 g, 15 mmol) were dissolved in chloroform (250 mL). The mixture was stirred at 0 oC for 20 minutes, and then methacryloyl chloride (1.04 g, 10 mmol) in 50 mL of chloroform was added dropwise. The reaction was kept at room temperature for 12 hours. The reaction mixture was concentrated and the crude product was purified by column chromatography using a mixed eluent of n-hexane/ethyl acetate (2:1, v/v) to give the product (1.13 g, yield: 65.2%). 1H NMR (300 MHz, CDCl3, δ, ppm): 6.10 (d, J =


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1.43 Hz, 1H), 5.60 (d, J = 1.67 Hz, 1H), 4.14 (t, J = 3.57 Hz, 2H), 3.74 (t, J = 6.51 Hz, 4H), 3.57 (t, J = 5.54 Hz, 2H), 1.98 (s, 3H). 13C NMR (75 MHz, CDCl3, ppm): δ 167.2, 136.1, 125.2, 70.0, 69.4, 65.1, 61.3, 17.9. Synthesis of acrylate-tethering camptothecin (ACPT). Camptothecin (2.0 g, 6.0 mmol) and TEA (1.01 g, 10.0 mmol) were dissolved in chloroform (350 mL). The mixture was stirred at 0 oC for 20 minutes, and then acryloyl chloride (0.594 g, 6.6 mmol) in 20 mL of chloroform was added dropwise. The reaction was kept at room temperature for 24 hours. The reaction mixture was concentrated and the crude product was purified by column chromatography using a mixed eluent of n-hexane/ethyl acetate (1:1, v/v) to give the product (1.6 g, yield: 80%). 1H NMR (300 MHz, CDCl3, δ, ppm): 8.40 (m, 1H), 8.21 (m, 1H), 7.94 (m, 1H), 7.82 (m, 1H), 7.67 (m, 1H), 7.23 (m, 1H), 6.52 (d, J = 1.78 Hz, 1H) and 5.98 (d, J = 1.64 Hz, 1H), 6.27 (dd, 1H), 5.71 and 5.43 (m, 2H), 5.29 (m, 2H), 2.4~2.1 (m, 2H), 1.01(t, J = 2.96 Hz, 3H). 13C NMR (75 MHz, CDCl3, ppm): δ 167.5, 166.5, 156.9, 152.7, 148.9, 145.8, 145.3, 131.9, 131.3, 130.4, 129.0, 128.4, 128.2, 127.9, 127.3, 126.6, 119.9, 97.5, 76.3, 65.4, 53.7, 31.9, 7.9. MS: [M-H+] = 403.12, found at 403.1286. Synthesis of methacrylate of lipoic acid (HEMA-LA). Hydroxyethyl methacrylate (1.31 g, 0.01 mol), DMAP (0.6 g, 0.005 mmol) and DCC (3.84 g, 0.02 mol) were dissolved in dichloromethane (70 mL). Lipoic acid (2.06 g, 0.01 mol) in 10 mL of dichloromethane was added dropwise into the mixture at room temperature, and the solution was stirred for 12 hours. The reaction mixture was then concentrated and the crude product was purified by column chromatography using a mixed eluent of n-hexane/ethyl acetate (4:1, v/v) to give the product (2.23 g, yield: 70.1%). The monomer is not stable at room temperature, and it should be stored at low temperature (-20 oC). 1H NMR (300 MHz, CDCl3, δ, ppm): 6.14 (d, J =


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1.56 Hz, 1H), 5.58 (t, J = 1.72 Hz, 1H), 4.32 (s, 4H), 3.54 (m, 1H), 3.12 (m, 2H), 2.53 (m, 1H), 2.33 (t, J = 7.40 Hz, 2H), 1.9~1.8 (m, 4H), 1.7~1.2 (m, 6H). 13C NMR (75 MHz, CDCl3, ppm): δ 173.1, 167.2, 136.0, 125.8, 62.2, 62.0, 40.2, 38.5, 34.6, 33.9, 29.0, 28.0, 17.9. Synthesis of Poly(2-(2-hydroethoxy) ethyl methacrylate) (PHEO2MA). HEO2MA (2.61 g, 0.015 mol), AIBN (5 mg, 0.03 mmol), CPADB (84 mg, 0.3 mmol) and THF (6 mL) were added into a 10 mL tube. After three freeze-vacuum-thaw cycles, the polymerization tube was sealed under vacuum. The polymerization was carried out at 70 oC for 6 h. The polymerization tube was cooled to room temperature, and then the reaction mixture was added dropwise into excess diethyl ether to obtain a precipitate. The precipitation process was repeated for three times with an isolated yield of 87.1%. The polymer was dried in a vacuum oven for 24 hours. Synthesis of P(HEO2MA)-b-P(HEMA-LA). PHEO2MA (Mn = 7700 g/mol, 770 mg, 0.1 mmol), HEMA-LA (1.272 g, 4.0 mmol), AIBN (1.64 mg, 0.01 mmol) and THF (8 mL) was added into a 10 mL tube. After three freeze-vacuumthaw cycles, the polymerization tube was sealed under vacuum. The polymerization was carried out at 70 oC for 6 h. The polymerization tube was cooled to room temperature, and then the polymer was obtained by precipitation into excess diethyl ether. The precipitation process was repeated for three times with an isolated yield of 30.9%. The obtained solid was dried in a vacuum oven at room temperature for 24 hours. Synthesis of P(HEO2MA)-b-P(HEMA-DHLA). P(HEO2MA)44-b-P(HEMA-LA)12 (Mn = 11500 g/mol, 115 mg, 0.01 mmol) was added into THF (10 mL). To this solution, NaBH4 (12 mg, 0.32 mmol) was added at 0 oC, then the reaction mixture was kept for 2 hours at room temperature. The final solution was dialyzed against


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deionized water for 24 hours, and it should be kept in the dark place. The product was obtained as a white solid by lyophilization with an isolated yield of 87.0%. Synthesis of Polymer Prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT). P(HEO2MA)44-b-P(HEMA-DHLA)12 (Mn = 11500 g/mol, 23 mg, 2.0 µmol), ACPT (19.2 mg, 48 µmol), THF (20 mL) and a drop of TEA were added into a 50 mL flask. The reaction was kept at 50 oC for 24 hours. After the reaction mixture was cooled to room temperature, the solution was added dropwise into an excess of diethyl ether to obtain the polymer. The precipitation process was repeated for three times. The precipitate was dried at room temperature in a vacuum oven for 24 hours with an isolated yield of 87.6%. Fabrication of Prodrug Nanoparticles. Polymer prodrug P(HEO2MA)-b-P(HEMA-DHLA-CPT) (12 mg) was dissolved in DMSO (1.0 mL) and stirred vigorously at room temperature for 1 hour. Deionized water (7.0 mL) was added into the solution through the syringe pump at a rate of 3.0 mL/h while stirring. After stirring for 5 h, the solution was loaded into a dialysis bag (MWCO 3500) and dialyzed against deionized water for 12 h, forming prodrug nanoparticles at a concentration of 1.5 mg/mL. The drug loading content (DLC) of the nanoparticles was calculated by UV quantitative analysis. In UV analysis, the UV absorbance at 365 cm-1 of a series of CPT solutions (0.005, 0.1, 0.05, 0.1, 0.25 mg/mL) in DMSO was measured. Figure S6 showed the plot of UV absorbance versus concentration of CPT in DMSO. Polymer prodrug (1.0 mg) was dissolved in DMSO (1.0 mL) and the concentration of CPT was obtained according to the standard curve. The drug loading content was obtained according to the followed formula: DLC(UV) = (weight of loaded CPT/weight of polymer prodrug) × 100% Critical micelle concentration (CMC) measurement.


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Pyrene was used as a fluorescence probe to determine the critical micelle concentration of prodrug nanoparticles. The aqueous solution of prodrug nanoparticles at a concentration of 1.5 mg/mL was diluted to various concentrations from 0.75 mg/mL to 8.125 × 10-5 mg/mL. Then 50 µL acetone solution of pyrene at a concentration of 6 × 10-5 M was added into the solution of prodrug nanoparticles at various concentrations. The excitation spectra of all samples were recorded. The emission wavelength was fixed at 374 nm while the excitation wavelength was fixed in the range of 300-370 nm. The I339/I332 values of all solutions were recorded, and CMC values were shown in Figure 3B. Release of CPT from Prodrug Nanoparticles. The drug release experiments were performed in PBS buffer at pH 5.0, 6.0 and 7.4, respectively. The specific experimental procedure was as follows: an aqueous dispersion of CPTloaded prodrug nanoparticles (2.0 mg/mL, 2.0 mL) was transferred to a dialysis bag with a membrane molecular weight cut-off of 3.5 KDa, and then the dialysis bag was immersed in 60 mL of PBS buffer solution at pH 5.0 or 6.0 or 7.4. At predetermined times, 2 mL of sample from the solution out of the dialysis bag was taken for estimating the amount of drug released, which was replaced with 2 mL of PBS buffer with identical pH value. UV absorbance of the dialysis solution at 365 cm-1 was monitored to determine the CPT releasing profile. This process could last for 4 days. Each measurement was done in triplicate, and the data was shown as the mean value plus a standard deviation (± SD). Cytotoxicity Evaluation. The cytotoxicity of CPT, P(HEO2MA)-b-P(HEMA-DHLA) and prodrug nanoparticles was evaluated by MTT assays. HeLa cells were cultured in a 96-well plate at a density of 5000 cells per well overnight using DMEM supplemented with 10% fetal bovine serum (FBS). The


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solutions of free CPT, P(HEO2MA)-b-P(HEMA-DHLA) and prodrug nanoparticles in PBS buffer (50 mM, pH 7.4) were diluted into samples with various concentrations. The cells were then incubated with samples at various concentrations for 24 hours at 37 oC and 5% CO2. Cells without treatment were used as control. The medium was removed and replaced with fresh DMEM media containing 0.5 mg/mL MTT reagent. After 4 hours, the cultured medium was removed. DMSO (150 µL) was then added, and absorbance of the solution in each well was detected at 490 nm by a microplate reader. For further analysis of cell apoptosis, Annexin V/PI assay was carried out. HeLa cells were cultured in a 24-well plate at a density of 105 cells per well overnight using DMEM with 10% FBS. Then the DMEM was removed, followed by addition of fresh DMEM containing CPT or prodrug nanoparticles at a CPT concentration of 5 µg/mL. After the cells were cultured for 4 h, the medium was replaced with DMEM with 10% FBS. The incubation was continued for 48 h. Then, Annexin V/PI staining for apoptotic cells was performed according to manufacturer’s protocols, and quantitative apoptotic measurements were performed by flow cytometric analysis. Cellular Uptake of the Prodrug Nanoparticles. HeLa cells were cultured in glass-bottom petri dishes at a density of 8000 cells per well. They were incubated for 24 h at 37 oC and 5% CO2 before treatment. Fresh DMEM medium (100 µL) containing CPT or prodrug nanoparticles at a CPT equivalent dose of 3.0 µg/mL was added into each well. After cells were incubated for 4 h, the cultured medium was removed. The cells were rinsed two times with PBS solution, and the cells were stained with calcein (green stain) for 15 minutes. Then the cells were rinsed with PBS solution to remove redundant calcein. After the above treatment, the cells were observed by confocal laser scanning microscope (CLSM) immediately. The CPT was excited at 405 nm and the emitted light was detected between 400


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and 500 nm. The calcein was excited at 495 nm and the emitted light was detected between 500 and 600 nm. For flow cytometric analysis, the cells were washed for three times with PBS solution, detached by trypsin treatment, and suspended in PBS for flow cytometry measurements. In Vivo Antitumor Efficacy of Free CPT and Prodrug Nanoparticles. The tumor models were established at the right flank of Male 7-week-old CD-1 (ICR) mice by inoculation with 107 4T1 cells suspended in 100 µL PBS. To exhibit the antitumor efficacy of prodrug nanoparticles, ICR mice bearing subcutaneous tumors (~ 50 mm3) were treated with PBS, free CPT and prodrug nanoparticles, respectively, at a dose of 5 mg equivalent CPT per kilogram mouse. Tumor size (V) was measured every two days, and tumor size was calculated from the equation V = a × b2/2 (a and b are the longest and shortest diameters of tumor, respectively.). After day 12, mice were sacrificed and tumors were excised according to standard protocol.

Supporting Information. GPC data for the obtained polymers, FTIR spectrum of P(HEO2MA)-b-P(HEMA-DHLA), DLS curves of prodrug nanoparticles, photographs of the representative tumors and standard curves of CPT in DMSO are available in Figure S1-6.

*Corresponding Author E-mail: [email protected], [email protected].


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Acknowledgments The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China under contrast Nos. 21525420 and 21774113. Notes The authors declare no competing financial interest.

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Graphical Table of Content

Efficient Synthesis of Polymer Prodrug by Thiol-Acrylate Michael Addition Reaction and Fabrication of pH-Responsive Prodrug Nanoparticles

Chao-Ran Xu†, Liang Qiu‡, Cai-Yuan Pan†, Chun-Yan Hong*,†, Zong-Yao Hao*,§


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Efficient Synthesis of Polymer Prodrug by Thiol-Acrylate Michael

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