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PEGylated poly(#-lipoic acid) loaded with doxorubicin as a pH and reduction dual responsive nanomedicine for breast cancer therapy Huailin Yang, Wei Shen, Wanguo Liu, Li Chen, Peng Zhang, Chunsheng Xiao, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01394 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 10, 2018

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PEGylated poly(α-lipoic acid) loaded with doxorubicin as a pH and reduction dual responsive nanomedicine for breast cancer therapy Huailin Yanga,b, Wei Shenb,c, Wanguo Liud, Li Chena,*, Peng Zhangb,*, Chunsheng Xiaob,*, Xuesi Chenb a

Department of Chemistry, Northeast Normal University, Changchun 130024, P. R. China Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China c University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, P. R. China d Department of Orthopaedic Surgery, China-Japan Union Hospital, Jilin University, Changchun 130033, P. R. China.

b

*

Corresponding authors E-mail addresses: [email protected] (L. Chen); [email protected] (P. Zhang); [email protected] (C. Xiao)

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ABSTRACT Disulfide-containing nanoparticles are promising vehicles for anticancer drug delivery. However, the preparation of disulfide-containing nanoparticles is usually to rely on complex synthetic procedures. In the present work, a PEGylated poly(α-lipoic acid) (mPEG-PαLA) copolymer was facilely synthesized and used for pH and reduction dual responsive drug delivery. The poly(α-lipoic acid) was prepared by thermal polymerization of α-lipoic acid without any catalyst or solvent, and then conjugated with methoxy poly(ethylene glycol) to form mPEG-PαLA copolymer. The obtained mPEG-PαLA copolymer was amphiphilic, which could self-assemble into nanoparticles (NPs) in aqueous solution. More interestingly, the mPEG-PαLA NPs showed high drug loading efficiency (87.7%) for cationic drug, doxorubicin (DOX). The DOX-loaded NPs (NPs-DOX) exhibited pH and reduction dual responsive drug release behaviors. Moreover, the flow cytometry analysis (FCA) and confocal laser scanning microscopy (CLSM) confirmed that the drug-loaded nanoparticles could be efficiently internalized and subsequently release DOX in 4T1 cancer cells. As a result, the NPs-DOX displayed favorable anti-proliferation efficacy in 4T1 cancer cells (measured by MTT assays). Furthermore, the NPs-DOX showed enhanced antitumor efficacy in a 4T1 tumor-bearing mice model, while with reduced side toxicities towards normal organs due to the prolonged circulation time and improved biodistribution in vivo. In a word, this work demonstrates that the PEGylated poly(α-lipoic acid) copolymer can be used as a biocompatible and stimuli-responsive nanocarrier for anticancer drug delivery, which may have potential clinical utility. Keywords: poly(α-lipoic acid), drug delivery, pH and reduction sensitive, nanomedicines, breast cancer


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1. INTRODUCTION In recent years, nano-drug delivery systems (nano-DDSs) have been widely used to overcome the poor selectivity and severe side effects of chemotherapy1-4. Based on the enhanced permeability and retention (EPR) effect, nanoparticles can effectively accumulate at tumor site due to their unique nano-size structure5-9. In order to increase the release of drugs in tumor site, nanoparticles are usually endowed with the ability to selectively release drugs in response to physiological stimuli, such as acid, hypoxia, redox, and enzymes10-14. Among these materials, reduction-sensitive nanoparticles containing disulfide bonds arouse great interest15-17. It is because that the low extracellular concentration (2–20 µM) of reducing glutathione (GSH) provides a relatively stable environment for disulfide bonds, while the higher GSH concentrations (2–10 mM) in cancer cells can induce cleavage of the disulfide bonds in nanoparticles, causing degradation of nanoparticle and release of loaded drugs17-20. Therefore, disulfide-containing nano-DDSs present great potential for highly efficient delivery of anticancer drugs15-18. However, the preparation of disulfide-containing nano-DDSs usually associates with complex synthetic procedures, such as development of new monomers for polymerization, post-polymerization modifications and cross-linking procedures, which may limit their potential clinical applications17-18, 20-21. Alpha-lipoic acid (αLA), a natural antioxidant synthesized in human body, has advantages of regulating blood sugar, suppressing appetite and resisting obesity22-25, and is commonly used for the treatment of diabetes, Alzheimer's disease and other diseases26-27. During the design of antitumor drug carriers, αLA is usually used as a cross-linker to crosslink the core of nanoparticles by disulfide bonds, which makes the nanoparticles more stable and could also mediate decrosslinking of the nanoparticles under high concentration of GSH to release drug into tumor cells28-31. Recently, a series of poly(α-lipoic acid)s bearing guanidinium side chain have been prepared by substrate-initiated polymerization and used as cell-penetrating poly(disulfide)s32-34. Actually, αLA can be polymerized into poly(α-lipoic acid) (PαLA) without any catalyst or solvent by heating to above its melting



point35-36. PαLA was synthesized as early as 10 years ago, yet until today, it is still rarely used to create interesting structures or implement specific functions. Several properties of PαLA make it advantageous for use in anticancer drug carriers: (i) Simple preparation process, a large amount of polymer can be produced by a simple heating procedure35; (ii) Favorable biocompatibility and biodegradability, moreover, the degradation products of PαLA show good biocompatibility and safety23, 37-38; (iii) Excellent reduction responsiveness endowed by a large number of disulfide bonds in the main chain39-40; (iv) The carboxyl groups on the side chains of PαLA can interact with cationic drugs through electrostatic interactions, greatly improving drug loading efficiency of the carriers and unloading the drug in an acidic environment41-44. Therefore, in this study, we synthesized an amphiphilic mPEG-modified PαLA (mPEG-PαLA) copolymer and used it to prepare reduction and pH dual responsive nanoparticles for anticancer drug delivery (Scheme 1). The presence of PEG could reduce the unspecific protein adsorption and diminish the recognition by immune system, then prolonging the blood circulation time of nanoparticles45-47. The disulfide bond-containing PαLA backbone is supposed to be degraded by intracellular reducing GSH and mediate reduction-responsive drug release. Meanwhile, carboxyl groups of PαLA were used to load cationic DOX via electrostatic and hydrophobic interactions and release the drugs in acidic intracellular environments. Our results revealed that this novel PαLA-based nano-DDS presented reduction and pH dual responsive drug release manner and could effectively kill tumor cells after cellular internalization. Moreover, in vivo results demonstrated the prolonged blood circulation time, reduced systemic toxicity, and enhanced antitumor effect of the prepared DOX-loaded nanomicelles.


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Scheme 1. Illustration of the synthesis and self-assembly of mPEG-PαLA, and the use of mPEG-PαLA nanoparticles for pH and reduction dual responsive drug delivery in vivo.



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2. MATERIALS AND METHODS 2.1 Materials α-Lipoic

acid

(αLA),

4-dimethylaminopyridine

poly(ethylene (DMAP),

glycol)

monomethyl

dihydrochloride

(DAPI),

ether

(mPEG,

Mn=2000

g

mol-1),

3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl,) and glutathione monoethyl ester (GSH-OEt) were purchased from Sigma-Aldrich LLC. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) was obtained from Beijing Huafeng United Technology Corporation (Beijing China). Dulbecco's modified Eagle's medium (DMEM, Gibco ) and fetal bovine serum (FBS, Gibco) were purchased from ThermoFisher Scientific (Shanghai, China). All the above-mentioned chemicals and reagents were used as received. N,N-Dimethylformamide (DMF), tetrahydrofuran (THF) and dimethylsulfoxide (DMSO) were obtained from

Sinopharm Chemical Reagent Co., Ltd (Shanghai, China) and purified by a solvent purification system (MB SPS-800, MBRAUN, Germany). The deionized water was prepared by the Milli-Q system (Millipore Co.,

Billerica, MA, USA). 2.2 Characterization 1

H NMR and

13

C NMR spectra were recorded on Bruker AV-300 NMR spectrometer in deuterated chloroform

(CDCl3) or deuterated dimethyl sulfoxide (DMSO-d6). FT IR spectra were performed on a Bio-Rad Win-IR Spectrometer. The molecular weights (Mn) and polydispersities (Ɖ) of obtained polymers were determined by gel permeation chromatography (GPC) equipped with a Waters 515 HPLC pump and a Waters 2414 Refractive Index Detector. Dynamic light scattering (DLS) measurement was performed on a WyattQELS instrument with a vertically polarized He-Ne laser (DAWN EOS, Wyatt Technology Co., USA). The scattering angle was fixed at 90°. Transmission electron microscopy (TEM) measurement was performed on a JEOL JEM-1011 transmission electron microscope (Tokyo, Japan) with an accelerating voltage of 100 kV. The fluorescence intensity measurement was performed on a Fluorescence Master System (Photon Technology International, USA). The ultraviolet-visible (UV-Vis) absorption spectrum was measured by UV-2401PC spectrophotometer (Shimadzu, Japan). 2.3 Synthesis of poly(α-lipoic acid) (PαLA)


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α-Lipoic acid (αLA) was able to self-polymerize into poly(α-lipoic acid) (PαLA) without any initiator when the reaction temperature increased above its melting point35. Specifically, α-lipoic acid (2.06 g, 10 mmol) was add to a dry flask under nitrogen flow and stirred at 80 °C for 2 h. The crude product was dissolved in 20 mL of THF and then poured in 200 mL ether to obtain a sticky white solid. The final product was obtained after filtration and vacuum drying (1.0 g, 48.5%). 2.4 Synthesis of mPEG-PαLA The methoxy poly(ethylene glycol)-grafted-poly(α-lipoic acid) (mPEG-PαLA) was synthesized by conjugation of mPEG onto PαLA backbone. Firstly, PαLA (824 mg, 1 mmol) was dissolved in 20 mL of THF, to which a mixture of mPEG (2.0 g, 1 mmol), EDC·HCl (383 mg, 2 mmol) and DMAP (61.08 mg, 0.5 mmol) dissolved in 20 mL of DMSO was added. After stirring at room temperature for 48 h, the reaction mixture was dialyzed against deionized water for 3 days using a dialysis bag (MWCO 3500 Da). The final product mPEG-PαLA was obtained as a white powder after lyophilization (2.2 g, 77.9%). 2.5 Preparation of mPEG-PαLA nanoparticles (NPs) and DOX-loaded mPEG-PαLA nanoparticles (NPs-DOX) NPs were prepared by the following method. mPEG-PαLA (80 mg) was dissolved in 4 mL of DMF, then 20 mL of phosphate buffer saline (PBS, pH=7.4) was added dropwise to the solution under gentle stirring in the dark. After 2 h, the DMF was removed by dialysis (MWCO 3500 Da) against deionized water for 12 h (the water was refreshed every 2 h). The NPs were obtained after lyophilization. NPs-DOX was prepared by a similar method. mPEG-PαLA (80 mg) and DOX·HCl (9.6 mg) were dissolved in 4.0 mL of DMF, then 20 mL of phosphate buffer saline (PBS, pH=7.4) was added dropwise to the solution under gentle stirring in the dark. After 2 h, free DOX and DMF were removed by dialysis (MWCO 3500 Da) against deionized water for 12 h. The DOX-loaded nanoparticles were obtained after lyophilization in the dark. The drug



loading content (DLC) and drug loading efficiency (DLE) of DOX were determined by fluorescence spectrometer and calculated according to the following formulas. DLC (%) = (weight of loaded DOX / weight of NPs-DOX) × 100% DLE (%) = (weight of loaded DOX / weight of feeding DOX) × 100% 2.6 Stability and GSH-responsiveness of NPs The stability and GSH-responsiveness of NPs (6 mL, 0.15 mg mL-1) were monitored by DLS at 37 °C in PBS buffer (100 mM, pH 7.4) and PBS buffer (100 mM, pH 7.4) containing 10 mM GSH, respectively. And the particle sizes of NPs were detected at predetermined times. The GSH treated NPs were also analyzed by TEM to further verify the degradation of NPs. 2.7 In vitro release of DOX Briefly, weighted DOX-loaded nanoparticles (NPs-DOX) was dispersed in 5.0 mL of PBS buffer solution and transferred into a dialysis bag (MWCO 3500 Da). Then, the dialysis bags were put in different release media (150 mL) in the dark. The release media were (i) PBS buffer (100 mM, pH 7.4), (ii) acetate buffer (100 mM, pH 5.5), (iii) PBS buffer (100 mM, pH 7.4) containing 1 mM GSH, and (iv) PBS buffer (100 mM, pH 7.4) containing 10 mM GSH. At a predetermined time, 2.0 mL of release media was withdrawn and 2.0 ml of original fresh solution was added. The concentration of released DOX was determined by a standard curve method on the basis of fluorescence measurements (excitation at 480 nm, emission at 592 nm). 2.8 Cellular uptake The cellular uptake and intracellular drug release behaviors of the NPs-DOX were determined by flow cytometry analysis (FCA) and confocal laser scanning microscopy (CLSM). For FCA, The mice breast cancer cells (4T1) were seeded in a 6-well plate with a density of 3.0 × 105 cells per well in 1.8 mL of DMEM containing 10% FBS at 37 °C under 5% CO2 atmosphere and cultured for 24 h. 200 µL


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of GSH-OEt aqueous solution (final concentration: 10 mM) was added to the GSH-OEt pretreated group, and medium was refreshed after 2 h. Next, NPs-DOX and free DOX (final concentration of DOX: 5.0 µg/mL) dissolved in 200 µL of DMEM were added and incubated for another 3 or 6 h. The medium was removed and 2.0 mL of PBS was added to each well to wash the cells for three times. Then, cells were detached with trypsin and collected after washing for two times with PBS. Finally, the cells were resuspended in 0.5 mL of PBS and the signals were measured by a Guava EasyCyte™ 12 Flow Cytometer (Millipore, Billerica, MA, USA). Cells without drug treatment were set as controls. For CLSM observation, The 4T1 breast cancer cells were seeded in a 6-well glass bottom culture dishes with a density of 1.0 × 105 cells per well in 1.8 mL of DMEM containing 10% FBS at 37 °C under 5% CO2 atmosphere and cultured for 24 h. 200 µL of GSH-OEt aqueous solution (final concentration: 10 mM) was added to the GSH-OEt pretreated group, and the medium was refreshed after 2 h. Next, NPs-DOX and free DOX (final concentration of DOX: 5.0 µg/mL) dissolved in 200 µL of DMEM were added and incubated for another 3 or 6 h. The media were removed and 2.0 mL of PBS was added to each well to wash the cells for three times. Cells were fixed in buffered formaldehyde (1.0 mL, 4% (W/V)) for 20 min and followed by washing for five times with PBS. Subsequently, the DAPI (blue) was used to stain the cell nuclei for 5 min. Finally, the cells were observed with a CLSM (LSM 780, Carl Zeiss). 2.9 Cytotoxicity assays The cytotoxicities of blank NPs were evaluated by MTT assay. The 4T1 cells and L929 cells were seeded in 96-well plates with a density of 8000 cells per well in 180 µL of DMEM containing 10% FBS at 37 °C under 5% CO2 atmosphere and cultured for 24 h. 20 µL of GSH-OEt aqueous solution (final concentration:10 mM) was added to the GSH-OEt pretreated group for 2 h. Next, the medium was replaced by 180 µL of fresh DMEM medium, and blank NPs dissolved in 20 µL of DMEM were added and incubated for another 72 h. Then, 20 µL of



MTT solution (5.0 mg/mL) was added. After incubation for 4 h, the medium was removed and 150 µL of DMSO was added to each well. After shaking for 5 min, the absorbance at 490 nm was measured using a microplate reader (Bio-Rad 680 microplate reader). Cells without NPs treatment were set as controls. The antitumor activity of free DOX and NPs-DOX were also evaluated by MTT assay. The 4T1 breast cancer cells were seeded in a 96-well plate with a density of 8000 cells per well in 180 µL of DMEM containing 10% FBS at 37 °C under 5% CO2 atmosphere and cultured for 24 h. 20 µL of GSH-OEt aqueous solution (final concentration: 10 mM) was added to the GSH-OEt pretreated group for 2 h. Next, the medium was replaced by 180 µL of fresh DMEM medium, NPs-DOX and free DOX dissolved in 20 µL of DMEM at different DOX concentrations were added and incubated for another 24 h or 48 h. Then, 20 µL of MTT solution (5 mg/mL) was added. After incubation for 4 h, the medium was removed and 150 µL of DMSO was added to each well. After shaking for 5 min, the absorbance at 490 nm was measured using a microplate reader (Bio-Rad 680 microplate reader). Cells without drug treatment were set as controls. 2.10 Pharmacokinetics Pharmacokinetics was characterized by measuring DOX levels in venous blood of SD rats. Specifically, SD rats (200～210 g) were randomly divided into 2 groups (3 in each group), and they were injected with free DOX (5.0 mg kg-1) and NPs-DOX (containing 5.0 mg kg-1 DOX) via tail vein, respectively. After that, retro-orbital blood collection was performed at predetermined times (0.25, 0.5, 1, 3, 6, 12, 24 h), then the blood was heparinized and centrifuged to obtain plasma. Next, 150 µL of plasma sample was deproteinized with 830 µL of methanol and 20 µL of daunorubicin hydrochloride (10 µg/mL, internal standard), vortexed for 15 min, and centrifuged at 8000 rpm for 10 minutes. Then 500 µL of the supernatant was collected and dried under a stream of nitrogen at 35 °C. At last, the dried sample was dissolved in the mobile phase and filtered by a 0.22 µm aperture organic phase filter for the following HPLC analysis of DOX level in supernatant. The data was analyzed by statistical moments using the


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software of Drug and Statistics (DAS, version 2.0), the elimination half-life (t1/2z) and the area under the curve (AUC) were calculated by the following formulas: t1/2z=0.693/Zeta (Zeta is the slope of the c-t curve tail)

AUC (ng ∗ h/mL) =

2.11 Evaluation of maximum tolerated dose (MTD) Female Kunming mice were randomly divided into 12 groups (n=5 per group), which were intravenously injected with DOX (5, 10, 15, or 20 mg kg-1), NPs (250, 500, 750, or 1000 mg kg-1), or NPs-DOX (5, 10, 15, or 20 mg kg-1 of equivalent DOX) for one time, then the mice were fed for 2 weeks. The body weights and survival rates of the mice were recorded daily. The MTD was defined as the concentration of drugs which caused less than 15% of mouse weight loss and no death of mice due to toxicity 48-49. 2.12 Ex vivo fluorescence imaging of DOX The in vivo fluorescence imaging analysis of DOX was as follows. Firstly, free DOX (5.0 mg kg-1) and NPs-DOX (containing 5.0 mg kg-1 DOX) were injected into 4T1 tumor bearing mice (tumor volume is about 100 mm3) via tail vein. After 3 or 10 h, the mice were sacrificed, the tumor and major organs (heart, liver, spleen, lung, and kidney) were excised, which were washed with PBS for three times and then used for ex vivo imaging using a Maestro Imaging System (Cambridge Research & Instrumentation, Inc.). 2.13 In vivo antitumor efficiency Female Balb/C nude mice (8 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and all procedures were approved by the Animal Care and Use Committee of Jilin University. The syngeneic mouse mammary tumor model was prepared by injection of 4T1 cells (2.0 × 106) suspended in 0.1 mL of PBS into the mammary gland of mice. When the tumors grew up to around 120 mm3, the



mice were randomly divided into 5 groups (5 mice for each group), which were administrated with PBS, free DOX (4 mg kg-1), free DOX (4 mg kg-1) plus free αLA (4.44 mg kg-1), blank NPs (15.2 mg kg-1, the amount of blank NPs was equal to the amount of NPs in NPs-DOX formulation) and NPs-DOX (containing 4.0 mg kg-1 DOX) via tail vein injection on days 0, 4, 8, and 12. The body weights and tumor sizes were measured every two days, and the tumor volume (V; mm3) was calculated as V = a × b2 / 2, where the a and b (mm) were the longest and shortest diameter of tumors, respectively. The tumor inhibition rate (%) = ( Vcontrol -Vsample ) / Vcontrol × 100%, where the Vcontrol and Vsample represented the tumor volumes in control and sample groups on day 18, respectively. 2.14 Histological analyses One week after the last injection, the mice were sacrificed. The tumor and major organs (heart, liver, spleen, lung, and kidney) were excised and fixed in buffered formaldehyde (4% (W / V) ) for 24 h, then they were embedded in paraffin and sliced at 5 µm for haematoxylin and eosin (H&E) staining and in situ cell apoptosis analyses (TUNEL). 2.15 Statistical analysis All experiments were performed for at least three times and the data were shown as mean ± standard deviation (SD). All results were analyzed by one-way ANOVA. *p < 0.05 was considered as statistically significant. **p < 0.01 and ***p < 0.001 were considered as highly significant.

3．．RESULTS AND DISCUSSION 3.1 Synthesis and characterization of mPEG-PαLA The synthesis of mPEG-PαLA was illustrated in Scheme 1. Firstly, PαLA was synthesized by ring-opening polymerization (ROP) of αLA monomer at 80 °C for 2 h. The structure of obtained PαLA was firstly characterized by 1H NMR spectrum. As shown in Figure S1, the proton peaks a and c adjacent to the sulfur atom of the monomer shift right to a1 and c1 after the ring-opening polymerization. Furthermore, the UV-Vis spectrum of PαLA suggested


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that there was no dithiolane characteristic peak at 330 nm (Figure S2), which proved that all monomer had already been ring-opening polymerized into PαLA31. Additionally, the structure of obtained PαLA was also confirmed by 13

C NMR (Figure S3) and FT IR characterization (Figure S4). Finally, the molecular weight (Mn) of PαLA was

determined to be 20800 g mol-1 (Ð = 1.66) by GPC analysis (Figure 1B). In summary, all the results showed the successful synthesis of PαLA. Afterwards, mPEG was conjugated to the side chains of PαLA via esterification reaction to obtain the final polymer, mPEG-PαLA. The structure of mPEG-PαLA was characterized by 1H NMR spectrum, 13C NMR spectrum, FT IR spectrum and GPC analysis. As shown in Figure 1A, the appearance of proton peaks h, i and j indicated that mPEG was successfully conjugated to the PαLA backbone. In addition, the integral ratio of the peak h to the peak g was close to 1:4, which indicated that 25% of the carboxyl groups had been modified with mPEG. As shown in Figure S3C, the i2 peak in 13C NMR of mPEG-PαLA indicated the formation of ester bond, which means that PEG was successfully attached to the side chain of PαLA. At the same time, the presence of the h2 peak indicated that there was still a part of free carboxyl groups, which is consistent with the results of 1H NMR spectrum. Additionally, in the FT IR spectrum, the two characteristic peaks at 1734 cm-1 (vibration of carbonyl stretching (C=O) in ester bonds) and 1701 cm-1 (vibration of carbonyl stretching (C=O) in carboxyl groups) can also be found (Figure S4C). In Figure 1B, the molecular weight (Mn) of mPEG-PαLA was determined to be 69100 g mol-1 (Ð = 1.23), which is significantly higher than that of mPEG (Mn = 3800 g mol-1) and PαLA (Mn = 20800 g mol-1). Taken together, these results indicated the successful synthesis of mPEG-PαLA. 3.2 Preparation of NPs and NPs-DOX The obtained mPEG-PαLA can self-assemble into nanoparticles in aqueous media due to the coexistence of hydrophilic mPEG chains and hydrophobic PαLA backbone. The critical micelle concentration (CMC) of the nanoparticles (NPs) was determined to be 0.00542 mg mL-1 by using benzoylacetone (BZA) as the probe (Figure



S5)50. The hydrodynamic radius (Rh) of the blank NPs measured by DLS was 39 ± 6 nm and the NPs presented irregular shapes as indicated by the TEM image (Figure 1C). Additionally, we have demonstrated that anionic polymers, such as poly(L-glutamic acid), can encapsulate positively charged DOX with high drug-loading efficiency41, 44. Therefore, the encapsulation of DOX by mPEG-PαLA micelles was then tested. As expected, the DLC and DLE of DOX were 20.8 wt% and 87.7%, respectively. The high drug loading efficiency should be ascribed to the strong electrostatic interactions between the negatively charged carboxyl groups on the polymer side chains and the positively charged amine group in DOX. Moreover, the DOX-loaded mPEG-PαLA nanoparticles (NPs-DOX) were spherical with a Rh of 48 ± 8 nm (Figure 1D). The slightly increased size of NPs-DOX also indicated the successful encapsulation of DOX in the nanoparticles.

1

Figure 1. (A) Typical H NMR spectrum of mPEG-PαLA in CDCl3. (B) GPC analyses of mPEG, PαLA and

mPEG-PαLA using DMF as the eluent. Sizes and morphologies of (C) NPs and (D) NPs-DOX measured by DLS


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technology and TEM images, respectively. 3.3 Stability and GSH-responsiveness of NPs In order to evaluate the stability of NPs under physiological conditions and the responsive property of NPs under simulated tumor intracellular environment, we tested the particle size changes of NPs under different conditions by DLS and TEM. Within 24 h, the particle size distribution of NPs in the control group did not show obvious change (Figure S6A). However, in the presence of 10 mM GSH, the radius of NPs changed obviously in 24 h, probably due to the rapid cleavage of disulfide bonds in the mPEG-PαLA backbone by reduced GSH (Figure S6A). TEM images also proved this assumption and there were no integrated nanoparticles observed in the solution (Figure S6B). Therefore, it is clear that NPs prepared by mPEG-PαLA have excellent colloidal stability under physiological conditions and could be degraded rapidly under reducing conditions in tumor cells. 3.4 In vitro Release of DOX To further investigate the stimuli-responsive drug release behavior of NPs-DOX, the in vitro drug release of NPs-DOX was performed at different pHs, with or without the presence of GSH. The results are shown in Figure 2. At pH 7.4, the cumulative release of DOX was significantly increased with the increased concentration of GSH. This should be ascribed to the GSH-mediated disintegration of NPs-DOX as discussed above. Meanwhile, the release of DOX also exhibited an acid-responsive release pattern, which should be due to the weakening of electrostatic interactions between carboxyl groups in polymer and the amine group in DOX, and increased hydrophilicity of DOX at acidic pH44. Moreover, it should be noted that the cumulative release of DOX in group pH 7.4 with 1 mM GSH was higher than that in group pH 5.5, indicating that the NPs-DOX was more sensitive to reduced GSH than to acidic pH. Altogether, these results indicated that NPs-DOX could be an ideal drug delivery system, which could prevent the undesired release of drug in the physiological environment, but accelerate the release of DOX in tumor cells with high GSH concentration (2–10 mM GSH) and acidic environment (pH 5.5).



Figure 2. The DOX release behavior of NPs-DOX in PBS: at pH 7.4, at pH 5.5, at pH 7.4 with 1 mM of GSH and at pH 7.4 with 10 mM of GSH. Data were presented as mean ± SD (n = 3). 3.5 Intracellular Release of DOX The cellular uptake and intracellular drug release behaviors of NPs-DOX were then evaluated in 4T1 cells by FCA and CLSM. As shown in Figure 3A, after 3 hours of co-cultivation, the free DOX treated group showed stronger fluorescence intensity (135.7 ± 5.3) (Figure 3B) compared to the NPs-DOX treated group, which was attributed to that the free DOX could rapidly diffuse into cells while the NPs-DOX entered cells via endocytic pathways. In addition, the DOX fluorescence signal was stronger in the GSH-OEt pretreated group (94.8 ± 4.5) compared to the non-treated group (65.5 ± 6.6) (Figure 3B). This result proved that intracellular DOX release from NPs-DOX was accelerated by GSH. After incubation with drug for 6 h, the fluorescence intensity of DOX in each group significantly increased, indicating the accumulation of NPs-DOX and the sustained release of DOX in the cells. The cellular uptake and intracellular drug release behaviors of NPs-DOX were further investigated by CLSM, and the results are shown in Figure 4. The nucleus was stained blue by DAPI and the fluorescence of DOX was red. The overlap of blue and red fluorescence turns pink, suggesting that the released DOX entered into the cell nuclei. The mean fluorescence intensities of DOX for all the tested groups were quantified and shown in Figure S7. It is clear that the GSH-OEt pretreated group displayed higher fluorescence intensity than the group without GSH-OEt pretreatment, which is consistent with the FCA results. In summary, the results of FCA and CLSM clearly indicated


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that NPs-DOX can be efficiently internalized into cancer cells and release DOX in the cancer cells.

Figure 3. Flow cytometry analyses for 4T1 cells incubated with free DOX or NPs-DOX for 3 h (A) or 6 h (C) with or without 10 mM GSH-OEt pretreatment. The mean fluorescence intensities of DOX in the tested groups at 3 h (B) or 6 h (D) (***p < 0.001). Data were presented as mean ± SD (n = 3).



Figure 4. Confocal laser scanning microscopy images of 4T1 cells after incubation with free DOX or NPs-DOX for 3 h (A) or 6 h (B) with or without 10 mM GSH-OEt pretreatment. Scale bar: 20 µm.


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3.6 Studies of in vitro cytotoxicity The biocompatibility of mPEG-PαLA NPs was evaluated by MTT assay in 4T1 cells and L929 cells. As shown in Figure 5A, after incubation with the different concentrations of mPEG-PαLA NPs for 72 h, the cell viability of the treated cells was as high as 93% even the concentration of NPs was at a high level of 1 mg/mL. This strongly proved that NPs were non-toxic to both cancer cells and non-cancerous cells; and the NPs had good biocompatibility for in vivo application. To verify the anticancer ability of NPs-DOX, different concentrations of NPs-DOX and 4T1 cells were co-cultured for 24 or 48 h, then the cell viability was measured by MTT assay. The results showed that the cytotoxicity of free DOX was higher than that of NPs-DOX after a 24 or 48 h incubation (Figure S8, Figure 5B), which may be because that free DOX entered the cells by free diffusion, while NPs-DOX entered the cells by endocytosis and released DOX slowly in the cells. Furthermore, as shown in Figure 5B and Figure S8, GSH-OEt pretreatment significantly facilitated the cytotoxicity of NPs-DOX. It is because that the increased intracellular GSH concentration accelerated the degradation of the internalized NPs-DOX and induced enhanced intracellular release of DOX.



Figure 5. In vitro cytotoxicity assays. (A) Cell viability of 4T1 cells and L929 cells after treatment with mPEG-PαLA NPs for 72 h. (B) Cell viability of 4T1 cells incubated with free DOX or NPs-DOX, pretreated with or

without 10 mM GSH-OEt for 48 h (**p < 0.01, ***p< 0.001). Data were presented as mean ± SD (n = 3). 3.7 Pharmacokinetic analysis The enhanced stability and nano-scaled size of nanoparticles can reduce the clearance of drugs from the blood and prolong the circulation time of drugs in the body. Herein, we investigated the pharmacokinetics of the NPs-DOX by injecting free DOX or NPs-DOX into the tail veins of SD rats to detect the plasma DOX concentrations at different time points. As shown in Figure 6, the DOX concentration in the plasma of the NPs-DOX treated group was significantly higher than that of the free DOX treated group. Specifically, NPs-DOX has a longer elimination half-life (t1/2z=1.42 h) than DOX (0.42 h) and the AUC of NPs-DOX was 19.3-fold higher than that of DOX in vivo. This suggests that NPs-DOX has a longer systemic circulation time, which may allow more accumulation of DOX in the tumor site and induce an outstanding anti-tumor efficacy of the DOX-loaded nanoparticles.


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Figure 6. In vivo pharmacokinetic analysis of free DOX and NPs-DOX in SD rats. Data were presented as mean ± SD (n = 3). 3.8 MTD Analysis To investigate the safety and biocompatibility of the nanoparticles, and to choose the ideal dose for in vivo implementation, MTDs of free DOX, NPs and NPs-DOX were evaluated by single intravenous injection of different concentrations of formulations into healthy Kunming mice. The survival rates and body weights of the mice were monitored for 14 days after intravenous administration of various formulations. The MTD was defined as the concentration of drugs which caused less than 15% of mouse weight loss and no death of mice due to toxicity. As shown in Figure 7A and 7B, the body weight of mice decreased by at most 8% when mice were injected with 5 mg kg-1 of free DOX, then the body weight gradually increased. However, when the free DOX dose was increased to 10 mg kg-1, the treated mice lost more than 15% of their body weights at day 8 and three mice died in 14 days, which is consistent with the literature that the MTD of DOX was determined to be between 5 and 10 mg kg-1 49,51. In contrast, the mice lost only 2% of their body weights and no mice died when the mice were injected with NPs-DOX at 10 mg kg-1 DOX equivalent. But the mice lost 19% of their body weight and one mouse died when treated with NPs-DOX at 15 mg kg-1 DOX equivalents (Figure 7C and 7D). Therefore, the MTD of NPs-DOX is between 10 and 15mg kg-1 DOX equivalent, which is about twice that of DOX. This may be because that the nanoparticles could protect DOX from leaking into the blood, prolong the systemic circulation time and promote



the accumulation of DOX in tumor sites by EPR effect, consequently reducing the systemic toxicities. From the results of Figure 7E and 7F, it can be seen that the body weights of the mice steadily increased and there was no death of mice at all tested concentrations, demonstrating good biocompatibility of NPs even at high dose of 1000 mg kg-1.

Figure 7. MTD studies of free DOX (A, B), NPs-DOX (C, D) and NPs (E, F) on body weight changes and survival rates in Kunming mice. Data were presented as mean ± SD (n = 5). 3.9 Ex vivo DOX fluorescence imaging In order to assess the in vivo distribution of NPs-DOX, tumors and major organs of 4T1 tumor-bearing mice were harvested at 3 h and 10 h after drug injection for ex vivo DOX fluorescence imaging. As shown in Figure 8A, free DOX was mainly concentrated in the liver and kidneys, and the rapid weakening of the fluorescence signal proved that free DOX in vivo was rapidly cleared through metabolism of the liver and kidneys, which may reduce the treatment effect and result in DOX-mediated damages to liver and kidneys. In contrast, the NPs-DOX treated group showed a stronger fluorescence signal compared with free DOX treated group, and the strongest fluorescence signal was detected at 10 h, which clearly demonstrated that NPs-DOX prolonged the circulation time of DOX in vivo. Furthermore, the fluorescence intensity of the tumor in the NPs-DOX treated group was significantly higher


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than that in the DOX treated group (Figure 8B). This reflects the ability of NPs-DOX to accumulate in tumor tissues due to the EPR effect and the prolonged systemic circulation time, which will enhance the anticancer effect of the nanoparticles.

Figure 8. (A) Biodistribution studies of free DOX and NPs-DOX in 4T1 tumor-bearing mice. (B) Average signals were counted from the major organs (heart, liver, spleen, lung and kidney) and tumors in 4T1 tumor-bearing mice after treatment with free DOX and NPs-DOX (***p < 0.001). Data were presented as mean ± SD (n = 3). 3.10 The evaluation of in vivo anticancer efficacy 4T1 tumor-bearing mice were intravenously injected with PBS, blank NPs, free DOX, αLA plus free DOX and NPs-DOX every 4 d to assess their antitumor effect in vivo. As shown in Figure 9A and 9B, tumor growth was effectively reduced in all groups treated with DOX-containing formulations compared to the control group treated with PBS, whereas the blank mPEG-PαLA micelles (see NPs group in Figure 9A) had no significant inhibitory effect on tumor growth. Moreover, NPs-DOX treated group demonstrated remarkably higher inhibition rate in tumor growth compared with free DOX treated group (Figure 9C). The higher tumor growth inhibition rate of the NPs-DOX treated group than that of the αLA plus free DOX treated group indicated that the NPs-DOX treated group mainly relied on the advantageous properties of the nanoparticles to enhance tumor inhibition effect rather than αLA (Figure 9A and 9C). The enhanced tumor inhibition efficacy of NPs-DOX may be explained by the



prolonged DOX circulation time in vivo and the EPR effect that could increase the accumulation of nanoparticles in tumor site. The intracellular reduction and pH responsive release of DOX may also contribute to the enhanced anticancer effect of the nanoparticles. The body weight changes of the mice were monitored to evaluate the systemic toxicity of the tested formulations. As shown in Figure 9D, the similar increase in body weights of mice in the PBS and NPs treated groups indicated that the empty micelles are non-toxic. There was less weight loss in the NPs-DOX treated group than in the free DOX and αLA plus free DOX treated groups, which may be due to the fact that NPs-DOX could protect the DOX from leakage into the blood to reduce toxicity. Moreover, lower extracellular concentration of GSH could also decrease DOX release from the nanoparticles before being internalized by cells, thereby reducing the side toxicity.


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Figure 9. In vivo antitumor efficacy and the body weight changes in mice. (A) Tumor volume changes in 4T1

tumor-bearing mice after treatments (***p < 0.001). (B) Images of the tumors at day 18. (C) Tumor inhibition rates of the DOX, NPs, αLA plus free DOX and NPs-DOX treated groups (***p < 0.001). (D) Changes of the body weights during treatment. (E) H&E staining (Scale bar: 100 µm) and TUNEL analyses (Scale bar: 50 µm) of tumors from the mice at the end of treatment. Data were presented as mean ± SD (n = 5). 3.11 Histological analyses



The antitumor effects and systemic toxicity of the DOX-loaded nanoparticles were further analyzed by H&E staining and TUNEL analyses. As shown in Figure 9E, no significant deformation or chromatin changes of the tumor cells were observed in the PBS or NPs treated group. However, different degrees of tumor tissue necrosis were observed in the DOX-containing formulations treated groups. Furthermore, NPs-DOX treated group presents fewer blue intact cell nuclei compared with other control groups, thus possesses more necrotic tumor cells (larger necrosis area), indicating that NPs-DOX is more effective in tumor growth suppression. In addition, the TUNEL results in Figure 9E also indicated that NPs-DOX was more effective to induce apoptosis of tumor cells than other tested formulations. In a word, both of H&E and TUNEL results further confirm that the mPEG-PαLA NPs should be a good candidate for cancer drug delivery in vivo. For systemic toxicity, the mice in the free DOX treated group exhibited significant myocardial atrophy and cardiomyocyte necrosis and apoptosis. In contrast, no significant necrosis and apoptosis was observed in the PBS or NPs-DOX treated groups, which demonstrated that NPs-DOX could effectively reduce systemic toxicity (Figure 10 and Figure S9). These results are consistent with the body weight evaluation results in Figure 9D.


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Figure 10. Histological (H&E staining) analyses of major organs from the mice at the end of treatment. Scale bar:

100 µm.

4. CONCLUSIONS In this work, a mPEG-grafted-poly(α-lipoic acid) (mPEG-PαLA) copolymer was facilely synthesized by chemical conjugation of mPEG onto the side chains of PαLA. The obtained mPEG-PαLA copolymer could self-assemble into nanoparticles, which displayed reduction responsive disintegration property due to the presence of disulfide linkages in the PαLA backbone. Moreover, the mPEG-PαLA NPs could encapsulate DOX with high loading efficiency through electrostatic interaction between the negatively charged carboxyl groups in the copolymer and the positively charged amine group in DOX. The formed DOX-loaded nanoparticles exhibited pH and reduction dual responsive drug release behavior. Furthermore, the DOX-loaded mPEG-PαLA NPs displayed effective antitumor activities both in vitro and in vivo. Meanwhile, the DOX-loaded mPEG-PαLA NPs exhibited reduced



side toxicities of DOX due to the prolonged circulation time and enhanced accumulation in tumor site. In summary, the present work highlights the promising use of mPEG-PαLA as biocompatible and stimuli-responsive drug delivery vehicles.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 1

H NMR, 13C NMR, UV-Vis and FT IR spectra of αLA and PαLA; 13C NMR and FT IR spectra of mPEG-PαLA;

The critical micelle concentration (CMC) of mPEG-PαLA NPs; Stability and GSH-responsiveness of mPEG-PαLA NPs; The mean fluorescence intensities of DOX for all the tested groups in CLSM images; The viability of 4T1 cells incubated with free DOX or NPs-DOX pretreated with or without 10 mM GSH-OEt for 24 h; TUNEL analyses of heart from the mice after treatment.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] ORCID Chunsheng Xiao: 0000-0001-7936-4146 Xuesi Chen: 0000-0003-3542-9256 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by the National Natural Science Foundation of China (51573184, 21474012 and 51273037), Jilin Province Science and Technology Development Plan (20180520207JH) and the Youth Innovation Promotion Association of CAS (2017266).


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