Synthesis of an Oxidation-Sensitive Polyphosphoester Bearing

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Synthesis of an Oxidation-Sensitive Polyphosphoester Bearing Thioether Group for Triggered Drug Release Jihong Wang, Dongdong Li, Wei Tao, Yang Lu, Xianzhu Yang, and Jun Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00101 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 9, 2019

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Synthesis of an Oxidation-Sensitive Polyphosphoester Bearing Thioether Group for Triggered Drug Release Jihong Wang,† Dongdong Li,‡ Wei Tao,† Yang Lu,† Xianzhu Yang,‡,,* Jun Wang, †

School of Chemistry and Chemical Engineering, Hefei University of Technology,

Hefei, Anhui, 230009, China ‡

Institutes for Life Sciences, School of Medicine, South China University of

Technology, Guangzhou, Guangdong 510006, China 

National Engineering Research Center for Tissue Restoration and Reconstruction,

South China University of Technology, Guangzhou 510006, China 

School of Biomedical Science and Engineering, South China University of

Technology, Guangzhou International Campus, Guangzhou 510006, P. R. China 

Guangzhou Regenerative Medicine and Health Guangdong Laboratory, 510005

Guangzhou, China

*Corresponding author: Prof. Xianzhu Yang ([email protected])

ACS Paragon Plus Environment

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ABSTRACT. In this work, novel amphiphilic diblock copolymers of polyethylene glycol

and

polyphosphoester

with

pendant

thioether

groups,

denoted

as

mPEG-b-PMSPEP, were synthesized through the ring-opening polymerization of functionalized cyclic phosphoester monomer using methoxy poly(ethylene glycol) and Sn(Oct)2 as the macroinitiator and catalyst, respectively. The successful synthesis was confirmed by 1H, 13C, 31P nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). These amphiphilic block copolymers self-assembled spontaneously in the aqueous solution, and the formed nanoparticles were sensitive to the oxidation that induced the hydrophobic to hydrophilic transition for its PMSPEP core under triggering of H2O2 and the subsequent dissociation of the nanoparticles. In addition, the reactive oxygen species (ROS) generated by light and the photosensitizer were also capable of carrying out the oxidation of these nanoparticles. Their oxidation profiles were systemically evaluated by 1H NMR. Finally, the mPEG-b-PMSPEP nanoparticles were used to co-encapsulate the photosensitizer chlorin e6 (Ce6) and anticancer drug paclitaxel (PTX), achieving the photo-accelerated PTX release via oxidation of the nanoparticles by the generated ROS under light irradiation. Meanwhile, the in vitro cytotoxicity assays indicated that these nanoparticles co-encapsulated with PTX and Ce6 showed a combined cell-killing effect toward MDA-MB-231 tumor cells, exhibiting great potential for drug delivery systems that realize the synergistic chemo-photodynamic therapy for cancer treatment. KEYWORDS: oxidation-responsive; polyphosphoester; photo-accelerated drug delivery; cancer therapy; synergistic therapy


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1. INTRODUCTION In the past decades, nanocarriers with on-demand drug release capability have attracted particular attention due to their great potential for improving the anticancer effect toward tumor cells while minimizing the toxicity to normal cells.1-6 Therefore, stimulus-sensitive polymeric nanocarriers have been explored to develop smart drug delivery systems because of their ability to release the encapsulated cargos with the triggering by specific stimuli.7-12 These internal or external stimuli included pH,13 redox potentials,14 overexpressed enzymes,15 ultrasound,16 magnetic field,17 and light.18 Among the various types of stimuli, reactive oxygen species (ROS)-sensitive drug delivery systems have been largely ignored. This is because the extremely low levels of hydrogen peroxide (H2O2), which is the main ROS in biological microenvironments, limit their practical use, even in cancerous cells and at the inflamed sites.19 Recently, several groups have found that a photosensitizer under light irradiation can trigger a structural change or dissociation of these ROS-sensitive nanocarriers.20,21 Therefore, the ROS-sensitive polymers can be widely developed for fabricating the photo-mediated drug delivery systems.22-24 Despite the promising progress, these ROS-sensitive polymers have been usually synthesized by step-growth polymerizations, resulting in the inability to control the molecular weight and polydispersity of these polymers.25 Taking all of these considerations into account, the development of a new synthetic method for obtaining ROS-sensitive polymer is still highly desirable. As biomedical polymers, polyphosphoesters (PPEs) with repeating phosphoester



linkages in the backbone have been widely used for biomedical applications.26,27 The PPEs can be synthesized by ring-opening polymerization (ROP) to obtain the polymer with a predictable molecular weight and low polydispersity.26 More importantly, the pentavalent nature of phosphorus in the backbone offers more flexibility for the synthesis of the stimulus-sensitive PPEs by adjusting its side group.28 For instance, redox-,29 temperature-,30 and pH-sensitive PPEs31 have been synthesized by the ROP methods. Thus, using this method, it is possible to synthesize ROS-sensitive PPE with a predictable molecular weight and low polydispersity. Herein, we report a novel type of ROS-sensitive PPE with pendant thioether groups that was synthesized by ROP of cyclic phosphoester monomers MSPEP. The kinetics of the polymerization of such cyclic phosphoester monomers were studied using a PEG macroinitiator and stannous octoate (Sn(Oct)2) as the catalyst. Furthermore, the oxidation profiles of the obtained ROS-sensitive diblock polymer and its hydrophobic to hydrophilic transition were investigated. We also demonstrated the potential of these ROS-responsive copolymers mPEG-b-PMSPEP as nanocarriers for the combination of photodynamic therapy (PDT) and chemotherapy. 2. EXPERIMENTAL SECTION Materials. 3-Methylthiopropanol (MSP) was obtained from Aladdin Chemical Co., Ltd. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (COP) was synthesized according to a literature report32 and purified by distillation under reduced pressure. mPEG-OH (Mn = 2000 gmol-1) and stannous octoate (Sn(Oct)2) were purchased from Sigma-Aldrich (Shanghai, China). Photosensitizer Ce6 was purchased from Frontier Scientific. PTX


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and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Dalian Meilun Biotechnology Co., LTD. Other solvents and chemicals were used as received. MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC) and were cultured in complete medium (DMEM with 10% fetal bovine serum (FBS)) at 37 C with 5% CO2. Characterization. 1H,

31P,

and

13C

NMR spectra were recorded on a Brucker

VNMRS 600 MHz NMR spectrometer at 25 C with deuterated chloroform (CDCl3) as the solvent and TMS as the internal reference. In addition, phosphoric acid (85%) was used as the external reference for 31P NMR analyses. The molecular weights and the molecular weight distributions were measured using a Waters gel permeation chromatography (GPC) system. The GPC system was composed of a Waters 1515 pump and a Waters 2414 refractive index detector, equipped with Waters Styragel high-resolution columns at 35 C. Chromatographic grade dimethyl formamide (DMF) was used as the flowing phase at a flow rate of 1.0 mLmin-1. The monomer conversion was determined by monitoring the disappearance of the peak centered at the elution volume of 30.1 mL, and the monomer concentrations at different reaction times were determined by the comparison of peak heights to those of a standard curve with known monomer concentrations. The equilibrium concentration of the monomer was obtained when the monomer was no longer consumed with further prolongation of the reaction time. The morphology of samples was analyzed using JEM-2100F transmission electron microscopy (TEM) at an accelerating voltage of 200 kV. The critical micellization concentrations (CMCs) of the amphiphilic copolymer were



determined according to the previously reported method.26 Synthesis of 3-(methylthio)propyl ethylene phosphate (MSPEP). An anhydrous tetrahydrofuran (THF) solution containing COP was added dropwise to THF solution containing the MSP and trimethylamine (TEA) at -5 C within 2 h. The molar ratio of COP:TEA:MSP was 1:1:1. After further reacting overnight, the precipitate triethylammonium chloride was filtrated by column chromatography in a Schlenk funnel under N2 atmosphere. Then, the filtrate was concentrated under reduced pressure to obtain the product (yield: 83%). Synthesis of diblock copolymer mPEG-b-PMSPEP. The diblock copolymer mPEG-b-PMSPEP was synthetized by ring-opening polymerization using mPEG-OH (Mn = 2000 gmol-1) and Sn(Oct)2 as the macroinitiator and catalyst. Typically, mPEG-OH (0.5 g, 0.25 mmol) and MSPEP (1.6 g, 7.5 mmol) were dissolved in anhydrous THF (5.0 mL) in a fresh flamed and nitrogen-purged flask in a glovebox. After dissolving, Sn(Oct)2 (50 mg) was added and further reacted at 45 C . For polymerization kinetics study, aliquots (40 μL) were taken out and diluted in 800 μL of DMF for GPC analyses at the predetermined time period. After 6 h of reaction, the solution was concentrated and precipitated into a cold diethyl ether/methanol mixture (10/1, v/v) twice. The obtained precipitate was dried under vacuum until constant weight at room temperature. Preparation of drug-loaded nanoparticles. The Ce6-loaded nanoparticles of the diblock copolymer were prepared through the nanoprecipitation method. Briefly, Ce6 (1.0 mg) and mPEG45-b-PMSPEP21 (10.0 mg) were dissolved in dimethylsulfoxide


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(DMSO) (1.0 mL) solution, and the mixed solution was added dropwise to ultra-purified water (10 mL) under stirring. After additionally stirring for 3 h, the solution was transferred to dialysis tubing (MWCO 14,000 Da) and dialyzed against ultra-purified water for 24 h. The unloaded Ce6 was removed through a 0.45-μm Millipore filter, and the Ce6-loaded nanoparticles were obtained and are denoted to as MS-NP/Ce6. Additionally, the Ce6 and PTX co-loaded nanoparticles, denoted as MS-NP/Ce6&PTX, were prepared by similar methods, except that the Ce6 (1.0 mg) was replaced with the mixture of Ce6 (1.0 mg) and PTX (1.0 mg). The drug loading contents (DLCs) and encapsulation efficacies (EEs) were calculated by the following equations: DLC (%) = EE (%) =

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶𝑒6 𝑜𝑟 𝑃𝑇𝑋 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 - 𝑙𝑜𝑎𝑑𝑒𝑑 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑥𝑙𝑒

× 100%

𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶𝑒6 𝑜𝑟 𝑃𝑇𝑋 𝑖𝑛 𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 × 100% 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝐶𝑒6 𝑜𝑟 𝑃𝑇𝑋 𝑎𝑑𝑑𝑒𝑑

For MS-NP/Ce6, the EE of Ce6 was 29.8%. For MS-NP/Ce6&PTX, the EEs of Ce6 and PTX were 28.1% and 46.7%, respectively, and the DLCs of Ce6 and PTX were 2.61% and 4.34%, respectively. Hydrogen peroxide sensitivity study. The blank nanoparticles (1.5 mL, 10.0 mgL-1) were mixed with the same volume of H2O2 solution at different concentrations. After incubation for 12 h at room temperature, the size of the samples in the aqueous solution was measured using a Malvern Zetasizer Nano ZS90 dynamic light scattering instrument with a 633-nm He-Ne laser and 90 collecting optics. In addition, the optical images of these nanoparticles were recorded by a digital camera. Moreover, the optical transmittances of the nanoparticle solution (10 mgmL-1)



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incubated with H2O2 solution at different concentrations for 12 h were measured at 500 nm using a UV-vis spectrophotometer (Hitachi, Japan). Thereafter, the obtained nanoparticles were lyophilized and recorded by NMR in CDCl3. ROS sensitivity study. The Ce6-loaded nanoparticles MS-NP/Ce6 were exposed to 660-nm laser irradiation at different power densities and times. Subsequently, the obtained nanoparticles were lyophilized and characterized by NMR in CDCl3. PTX release with or without light irradiation. The aqueous solution of MS-NP/Ce6&PTX was suspended in phosphate buffer (PB buffer, 0.02 M) at a PTX concentration of 100 μg/mL. The solution (1.0 mL) was exposed to 660-nm laser irradiation (1.0 W/cm2, 10 min) and then was transferred into the dialysis tubing. Next, a PB buffer (20 mL) was added to immerse the tubing at 37 C with gentle shaking (100 rpm). The external PB buffer was collected at predetermined intervals and lyophilized for HPLC analysis to determine the concentration of PTX according to a previously reported method.33 In vitro cytotoxicity assays. MDA-MB-231 cells were seeded in 96-well plates at a cell density of 5000 cells per well and were further incubated overnight. Fresh complete

medium

containing

MS-NP/Ce6&PTX

nanoparticles

or

control

formulations was added at different PTX concentrations to replace the original medium in the absence or presence of Vitamin C (VC, 1.0 mM). Vitamin C was used as a ROS scavenger to eliminate intracellular ROS in partial cells. After incubation for 12 h, the cells were washed with PBS twice and then were exposed to 660-nm laser irradiation (1.0 W/cm2, 10 min). After further incubation for 36 h, the cell


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viabilities were detected by the MTT assay according to the standard protocol. Statistical Analysis. To measure significant differences among each groups, statistical analyses were performed using Student’s t-test. p values < 0.05, p values < 0.01 were considered to be statistically significant.

3. RESULTS AND DISCUSSION Synthesis and characterization of diblock copolymers. To obtain ROS-sensitive amphiphilic diblock polymers of mPEG and PPE, we first synthesized the cyclic phosphoester monomer MSPEP with a thioether group. The MSPEP was synthesized by esterification of MSP and COP (Scheme 1A), similar to the synthesis of other cyclic phosphoester monomers.

Scheme 1. Synthetic routes of MSPEP monomer (A) and mPEG-b-PMSPEP diblock polymer (B). The 1H and

13C

NMR spectra of the obtained MSPEP monomer are shown in

Figure 1A and 1B, respectively. All of the resonances were assigned as illustrated. The 1H NMR (Figure 1A)  (600 MHz, CDCl3, ppm): 4.42 (m, 2H, -POCH2CH2O-), 4.33 (m, 2H, -POCH2CH2O-), 4.22 (dt, J=8.7, 6.2 Hz, 2H, -POCH2CH2CH2S-), 2.57 (m, 2H, -POCH2CH2CH2S-), 2.07 (m, 2H, -CH2SCH3), 1.96 (m, 3H, -CH2SCH3). 13C



 (151 MHz, CDCl3, ppm): 69.89 (d, J=6.2 Hz,

NMR (Figure 1B)

-OCH2CH2CH2S-), 68.71 (d, J=2.5 Hz, -POCH2CH2O-), 32.50 (s, -CH2CH2SCH3), 32.36 (d, J=6.3 Hz, -CH2CH2CH2SCH3), 18.00 (m, -CH2SCH3). In addition, the single peal observed from

31P

NMR at 14.5 ppm (Figure 1C) suggests the successful

formation of MSPEP monomer. The electrospray ionisation mass spectrometry (ESI-MS) of MSPEP further demonstrates the successful synthesis of the MSPEP monomer (Figure S1).

Figure 1. 1H (A), 13C (B), and 31P (C) NMR spectra of MSPEP (in CDCl3). Subsequently, we synthesized a diblock copolymer mPEG-b-PMSPEP by ring-opening polymerization of MSPEP using mPEG-OH as the macroinitiator and stannous octoate as the catalyst. The synthesis pathway is shown in Scheme 1B, and


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the copolymers with various degrees of polymerization (DP) were synthesized by tuning the feeding ratio of the monomers and the mPEG-OH macroinitiator (Table S1). The obtained products were analyzed by GPC and NMR spectra. Figure 2A indicates the molecular weight and the molecular weight distribution of these produces (Table S1) obtained by GPC measurement. All of these diblock polymers exhibited decreased retention times compared to the mPEG45-OH macroinitiator, and all of the resonances in their 1H NMR spectra were assigned to the corresponding protons (Figure 2B). These results indicated the successful synthesis of the diblock copolymers. Furthermore, we calculated the DP of PMSPEP from its 1H NMR based on the integration ratio at 2.17 ppm (g) and 3.62 ppm (b), assigned to the methyl protons of MSPEP units and methylene protons of PEG, respectively. Therefore, the obtained copolymer were denoted as mPEG45-b-PMSPEP8, mPEG45-b-PMSPEP12, and mPEG45-b-PMSPEP21 (the subscript number represents DP), respectively. In addition, the 31P NMR spectrum of mPEG45-b-PMSPEP12 shown in Figure 2C gave a strong resonance at -4.30 ppm, further confirming the successful synthesis.



A

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mPEG45-b-PMSPEP21 mPEG45-b-PMSPEP12 mPEG45-b-PMSPEP8 mPEG45

20

22

24

26

28

Elution volume (mL)

O

B

b

a

O

b

c

P

O

45

O

O

O d

e

f

S yg

H

g f

a

c d

e

y=8 y = 12

y = 21 5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

ppm

C

10

8

6

4

2

0

-2

-4

-6

-8

ppm

-10 -12 -14 -16 -18 -20

Figure 2. (A) GPC chromatograms of mPEG45-b-PMSPEP and mPEG45-OH. (B) 1H NMR spectra of diblock copolymer mPEG45-b-PMSPEP (in CDCl3). (C)

31P

NMR

spectra of diblock copolymer mPEG45-b-PMSPEP12. The polymerization kinetics of MSPEP initiated with co-initiation of mPEG45-OH and Sn(Oct)2 were studied. The polymerization reaction was performed in THF solvent and was followed by GPC analyses at various time periods. It is clearly observed in Figure 3A that MSPEP monomer was consumed with the increase in the


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molecular weights of mPEG-b-PMSPEP. The relationship between MSPEP conversion versus the number average molecular weight (Mn) and dispersity (Ð) of mPEG-b-PMSPEP is shown in Figure 3B. It was found that the conversion of MSPEP reached 18.6% and 39.1% in 0.5 h and 1 h (Figure 3B) and increased to 77.6% after 6 h of reaction, which was relatively slow compared to the previous reported cyclic phosphoester monomers. The Mn of mPEG-b-PMSPEP follows a linear relationship with MSPEP conversion, indicating that a limited amount of transesterification reactions occurred during the polymerization. Therefore, the Ð of the polymer was below 1.25 even at 6 h.

Figure 3. (A) GPC chromatograms of the samples at various time intervals for the ring-opening polymerization of MSPEP in THF at a monomer-to-initiator ratio of 30:1. (B) Plot of Mn versus monomer conversion for the polymerization of MSPEP using Sn(Oct)2 as the initiator, obtained from 1H NMR analyses. Self-Assembly

and

Oxidation

Sensitivity

of


mPEG-b-PMSPEP.

The


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amphiphilic property of the mPEG-b-PMSPEP ensured its spontaneous self-assembly into polymeric aggregates in aqueous solution. To demonstrate this, the CMCs of these mPEG-b-PMSPEP copolymers were determined using the pyrene probe according to our previously reported methods.26 From the sigmoidal shape curve (Figure 4A), the CMC values were calculated as 3.7×10-1, 1.1×10-2, and 3.2×10-2 mg/mL of mPEG45-b-PMSPEP8, mPEG45-b-PMSPEP12, and mPEG45-b-PMSPEP21, respectively. It was easily found that the CMC values of these mPEG-b-PMSPEP copolymers decreased with the increase of the chain length of hydrophobic PMSPEP block,

resulting

in

the

higher

thermodynamic

stability.

Therefore,

mPEG45-b-PMSPEP21 was used for the subsequent experiment. The amphiphilic mPEG45-b-PMSPEP21 can form micellular nanoparticles in an aqueous solution, the nanoparticle exhibited good stability in the PBS or 10% FBS (Figure S2). As reported, the hydrophobic thioether is oxidized to form hydrophilic sulfoxide or sulfone groups, resulting in the shrinkage of the formed nanoparticles. To investigate

the

oxidation-responsive

behavior

of

the

mPEG45-b-PMSPEP21

copolymers, the turbidity (Figure 4B) was monitored after incubation in various concentrations of H2O2 for 12 h. The transmittance of the nanoparticle solution increased to ~100% at a H2O2 concentration of 37.5 mM or more, which could also be clearly observed in the final image of the nanoparticle solution (Figure 4C). In addition, the size changes (Figure 4D) of the mPEG45-b-PMSPEP21 nanoparticles were also examined. Clearly, the mPEG45-b-PMSPEP21 nanoparticle was shrunk with the increase in the H2O2 concentration, which was further confirmed by the


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transmission electron microscope (TEM) images. These results may be due to the switch of the hydrophobic thioether group into the hydrophilic sulfoxide or sulfone group at the high H2O2 concentration.

Figure 4. (A) The intensity ratio (I339/I336) as a function of mPEG-b-PMSPEP concentration.

(B)

Influence

of

various

concentrations

of

H2O2

on

the

oxidation-responsive behavior of mPEG45-b-PMSPEP21. (C) Images of the nanoparticle solution incubation with different concentrations of H2O2. (D, E) The



diameter change (D) and TEM images (E) of the nanoparticles after incubation with different concentrations of H2O2. Scale bar = 200 nm. To verify the above speculation, the samples were analyzed by 1H NMR (Figure 5A). Three peaks at 1.90, 2.10, and 2.60 ppm, ascribed to methyl and methylene groups around the thioether group (e, f, and g), respectively, disappeared gradually with the increase in the H2O2 concentration. Meanwhile, the gradually emerged peaks at 2.20 (e’), 2.65 (g’), and 3.97 (f’) ppm further verified the oxidation of sulfur atoms. The extent of oxidation was then quantitatively estimated by plotting the integrals of methyl g’ against methyl g, and the results are shown in Figure 5B. The extent of the oxidation was calculated as 17.5% and 41.5% for mPEG45-b-PMSPEP21 after 12 h of incubation with H2O2 at a concentration of 1.2 and 4.7 mM, respectively, and almost 100% of the thioether group was oxidized at a concentration of 18.8 mM or more. This H2O2 concentration-dependent oxidation behavior is consistent with the observations of turbidity measurements.


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Figure 5. (A) 1H NMR spectra of the oxidation of mPEG45-b-PMSPEP21 at different H2O2 concentrations. (B) Extent of oxygenation of mPEG45-b-PMSPEP21 at various concentrations of H2O2 (25 C, 12 h). Apart from the H2O2, other types of ROS, e.g., the singlet oxygen generated by photosensitizers under the irradiation of specific wavelength light, may also be capable of oxidizing the thioether group of the PMSPEP block. To demonstrate this, the photosensitizer chlorin e6 (Ce6) was encapsulated into the mPEG45-b-PMSPEP21 nanoparticle (MS-NP/Ce6). The MS-NP/Ce6 was exposed to 660-nm laser at the power density of 2.0 W/cm2 for different times, and the sample was lyophilized and analyzed by 1H NMR. As shown in Figure 6A, three peaks at 1.90, 2.10, and 2.60 ppm disappeared gradually, and the peaks at 2.20 (e’), 2.65 (g’), and 3.97 (f’) ppm gradually emerged with the extension of the irradiation times, which is similar to the



H2O2-mediated oxidation behavior (Figure 5A). Subsequently, the oxidation behavior of MS-NP/Ce6 was further determined at different power densities. It was clearly observed that the extent of oxidation increased with the increase in the power density (Figure 6B). More interestingly, the extent of oxidation for mPEG45-b-PMSPEP21 was further enhanced in the oxygen-saturated aqueous solution, which could be attributed to more efficient ROS generation.34 Both results demonstrated that light with the assistance of Ce6 can oxidize the thioether group by the generated ROS, achieving the hydrophobic to hydrophilic transition for the PMSPEP block. Thereby, and the final disassembly of the nanoparticles was realized, which could be verified by the TEM images of the MS-NP/Ce6 following these treatments (Figure 6C). It is well known that stimulus-sensitive disassembly of nanoparticles could be used in smart drug delivery systems to control drug release. To demonstrate the potential use, PTX, as a model drug, and Ce6 were simultaneously encapsulated into the mPEG45-b-PMSPEP21 nanoparticles (MS-NP/Ce6&PTX). The release profile of MS-NP/Ce6&PTX with or without 660-nm laser irradiation was first examined. As shown in Figure 6D, the PTX release was markedly accelerated after receiving the light irradiation (660 nm, 1.0 W/cm2, 10 min). For instance, only 44.5% of PTX was released from MS-NP/Ce6&PTX without illumination, whereas the released PTX was significantly increased to 73.9% after receiving 660-nm laser irradiation.


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Figure 6. (A) 1H NMR spectra of the oxidation of mPEG45-b-PMSPEP21 at different irradiation

times

under

660-nm

laser.

(B)

Oxygenation

efficiency

of

mPEG45-b-PMSPEP21 with different conditions. (C) TEM of MS-NP/Ce6 at different conditions. Scale bar = 200 nm. (D) PTX release profiles of MS-NP/Ce6&PTX with and without 660-nm laser irradiation. To further demonstrate the advantage of such ROS-sensitive nanoparticles for cancer therapy, the cytotoxicity of MS-NP/Ce6&PTX against MDA-MB-231 cells was evaluated. MDA-MB-231 cells were incubated with MS-NP/Ce6&PTX and then treated with or without NIR irradiation. Additionally, VC, as a ROS scavenger, was also added in partial cells to eliminate intracellular ROS. As shown in Figure 7A, MS-NP/Ce6&PTX without the 660-nm laser irradiation exhibited the lowest



anticancer effect at each concentration, whereas treatment with MS-NP/Ce6&PTX plus light irradiation displayed the highest anticancer efficacy. In contrast, the addition of VC moderately reduced the anticancer efficacy of MS-NP/Ce6&PTX (L+), implying that the ROS generated under light irradiation were also involved in the superior anticancer effect of MS-NP/Ce6&PTX (L+). It should be noted that the blank MS-NP did not exhibit cytotoxicity to the cancer cells (Figure 7B) even at the higher concentration. Collectively, these results demonstrated that the ROS-sensitive nanoparticles could not only realize photo-triggered intracellular drug release though the hydrophobic to hydrophilic transition for the PMSPEP core but also combine chemotherapy and photodynamic therapy, which could be used for synergistic chemo-photodynamic therapy in the future.

Figure 7. (A) The cell viabilities of MDA-MB-231 cells after treatment with MS-NP/Ce6&PTX (L-), MS-NP/Ce6&PTX and MS-NP/Ce6&PTX + VC (L+). The MS-NP/Ce6&PTX (L+) and MS-NP/Ce6&PTX + VC (L+) groups received the 660-nm light irradiation (1.0 W/cm2, 10 min). (B) The cell viabilities of MDA-MB-231 cells after treatment with blank MS-NP.


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Biomacromolecules

4. CONCLUSION The amphiphilic diblock copolymers of PEG and PMSPEP containing a thioether group were synthesized through ring-opening polymerization. The structure, self-assembly behavior, and oxidation sensitivity of these diblock copolymers were well characterized by various methods. It was found that the nanoparticles prepared by this oxidation-sensitive copolymer can be dissociated by H2O2 or the photosensitizer plus light by the hydrophobic to hydrophilic transition for the PMSPEP core. As such, the photo-accelerated PTX release effect could be found by these nanoparticles co-encapsulating Ce6 and PTX, resulting in a combined anticancer effect in vitro. In the future, such oxidation-sensitive amphiphilic copolymers could be explored as a co-delivery system of a photosensitizer and a chemotherapeutic drug for remote on-demand drug delivery and synergistic chemo-photodynamic therapy.

ASSOCIATED CONTENT Supporting Information. Mass spectrometry of MSPEP monomer, colloidal stability of the mPEG45-b-PMSPEP21-based nanoparticles, and composition and molecular weight of block copolymer mPEG-b-PMSPEP. This material is available free of charge via the Internet at http://pubs.acs.org.



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AUTHOR INFORMATION Corresponding Author *Email: [email protected]

Author Contributions Jihong Wang and Dongdong Li contributed equally to this work. Notes The authors declare no competing ﬁnancial interest Acknowledgements This work was supported by the National Natural Science Foundation of China (51822302, 51773067), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2017B030306002), outstanding Scholar Program of Guangzhou

Regenerative

Medicine

and

Health

Guangdong

Laboratory

(2018GZR110102001), and the Fundamental Research Funds for the Central Universities.

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Synthesis of an Oxidation-Sensitive Polyphosphoester Bearing

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