Redox Responsive Polymeric Prodrug and

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One-Pot Synthesis of pH/Redox Responsive Polymeric Prodrug and Fabrication of Shell Cross-Linked Prodrug Micelles for Antitumor Drug Transportation Lei Li, Dian Li, Mingzu Zhang, Jinlin He, Jian Liu, and Peihong Ni Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00421 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Bioconjugate Chemistry

One-Pot Synthesis of pH/Redox Responsive Polymeric Prodrug and Fabrication of Shell Cross-Linked Prodrug Micelles for Antitumor Drug Transportation Lei Li1, Dian Li1, Mingzu Zhang1, Jinlin He1, Jian Liu2, Peihong Ni1,* 1

College of Chemistry, Chemical Engineering and Materials Science, State and Local Joint Engineering Laboratory for Novel Functional

Polymeric Materials, Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Suzhou Key Laboratory of Macromolecular Design and Precision Synthesis, Soochow University, Suzhou 215123, P. R. China 2

Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou, 215123, P. R. China

Supporting Information ■ ABSTRACT: Shell cross-linked (SCL) polymeric prodrug micelles have the advantages of good blood circulation stability and high drug content. Herein, we report on a new kind of pH/redox responsive dynamic covalent SCL micelles, which was fabricated by selfassembly of a multifunctional polymeric prodrug. At first, a macroinitiator PBYP-ss-iBuBr was prepared via ring-opening polymerization (ROP), wherein PBYP represents poly[2-(but-3-yn-1-yloxy)-2-oxo-1,3,2dioxaphospholane]. Subsequently, PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA) prodrug was synthesized by one-pot method with a combination of atom transfer radical polymerization (ATRP) and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction using a doxorubicin (DOX) derivative containing azide group to react with the alkynyl group of the side chain in the PBYP block, while DMAEMA and FBEMA are the abbriviations of N,N-(2-dimethylamino)ethyl methacrylate and 2-(4-formylbenzoyloxy)ethyl methacrylate, respectively. The chemical structures of the polymer precurcors and the prodrugs have been fully characterized. The SCL prodrug micelles were obtained by self-assembly of the prodrug and adding cross-linker dithiol bis(propanoic dihydrazide) (DTP). Compared with the shell uncross-linked prodrug micelles, the SCL prodrug micelles can enhance the stability and prevent the drug from leaking in the body during blood circulation. The average size and morphology of the SCL prodrug micelles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The SCL micelles can be dissociated under moderately acidic or/and reductive microenvironment, that is, endosomal/lysosomal pH medium or high GSH level in the tumorous cytosol. The results of DOX release also confirmed that the SCL prodrug micelles possessed pH/reduction responsive properties. Cytotoxicity and cellular uptake analyses further revealed that the SCL prodrug micelles could be rapidly internalized into tumor cells through endocytosis and efficiently release DOX into the HeLa and HepG2 cells, which could efficient inhibit the cell proliferation. This study provides a fast and precise synthesis method for preparing multifunctional polymer prodrugs, which hold great potential for optimal antitumor therapy. enhanced permeation and retention (EPR) effect.17,18 For enhancing the efficiency of drug delivery and achieving rapid release at tumor site, a series of smart prodrugs containing cleavable bonds have been designed, which can be rapidly triggered and subsequent make prodrug micelles disassembly in the tumor environment by internal stimuli-responsive conditions such as pH and redox.19-21 Many stimuli-responsive prodrug micelles can release drug in response to intrinsic biological stimuli-responsive conditions, such as lysosomal pH and cytoplasmic glutathione (GSH).22,23 This is due to the pH difference between healthy tissues (pH∼7.4) and intracellular environment of tumors, especially in the endosomal and lysosomal with pH about 4.5-6.5.24-26 The cytosolic concentration of GSH is higher three orders of magnitude than the extracellular concentration, leading to the cleavage of acylhydrazone and disulfide bond and allowing to construct drug delivery, which contain the disulfide linkage of main chain of reduction-responsiveness.27,28 Polymeric prodrug micelles have been widely researched, which can be quantitatively control the drug loading content and reduce premature drug release compared with the approach of physical encapsulation of drugs.29,30 It is worth mentioning that the polymers conjugated with different anticancer drugs should have unique physical and chemical prop

■ INTRODUCTION In the past few decades, cancer therapy has become extremely important in the world. The current treatment modalities for tumors mainly include surgery, chemotherapy, radiotherapy, and immunotherapy, among which chemotherapy is still the most widely used method to treat various tumors.1,2 However, small molecule chemotherapeutic drugs have poor water solubility, unsatisfactory biodistribution and pharmacokinetics, and severe side effects, which greatly hinder their applications in the treatment of tumors.3 To overcome these limitations, nanotechnology-based drug delivery systems such as micelles,4-6 liposomes,7,8 nanogels,9-11 and vesicles12,13 were used to enhance water solubility, improve drug loading capacity, sustain controlled release and prolong circulation in the blood.14 Recently, polymeric prodrug micelles have attracted more attention due to their unique property and structure, such as their nanoscale size and relatively high stability.15,16 Prodrug micelles may not only help to decrease systemic toxicity of chemotherapy and the premature drug release, but also improve drug uptake and drug pharmacokinetics in the tumor site via the

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Scheme 1. Synthesis routes and self-assembly of PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA) polymeric prodrug.

erties.3,31 Hence, some acid-labile linkages such as hydrazone,32 acetal,33 and imine,34 have been used to conjugate drugs onto polymeric side chains. The cleavage of these linkages enables the drug release in tumor sites. Until now, a number of reductionresponsive prodrug micelles or nanoparticles with disulfide bonds have been designed for intelligent drug delivery, which can be cleaved in response to high GSH concentration at tumor site.35,36 Prodrug micelles will face a series of barriers prior to reaching the tumor site in vivo. For example, during circulation, the extensive dilution and high salt concentration environment would cause the dissociation and/or aggregation of the prodrug micelles. One of the effective ways to solve this problem is to build crosslinked micelles, including shell cross-linking (SCL)37,38 and core cross-linking (CCL).39,40 If these cross-linking bonds possess the stimuli-responsiveness, they will be stable in extracellular media and destroyed in the tumor cell environment. Our group reported folate-conjugated and core cross-linked (ACCL-FA) micelles with acid-cleavable acetal groups, which was prepared by the CuAAC “click” reaction between the two azide groups on the clickable tetraethylene glycol (N3-a-TEG-a-N3) and the alkynyl groups on the polyphosphoester PBYP-b-PEEP-FA.41 In another study, folic acid (FA)-conjugated copolymer P(EAEP-AP)-LA-FA was obtained by Michael addition polymerization and esterification. It could self-assembly into core-shell structure to encapsulate DOX in water and reversible core cross-linked by lipoyl groups.39 Compared with the core cross-linked nanoparticles, the shell cross-linked (SCL) nanoparticles have benefit for the stability of corona of shell to reduce premature drug release and maintain high structural stability.38,42 Liu and co-workers have prepared SCL micelles with hydrophobic cores conjugated with photocaged chemotherapy and coronas functionalized with ligands at pH 6.2 upon addition of difunctional cross-linker, which can efficiently prevent drug premature release during circulation and maintain the stability of prodrug micelles.37

Polyphosphoesters (PPEs)-based materials have great potential applications in gene delivery, in vivo imaging, drug delivery, and tissue engineering.43-45 Due to their adjustable properties, facile functionalization, as well as favorable biodegradability and biocompatibility, PPEs have many repeating phosphoester linkages in the main chains of polymers, allowing the introduction of various functional groups.46-48 In recent years, in the field of polymer synthesis, ROP, ATRP, and CuAAC “click” chemistry have been widely used. However, for the pH/redox responsive polymeric prodrugs, the prepared process of one-pot method through a combination of ATRP and CuAAC “click” chemistry is rarely reported. In addition, compared with the previous methods, one-pot method has obvious advantages in convenient synthesis and easy control of polymer prodrug composition.49-51 Hence, in this work, we designed and synthesized a welldefined pH/redox responsive shell cross-linked PBYP-hyd-DOXss-P(DMAEMA-co-FBEMA) polymeric prodrug by one-pot method with a combination of ATRP and CuAAC “click” chemistry. Afterwards, the amphiphilic polymeric prodrug could selfassembly into micelles with a core-shell structure. Then the SCL prodrug micelles were prepared through addition of difunctional cross-linker in the presence of pH 6.2 and aniline as catalyst, as shown in Scheme 1. The obtained SCL micelles can be dissociated under dual-stimuli conditions, namely, acidic pH-triggered cleavage of acylhydrazone bonds into aldehyde and hydrazide, and thiol-triggered cleavage of disulfide linkages in the outer coronas. Once the prodrug micelles were internalized into the cancer cells, the following change process will occur. First, the shell cross linker of prodrug micelles containing acylhydrazone were cleaved under the conditions of endosomal/lysosomal pH, and disulfide linkages were cleaved in high GSH concentration of the cytosol. Then the disulfide bonds of main chains in prodrugs were cleavage, leading to the disassembly of prodrug micelles. Finally,


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the cleavage of hydrazone bond of prodrug could efficiently and rapid release drug under the condition of mildly acidity. Therefore, this work may provide a facile way to prepare pH-/redoxresponsive SCL prodrug micelles, which have highly promising potential for drug delivery.

rescence spectrophotometer (Cary Eclipse, Agilent Technologies) at the excitation wavelength of 335 nm and an emission wavelength of 350 to 550 nm, with both bandwidths set at 2.5 nm. From the pyrene emission spectra, the intensity ratio (I3/I1) of the third band (382 nm, I3) to the first band (371 nm, I1) was analyzed as a function of the polymer concentration. The CAC value was defined as the point of intersection of the two lines in the plot of fluorescence versus polymer concentration.55,56

■ EXPERIMENTAL SECTION Materials. The following agents were purchased and used without further purification: 2-hydroxyethyl disulfide, (SigmaAldrich), 2-bromoisobutyryl bromide (BIBB, Aldrich), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, J&K Chemical), 1, 8-diazabicyclo[5.4.0]-undec-7-ene (DBU, 98%, J&K Chemical), 4-dimethylamino pyridine (DMAP, 99%, 9 Ding chemistry), N,N′-dicyclohexylcarbodiimide (DCC, 99%, Alfa Aesar), doxorubicin hydrochloride (DOX⋅HCl, 99%, Beijing Zhongshuo Pharmaceutical Technology Development), 3, 3′dithiopropionic acid dimethylester (98%, TCI), 4-formylbenzoic acid (99%, Sinopharm Chemical Reagent), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT, 98%, Sigma-Aldrich), and sodium azide (NaN3, Sinopharm Chemical Reagent). DMAEMA and HEMA (Sigma-Aldrich) were separately passed through the column of activated basic alumina to remove the inhibitors. 2-(But-3-yn-1-yloxy)-2-oxo-1,3,2dioxaphospholane (BYP) was prepared and purified according to the previous literature.45,52 Cuprous bromide (CuBr, 95%, Sinopharm Chemical Reagent) was purified by washing three times with acetone, glacial acetic acid, and ethanol in turn, followed by drying under vacuum at 30 °C. DOX-hyd-N3 was prepared according to the previously reported methods.53,54 N,NDimethylformamide (DMF) was dried over CaH2 and distilled under reduced pressure before use. Dichloromethane (CH2Cl2) were dried and purified before use. Milli-Q water (18.2 MΩ cm-1) was generated using a water purification system (Simplicity UV, Millipore). All cell culture related reagents were purchased from Invitrogen/Life Technologies.

Synthesis of PBYP-ss-iBuBr. The macroinitiator PBYP-ssiBuBr was prepared by ROP with different feed ratios of BYP using DBU as catalyst. A typical procedure was carried out as follows: To a dried round flask, HO-ss-iBuBr (0.112 g, 0.368 mmol) and BYP (1.756 g, 11.04 mmol) in anhydrous CH2Cl2 (5 mL) was added under a nitrogen atmosphere. Then DBU (57 mg, 0.375 mmol) and 1mL of anhydrous CH2Cl2 were injected into the round flask via a syringe, and the reaction mixture was kept under stirring at 25 °C for 30 min. After the polymerization, the product was precipitated three times in diethyl ether/methanol (10/1, v/v). Finally, the precipitate was collected and dried under vacuum to a constant weight at 30 oC. One-Pot Synthesis of PBYP-hyd-DOX-ss-P(DMAEMAco-FBEMA) Polymeric Prodrug. A typical procedure was conducted as follows: To a Schlenk tube containing CuBr (6.4 mg, 0.045 mmol, 4 equiv), a DMF solution containing DMAEMA (353 mg, 2.25 mmol, 200 equiv.) and FBEMA (441.8 mg, 1.68 mmol, 150 equiv) was added. Then PBYP-ss-iBuBr (100 mg, 0.011 mmol), PMDETA (15.6 mg, 0.090 mmol, 8 equiv) and DOX-hyd-N3 (60 mg, 0.081 mmol) were also added to the Schlenk tube. The mixed reactant was degassed by freeze pumpthaw cycles and stirred at 70 °C for 24 h. The polymerization was quenched by quickly cooling the Schlenk tube in a ice-water bath. Subsequently, the product was dialyzed (MWCO 7000) against Milli-Q water for 24 h, with the purpose of removing the unreacted monomers and copper catalysts. The purified product was obtained by lyophilization method. (425 mg, yield: 44.5%)

Characterizations. 1H NMR and 13C NMR spectra were recorded on the 400 MHz spectrometer (INOVA-400) using deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as internal standard. The number-average molecular weight (M̅n) and molecular weight distribution (PDI) of PBYP-ssiBuBr were analyzed by gel permeation chromatography (GPC) instrument (HLC-8320, TOSOH) using polystyrene as the standard and DMF as the eluent. The DOX-hyd-N3 and polymeric prodrugs were determined by high performance liquid chromatography (HPLC) (UltiMate 3000, Thermo Fisher Scientific) at 30 °C with acetonitrile/Milli-Q water (50/50, v/v) as the mobile phase at a flow rate of 1.0 mL min-1. The ultraviolet-visible (UV-vis) absorption spectra were recorded at 480 nm on a UV-vis spectrophotometer (UV-3150, Shimadzu), and the fluorescence spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Agilent Technologies). The morphologies of the polymeric prodrug micelles were observed by a transmission electron microscopy (TEM) instrument (HT7700, Hitachi) operated at an accelerating voltage of 120 kV.

Preparation of PBYP-hyd-DOX-ss-P(DMAEMA-coFBEMA) Shell Cross-Linked Micelles. The PBYP-hydDOX-ss-P(DMAEMA-co-FBEMA) (25 mg) was dissolved in DMSO (2 mL). Then 15 mL of NaOH-KH2PO4 buffer solution (50 mM, pH 6.2) was added dropwise with 2 mL/h of flow rate using microinjection pump at room temperature. After stirring for 4 h, DMSO was removed by dialysis (MWCO 7000) against NaOH-KH2PO4 buffer solution for 24 h to afford the uncrosslinked (UCL) prodrug micelles. Shell cross-linked prodrug micelles were prepared by addition of DTP crosslinker (DTP/FBEMA molar ratio = 1: 3) in the presence of 10 mM aniline. The solution was stirred for 24 h at room temperature and then dialyzed against pH 7.4 PB solution for 24 h to remove unreacted cross-linker and aniline. The obtained SCL prodrug micelles were measured by DLS and TEM. In Vitro Drug Release from SCL Prodrug Micelles. PBYPhyd-DOX-ss-P(DMAEMA-co-FBEMA) (50 mg) was dissolved in DMSO, then 30 mL NaOH-KH2PO4 buffer (50 mM, pH 6.2) was added dropwise with 2 mL/h of flow rate using microinjection pump at room temperature. After stirring for 4 h, the solution was dialyzed against NaOH-KH2PO4 buffer (50 mM, pH 6.2) for 24 h with the purpose of removing DMSO. DTP cross-linker (DTP/FBEMA molar ratio = 1/3) was added in the presence of 10 mM aniline, and the mixed solution was stirred for 24 h. Then, the SCL prodrug micelles was dialyzed against pH 7.4 PB solution. Finally, the solution was filtered through a Φ 0.45 µm Millipore

Self-assembly Behavior. The critical aggregation concentrations (CAC) were determined by the fluorescence probe method using pyrene as the hydrophobic probe. Typically, a predetermined pyrene solution in acetone was added into a series of ampoules, respectively. Then, acetone was evaporated and replaced with prodrug micelles at different concentrations in the range of 500 to 2×10-4 mg L-1. The final concentration of pyrene in each ampoule was 6×10-6 mol L-1. The samples were sonicated for 20 min, stirred at room temperature for 48 h, and analyzed on fluo-



filter and some of which was finally lyophilized for calculating the DOX content. The in vitro DOX release behavior of the prodrug micelles was studied by dialysis method as follows: each of 5 mL of prodrug micelles was transferred to dialysis membrane (MWCO 7000), and then the dialysis membrane was placed into a centrifuge tube with 25 mL of different buffer solutions, in which the conditions were set to two phosphate buffer solutions (pH 7.4 and pH 7.4 with 10 mM GSH) and other two acetate buffer solutions (pH 5.0 and pH 5.0 with 10 mM GSH). All the centrifuge tubes were kept constantly shaking with a speed of 160 rpm at 37.5 °C. At predetermined intervals, 5 mL of the solution was taken out and replenished with an equal volume of the corresponding fresh buffer solution. The fluorescence spectrophotometer was employed to determine the content of the released DOX. The excitation wavelength was set at 480 nm while emission spectra were recorded with a 2.5 nm slit width over a wavelength from 520 to 650 nm. The DOX content (CDOX, wt%) was calculated according to eq (1):

CDOX ( wt %) =

CUV-vis ×100 CDPD

Synthesis and Characterization of PBYP-hyd-DOX-ssP(DMAEMA-co-FBEMA) Prodrug. In this study, the polymeric prodrug PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA) was synthesized via a combination of ROP, ATRP, and CuAAC “click” chemistry as shown in Scheme 1. First, an azidofunctionalized acid-labile DOX derivative containing hydrazone group (designated as DOX-hyd-N3) was synthesized. Second, PBYP-ss-iBuBr was synthesized by ROP using HO-ss-iBuBr as an initiator. Finally, the prodrug was obtained via one-pot method with a combination of ATRP and CuAAC “click” chemistry. The chemical structures of HO-ss-iBuBr, DTP, and FBEMA were verified by 1H NMR and 13C NMR analysis, respectively, as shown in Figure S1(A), Figure S4, Figure S5, Figure S2, and Figure S3 in the Supporting Information. The chemical structure of DOX-hyd-N3 was characterized by 1H NMR analysis, as shown in Figure S6 and S7 of the Supporting Information (SI), which indicate that DOX-hyd-N3 has been synthesized successfully. From the results of the 1H NMR spectrum of FBEMA in Figure S2, there are three chemical shifts at δ 10.11 ppm (peak a), δ 8.19 ppm (peak b), and δ 7.97 ppm (peak c), which can be ascribed to the protons of 4-formylbenzoic acid. In addition, the characteristic signals at δ 1.95 ppm, δ 4.51 ppm, δ 4.62 ppm, and δ 5.60-6.15 ppm belong to the protons of -O-CO-C(CH2)-CH3, CH2-CH2-OCO-, -CH2-CH2-OCO-, and-CO-C(CH2) in FBEMA monomer, respectively. Then, the well-defined PBYP-ss-iBuBr homopolymer was prepared via ROP. The chemical structures, the molecular weights, and molecular weight distributions of PBYPss-iBuBr were characterized by 1H NMR and GPC measurements. The 1H NMR spectrum of PBYP50-ss-iBuBr homopolymer is shown in Figure S1(B). The resonances appeared at δ 2.11 ppm (peak k), δ 2.62 ppm (peak i), δ 4.18 ppm (peak j) are separately ascribed to the protons (HC≡C-CH2-, HC≡C-CH2-CH2-, and HC≡C-CH2-CH2-) of the alkynyl group, and the resonances appeared at δ 4.30 ppm is ascribed to the characteristic resonance signal of methylene in the backbone (-O-CH2-CH2-O-) of PBYP. Furthermore, some characteristic resonance signals can be observed at δ 1.95 ppm (peak e), δ 2.99 ppm (peak b and c), δ 3.80 ppm (peak a), and δ 4.45 ppm (peak d), which belong to the protons of HO-ss-iBuBr.

(1)

where CUV-vis represents the concentration of DOX measured by UV-vis, while CDPD is the concentration of prodrug micelles. In Vitro Cytotoxicity Test. A standard MTT assay was employed to evaluate the cytotoxicity of SCL-DPD prodrug micelles. HeLa cells and HepG2 cells were separately seeded in 96-well plates at a density of about 5×104 cells per well and cultured in DMEM culture medium with 10% serum and 1% penicillin/streptomycin in an incubator at 37 °C under a 5% CO2 atmosphere for 24 h. The prodrug micelles with different concentrations were added into each well, and then incubated with the cells for another 48 h using free DOX as the control. Afterwards, 25 µL of the MTT stock solution (5 mg mL-1 in PBS) was added to each well and incubated for another 4 h. The DMEM medium was removed and 150 µL of DMSO was added to each well. The optical density (OD) of each well was measured on a microplate reader (Bio-Rad 680) at 570 nm. The cell viability was calculated according to eq (2):

Cell viability(%) =

OD treated ×100 ODcontrol

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(2)

where ODtreated and ODcontrol stand for the OD values of the wells treated with prodrug micelles and the control wells without prodrug micelles, respectively. The data are gathered and processed as the average values with standard deviations. Cellular Uptake and Intracellular Release of DOX. Cellular uptake and intracellular drug release studies of free DOX and SCL-DPD prodrug micelles in HeLa cells were real-time monitored using the live cell imaging system (Cell’R, Olympus, Japan). Typically, HeLa cells were seeded in a Φ 35 mm glass Petri dish at 1.0×105 cells and cultured in high-glucose DMEM culture medium at 37 °C under a 5% CO2 atmosphere for different times. Subsequently, the culture medium was removed by washing three times using phosphate buffer saline (PBS) and then the cell nucleus was stained with H 33342 for 15 min. The culture medium was then replaced by a DMEM medium containing free DOX or SCLDPD1 (4 mg L-1 of DOX). The images were captured at excitation wavelengths of 480 nm (red) and 340 nm (blue) for 6 h.

Figure 1. GPC traces of PBYP30-ss-iBuBr and PBYP50-ss-iBuBr homopolymers.

■ RESULTS AND DISCUSSION

The GPC traces of PBYP30-ss-iBuBr and PBYP50-ss-iBuBr homopolymers are shown in Figure 1. The two homopolymers


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exhibit unimodal distribution with relatively narrow polydispersity indexes (PDIs). The GPC curve of PBYP50-ss-iBuBr shifts toward lower elution time compared to that of PBYP30-ss-iBuBr in Figure 1, indicating that its M̅n was higher than that of PBYP30ss-iBuBr. The detailed information about the molecular weights and PDIs of the homopolymers are listed in Table 1. The molecular weight (M̅n, NMR) of PBYP-ss-iBuBr were calculated according to the 1H NMR analysis by the following eqs (3) and (4): Ae

Ai

M n,NMR

=

3Ai Ae

=

3

The polymerization degree of PBYP-hyd-DOX-ssP(DMAEMA-co-FBEMA) were calculated according to the 1H NMR analysis by the following eqs (5) and (6): Ab Ak

Ab Ai

(3)

2x

(5)

3y

=

x

(6)

z

where Ab, Ak, and Ai are the integral values of the peaks b, k, and i in Figure 2, respectively. The x, y, and z are the polymerization degree of BYP, DMAEMA, and FBEMA monomers.

n

× 176.02 + 303.23

=

(4) Table 1. Molecular weights and molecular weight distributions (PDIs) of PBYP-ss-iBuBr homopolymers.

where Ae and Ai were the integral values of the peaks e and i in Figure S1(B), respectively. 176.02 was the molecular weight of one repeating unit of PBYP, 303.23 was the molecular weight of the HO-ss-iBuBr, and n was the polymerization degree of PBYP.

M̅n, NMR a)

M̅n, GPC b)

(g mol-1)

(g mol-1)

PBYP30-ss-iBuBr

5600

16800

1.17

PBYP50-ss-iBuBr

9100

19000

1.20

Sample

Then, two polymeric prodrugs were prepared, which were PBYP30-hyd-DOX-ss-P(DMAEMA38-co-FBEMA17) and PBYP50hyd-DOX-ss-P(DMAEMA40-co-FBEMA32). For the sake of simplicity, we designated the two prodrugs as DPD1 and DPD2, respectively. Figure 2 shows the 1H NMR spectrum of DPD1 prodrug. The characteristic signals at around δ 4.03 ppm (peak g), δ 2.51 ppm (peak j), and δ 2.22 ppm (peak k) can be assigned to the protons of -CH2-CH2-N-(CH3)2, -CH2-N-(CH3)2, and -N(CH3)2 in DMAEMA units, respectively. The resonances appeared at δ 4.51 ppm (peak i), δ 7.97 ppm (peak l), δ 8.19 ppm (peak m), and δ 10.10 ppm (peak n), which are ascribed to the protons of the FBEMA. The resonances appeared at δ 1.23 ppm (peak f) and δ 0.92 ppm (peak h) can be attributed to the characteristic signal of CH2-C-(CH3)-CO- and -CH2-C-(CH3)-CO- in the main chain of P(DMAEMA-co-FBEMA), respectively. Furthermore, a new peak attributed to the proton of the triazole ring is clearly detected at δ 7.55 ppm (peak b′) in Figure 2. Therefore, these results confirmed that DOX-hyd-N3 had been successfully grafted onto the BYP segments of PBYP-ss-iBuBr by one-pot method.

a)

PDI b)

1

Calculated by H NMR spectra.

b)

Determined by GPC with DMF as the eluent and polystyrene as the standard.

Figure 3. HPLC analyses results of (A) free DOX, (B) DOX-hydN3, and (C) DPD1 prodrug. HPLC analyses were performed with acetonitrile/water (50/50, v/v), as the mobile phase at 30 °C with a flow rate of 1.0 mL min-1.

To further prove that DOX-hyd-N3 has been conjugated onto the side chains of DPD prodrug, HPLC and UV-vis measurements were applied. HPLC traces and UV-vis spectra are shown in Figure 3 and Figure S8, respectively. We can find that the DOX-hydN3 elutes at 2.50 min, while DPD1 prodrug elutes at 1.51 min. There are no traces at 2.50 min and 5.25 min in Figure 3(C), indicating that the polymeric prodrug has been purified without free DOX and residual DOX-hyd-N3. Furthermore, the top peak of DOX in DPD1 prodrug has about 56 nm of red shift compared

Figure 2. 1H NMR spectrum of PBYP30-hyd-DOX-ssP(DMAEMA38-co-FBEMA17) prodrug in CDCl3.



with free DOX in Figure S8 due to the bonding of the drug with the polymer. The DOX contents (CDOX, wt%) were determined by UV-vis spectroscopy and all the results are listed in Table 2. These results also confirmed that the DPD prodrug had been synthesized successfully.

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coronas are invisible under TEM observation, while they can be extended into the water phase in DLS measurement. For verifying the successful preparation of shell cross-linked prodrug micelles, DMF was added into the prodrug micelles, and then the changes of prodrug micelles morphologies were analyzed by TEM. From Figure 5(A and B), we can find that the morphologies of SCL-DPD1 prodrug micelles have almost no obvious change, indicating that the SCL-DPD1 micelles maintain a good stability in the presence of DMF solvent. However, the UCLDPD1 micelles system shows irregular spherical structure in Figure 5(C and D), indicating that the UCL-DPD1 micelles dissociate in the presence of DMF solvent. Therefore, the SCL prodrug micelles possess a better stability than UCL prodrug micelles.

Self-assembly of the Polymeric Prodrugs. The critical aggregation concentration (CAC) represents the thermodynamic stability of micelles in aqueous medium. When the concentration of prodrug are higher than the CAC value, they can self-assemble into micelles with PBYP grafted DOX as the core and hydrophilic P(DMAEMA-co-FBEMA) as the shell in aqueous solution. The CAC value of uncrosslinked (UCL) micelle was determined by the steady-state fluorescence probe method using pyrene as the probe, which was 95.4 mg L-1, as shown in Figure S9. SCL-DPD micelles were fabricated upon introduction of DTP cross-linker, which could react with pendent aldehyde moieties to generate acylhydrazone linkages within the hydrophilic shell. The obtained SCL-DPD micelles were characterized by DLS and TEM. The prodrug micelle sizes ( D Z ), polydispersity index (PDI), theoretical and actual DOX contents are summarized and the results have been listed in Table 2.

Table 2. Sizes ( D Z ), size PDI, theoretical and actual DOX content values of DPD prodrug micelles Prodrugs

DZ (nm)

a)

Size a) PDI

DOX content (theor. wt %)

DOX content b) (wt %)

SCL-DPD1

144

0.231

20

15.2

SCL-DPD2

162

0.278

20

17.3

a)

Determined by DLS measurement.

b)

Determined by UV-vis measurement.

Figure 5. TEM images marked separately by two bars (500 nm and 200 nm), (A) and (B) the SCL-DPD1 micelles with DMF; (C) and (D) the uncrosslinked UCL-DPD1 micelles with DMF. All the prodrug micelles concentrations were 0.5 mg mL-1.

Figure 4. (A) TEM images of SCL-DPD1 micelles (scale bar 200 nm) and (B) the micelles size distribution curve corresponding to the TEM samples. (The concentration of the SCL-DPD1 micelles was 0.5 mg mL-1) As shown in Figure 4(A), we can find that the TEM images of SCL-DPD1 micelles are mainly spherical structure and the average size is about 100 nm. In addition, the corresponding micelles size distribution displays the unimodal pattern in Figure 4(B) and the average diameter of SCL-DPD1 micelles is about 144 nm by DLS measurement. The average particle size observed by TEM is a little smaller than those obtained from DLS measurement. This is because of the shrinkage of the micellar shell during the preparation of TEM sample. The hydrophilic shell

Figure 6. TEM images of SCL-DPD1 prodrug micelles at different conditions: (A) and (C) before and after incubation with at pH 7.4 for 48 h; (B) after incubation with pH 5.0 for 48 h; (D) after incubation with 10 mM GSH for 48 h, respectively.


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Figure 7. Reduction- and pH-induced size change of SCL-DPD1 prodrug micelles under different conditions of (A) pH 7.4, (B) pH 5.0, and (C) pH 7.4 with 10 mM GSH as determined by DLS measurement.

phology under physiological condition and disassemble rapidly in the presence of acidic microenvironments and high GSH concentration.

The size changes of SCL-DPD1 prodrug micelles was monitored by TEM measurement. As shown in Figure 6(A and C), the morphologies of the prodrug micelles have no obvious change under pH 7.4 for 48 h. However, the irregular aggregates in the SCL-DPD1 prodrug micelles were clearly observed under the condition of pH 5.0 or 10 mM GSH at 48 h intervals in Figure 6(B and D), respectively. Therefore, the results also verify that SCL-DPD prodrug micelles can maintain mainly spherical mor-

For further evaluating the pH- and reduction-responsiveness of SCL prodrug micelles, the size change of prodrug micelles were monitored by DLS and TEM measurements in the presence of pH 5.0 or 10 mM GSH at different time intervals. As shown in Figure 7(A), there was almost no obvious change in micelles size



over 48 h under the condition of pH 7.4, indicating that the SCLDPD1 prodrug micelles kept stability under physiological conditions. In Figure 7(B), we can find that the size of SCL prodrug micelles increased to about 500 nm at pH 5.0 for 10 h. Even then, the size distribution of SCL prodrug micelles show a relatively wide at pH 5.0 over 48 h. This phenomenon might be due to the partial cleavage of the acylhydrazone linkages and hydrazone bonds in the SCL prodrug micelles, which could induce the hydrophilic chains diffusion and the hydrophobic segments aggregation over 48 h.57,58

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The in vivo release of DOX from SCL-DPD1 prodrug micelles were explored under mildly acidic pH and/or reductive conditions in Figure 8. The significant acceleration of DOX release were actuated upon either introducing 10 mM GSH or adjusting the solution pH to 5.0, approximately 46.1% and 43.5% of DOX were released from the SCL-DPD1 prodrug micelles after 70 h. Then, the DOX could be released rapidly when dual stimuli factors were applied to the prodrug micelles simultaneously, in which approximately 70.0% of DOX was released at pH 5.0 with 10 mM GSH. This is due to the cleavage of acylhydrazone and hydrazone linkages in the SCL prodrug micelles under acidic and reductive microenvironment, it can lead to the SCL-DPD1 prodrug micelles rapid disintegration. During circulation, considering the premature DOX release would lead to the unfavorable drug leakage, SCL-DPD1 prodrug micelles can display its potential to load DOX in the drug delivery system.

From the results of Figure 7(C), we can find that the size of SCL-DPD1 prodrug micelles also increased to 500 nm, or even larger. The size of change also demonstrates that the disulfide bond in the cross-linkers and prodrug micelles could be cleaved under the condition of high GSH concentration, leading to the aggregation of hydrophobic segments and an increase of prodrug micelles sizes. The results by DLS measurement are consistent with the TEM measurement in Figure 6. These results also confirm that the SCL prodrug micelles can be of both pH- and reduction-sensitive behavior.

In Vitro Cytotoxicity. Good biocompatibility is a vital requirement for micelles used for drug delivery. Herein, MTT assays were performed to study the cytotoxicity of the SCL PBYP-ss-P(DMAEMA-co-FBEMA) micelles against normal cells (L929 cells) and cancer cells (HeLa and HepG2 cells). In Figure S10, the polymeric micelles have a negligible impairment in the cell viability with three kind cells after 48 h, even if the concentration were up to 250 mg L-1, the cell viabilities of L929, HeLa, and HepG2 cells were all above 85% after incubation with the block copolymer, indicating the polymer possesses a good biocompatibitity as a drug carrier.

In Vitro Drug Release. In this study, for estimating drug release behavior of prodrug micelles at the various media, the in vitro cumulative release of DOX from UCL-DPD1 and SCLDPD1 prodrug micelles were carried out under the various media. Four different media were selected as follows: (i) pH 7.4, (ii) pH 5.0, (iii) pH 7.4 with 10 mM GSH, (iv) pH 5.0 with 10 mM GSH, which could be used to mimick the microenvironment of blood or tumorous site. As Figure 8 shown, the final cumulative release of DOX from UCL-DPD1 and SCL-DPD1 prodrug micelles were approximately 25.5% and 11.0% at pH 7.4. Compared with UCLDPD1 prodrug micelles, SCL-DPD1 prodrug micelles could avoid premature drug leakage effectively at pH 7.4. The SCL structure could act as a diffusion barrier to prevent premature drug leaking at physiological conditions.

Figure 8. In vitro DOX release curves for UCL DPD1 micelles pH 7.4, SCL DPD1 micelles pH 7.4, SCL DPD1 micelles pH 5.0, SCL DPD1 micelles pH 7.4 with 10 mM GSH, and SCL DPD1 micelles pH 5.0 with 10 mM GSH. The prodrug micelles concentrations were 500 mg L-1.

Figure 9. Cell viabilities of (A) HeLa cells and (B) HepG2 cells, treated with SCL-DPD1 prodrug micelles and free DOX with different DOX dosages for 48 h of incubation.


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For further evaluating the prodrug’s antitumor efficency, the antiproliferation activity of SCL-DPD1 prodrug micelles against HeLa cells and HepG2 cells were investigated using MTT assays, respectively. As shown in Figure 9(A), the cell viabilities of HeLa cells were decreased gradually when DOX concentration increased from 0.078 mg L-1 to 40 mg L-1. The half-maximal inhibitory concentration (IC50) values of free DOX and SCL-DPD1 prodrug micelles against HeLa cells were determined to be 0.44 mg L-1 and 0.92 mg L-1, respectively, in Figure 9(A). The result of Figure 9(B) is similar to that of Figure 9(A). The IC50 values of free DOX and SCL-DPD1 prodrug micelles aganist HepG2 cells were 0.44 mg L-1 and 1.57 mg L-1, respectively. The SCL-DPD1 prodrug micelles have a higher IC50 value compared with free DOX in Figure 9. The cells viabilities aganisit HeLa and HepG2 cells with free DOX were lower than the prodrug micelles with equal DOX concentration after 48 h incubation, which could indicate that the shell corona of SCL-DPD1 prodrug micelles would not impair normal cell before reaching cancer cell. It is remarkable that the SCL prodrug micelles is probably due to effective cellular uptake via the endocytosis pathway, it can be activated in the mildly acidic and reductive endosomal/lysosomal compartments to exert the cytostatic effects.59,60 Therefore, these cytotoxicity assays confirm that the SCL-DPD prodrug micelles have the ablity to inhibit the proliferation aganist HeLa and HepG2 cells as well as reduce the side effects of the drug.

33342 (blue). The DOX fluorescence in HeLa cells was monitored incubation with the SCL-DPD1 prodrug micelles after 0.5 h, indicating that SCL-DPD1 produrg was internalized quickly by HeLa cells and DOX was released from the SCL-DPD1 prodrug efficiently inside HeLa cells. As Figure 10(A) shown, the red fluorescence intensity of DOX was observed in the cytoplasm and nucleic regions with increasing the incubation time. Besides, there are strong red DOX flurescence intensity in the nucleic regions after 6 h incubation compared with that of free DOX in Figure 10(B). This is because of free DOX can be transported through the cell membrane via passive diffusion rapidly, and then some free DOX could be pumped out of the HeLa cells quickly,20,61,62 from which the accumulated DOX fluorescence in cytoplasm and nucleus eventually reached a very low level after incubation with 6 h. All these results verify that the DOX was released from the SCL-DPD1 prodrug and mainly accumulated in the HeLa cell nuclei. ■ CONCLUSIONS In summary, we have prepared a novel pH/redox responsive PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA) prodrug using PBYP-ss-iBuBr as macroinitator by a combination of ATRP and CuAAC “click” chemistry. The amphiphilic prodrug can selfassembly into polymeric micelles in aqueous solution, and the size of prodrug micelles is 144 nm. Shell cross-linked can improve the stability of prodrug micelles and reduce the side effect of DOX. The results of DLS and TEM verified that SCL-DPD micelles could keep relatively stability under physiological conditions and disassemble rapidly under acidic medium or/and high GSH concentration. DOX was released from SCL-DPD prodrug micelles in the presence of pH 5.0 and 10 mM GSH media with the accumulative release amount up to 70.0%. The results of MTT assays indicated that the SCL-DPD prodrug micelles had antiproliferation activity against HeLa cells and HepG2 cells. These SCL-DPD prodrug micelles also could be internalized into HeLa cells through endocytosis, and DOX was released from SCL-DPD prodrug micelles due to acidic medium and high GSH concentration inside the tumor cells, which could inhibit the cell proliferation efficiently. Therefore, a novel pH/redox responsive SCL-DPD prodrug micelles are highly potential in cancer chemotherapy. ■ ASSOCIATED CONTENT Supporting Information Synthetic and experimental procedure (synthesis of DTP, FBEMA, and HO-ss-iBuBr). 1H NMR spectra of 6azidehexanohydrazine, DOX-hyd-N3, and dithiodipropionic acid dihydrazide (DTP). 13C NMR spectrum of DTP in DMSO-d6. UVvis spectra of free DOX and PBYP30-hyd-DOX-ssP(DMAEMA38-co-FBEMA17) prodrug. The CAC of PBYP30-hydDOX-ss-P(DMAEMA38-co-FBEMA17) prodrug micelles in pH 7.4 buffer solution. Cell viability of L929 cells, HeLa cells, and HepG2 cells treated with the SCL PBYP-ss-P(DMAEMA-coFBEMA) polymeric micelles at different concentrations for 48 h of incubation.

Figure 10. Live cell imaging system images of HeLa cells incubated with (A) SCL-DPD1 micelles and (B) free DOX for different times. The DOX dosage was 4 mg L-1. For each panel, images from left to right show cell nuclei stained by H 33342 (blue), DOX fluorescence in cells (red), and overlays of the blue and red images. The scale bars are 50 µm in all images.

■ AUTHOR INFORMATION Corresponding Author

Cellular Uptake. It is crucial for prodrug micelles to rapid release therapeutic agents inside the tumor cells. The cellular uptake behavior of SCL-DPD1 prodrug micelles was investigated against HeLa cells by fluorescence microscopy of the live cell imaging system. As shown in Figure 10, the cell nuclei was stained with H

* Tel: +86 512 65882047; E-mail: [email protected] ORCID Peihong Ni: 0000-0003-4572-3213 Jinlin He: 0000-0003-3533-2905



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Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS We gratefully acknowledge the financial supports from the National Natural Science Foundation of China (21374066), the Major Program of the Natural Science Project of Jiangsu Higher Education Institutions (15KJA150007), the Natural Science Foundation of Jiangsu Province (BK20171212), a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, and Soochow-Waterloo University Joint Project for Nanotechnology from Suzhou Industrial Park. Mr. Lei Li would like to thank the financial support from the Innovative Graduate Research Program of Jiangsu Province (KYCX17_1981). ■ REFERENCES (1) Xiao, B., Ma, L. J., Merlin, D. (2017) Nanoparticle-mediated co-delivery of chemotherapeutic agent and siRNA for combination cancer therapy. Expert Opin. Drug Delivery 14, 65-73. (2) Dai, W. B., Wang, X. Y., Song, G., Liu, T. Z.,He, B., Zhang, H., Wang, X. Q., Zhang, Q. (2017) Combination antitumor therapy with targeted dual-nanomedicines. Adv. Drug Delivery Rev. 115, 23-45. (3) Du, X. Q., Sun, Y., Zhang, M. Z., He, J. L., Ni, P. H. (2017) Polyphosphoester-camptothecin prodrug with reduction-response prepared via Michael addition polymerization and click reaction. ACS Appl. Mater. Interfaces 9, 13939-13949. (4) Zhong, Y. N., Goltsche, K., Cheng, L., Xie, F., Meng, F. H., Deng, C., Zhong, Z. Y., Haag, R. (2016) Hyaluronic acid-shelled acid-activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo. Biomaterials 84, 250-261. (5) Wen, H. Y., Dong, H. Q., Xie, W. J., Li, Y. Y., Wang, K., Pauletti, G. M., Shi, D. L. (2011) Rapidly disassembling nanomicelles with disulfide-linked PEG shells for glutathionemediated intracellular drug delivery. Chem. Commun. 47, 35503552. (6) Pramanick, S., Kim, J., Kim, J., Saravanakumar, G., Park, D., Kim, W. J. (2018) Synthesis and characterization of nitric oxidereleasing platinum (IV) prodrug and polymeric micelle triggered by light. Bioconjugate Chem. 29, 885-897. (7) Allen, T. M., Cullis, P. R. (2013) Liposomal drug delivery systems: from concept to clinical applications. Adv. Drug Delivery Rev. 65, 36-48. (8) Pattni, B. S., Chupin, V. V., Torchilin, V. P. (2015) New developments in liposomal drug Delivery. Chem. Rev. 115, 1093810966. (9) Oh, J. K., Lee, D. I., Park, J. M. (2009) Biopolymer-based microgels/nanogels for drug delivery applications. Prog. Polym. Sci. 34, 1261-1282. (10) Wu, W., Yao, W., Wang, X., Xie, C., Zhang, J. L., Jiang, X. Q. (2015) Bioreducible heparin-based nanogel drug delivery system. Biomaterials 39, 260-268. (11) Wei, X., Senanayake, T. H., Warren, G., Vinogradov, S. V. (2013) Hyaluronic acid-based nanogel-drug conjugates with enhanced anticancer activity designed for the targeting of CD44positive and drug-resistant tumors. Bioconjugate Chem. 24, 658668. (12) Liu, Q. M., Zhu, H. S., Qin, J. Y., Dong, H. Q., Du, J. Z. (2014) Theranostic vesicles based on bovine serum albumin and poly(ethylene glycol)-block-poly(L-lactic-co-glycolic acid) for magnetic resonance imaging and anticancer drug delivery. Biomacromolecules 15, 1586-1592.


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TOC Graphic

Illustration of shell cross-linked prodrug micelles for efficient intracellular release of hydrophobic anticancer drugs triggered by the acidic and reductive microenvironment inside the tumor tissue.



Illustration of shell cross-linked prodrug micelles for efficient intracellular release of hydrophobic anticancer drugs triggered by the acidic and reductive microenvironment inside the tumor tissue. 290x182mm (300 x 300 DPI)

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Scheme 1. Synthesis routes and self-assembly of PBYP-hyd-DOX-ss-P(DMAEMA-co-FBEMA) polymeric prodrug. 329x176mm (300 x 300 DPI)



Figure 1. GPC traces of PBYP30-ss-iBuBr and PBYP50-ss-iBuBr homopolymers. 228x175mm (300 x 300 DPI)


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Figure 2. 1H NMR spectrum of PBYP30-hyd-DOX-ss-P(DMAEMA38-co-FBEMA17) prodrug in CDCl3. 272x182mm (300 x 300 DPI)



Figure 3. HPLC analyses results of (A) free DOX, (B) DOX-hyd-N3, and (C) DPD1 prodrug. HPLC analyses were performed with acetonitrile/water (50/50, v/v), as the mobile phase at 30 °C with a flow rate of 1.0 mL min-1. 161x140mm (300 x 300 DPI)


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Figure 4. (A) TEM images of SCL-DPD1 micelles (scale bar 200 nm) and (B) the micelles size distribution curve corresponding to the TEM samples. (The concentration of the SCL-DPD1 micelles was 0.5 mg mL-1) 232x104mm (300 x 300 DPI)



Figure 5. TEM images marked separately by two bars (500 nm and 200 nm), (A) and (B) the SCL-DPD1 micelles with DMF; (C) and (D) the uncrosslinked UCL-DPD1 micelles with DMF. All the prodrug micelles concentrations were 0.5 mg mL-1. 172x171mm (300 x 300 DPI)


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Figure 6. TEM images of SCL-DPD1 prodrug micelles at different conditions: (A) and (C) before and after incubation with at pH 7.4 for 48 h; (B) after incubation with pH 5.0 for 48 h; (D) after incubation with 10 mM GSH for 48 h, respectively. 157x156mm (300 x 300 DPI)



Figure 7. Reduction- and pH-induced size change of SCL-DPD1 prodrug micelles under different conditions of (A) pH 7.4, (B) pH 5.0, and (C) pH 7.4 with 10 mM GSH as determined by DLS measurement. 372x391mm (300 x 300 DPI)


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Figure 8. In vitro DOX release curves for UCL DPD1 micelles pH 7.4, SCL DPD1 micelles pH 7.4, SCL DPD1 micelles pH 5.0, SCL DPD1 micelles pH 7.4 with 10 mM GSH, and SCL DPD1 micelles pH 5.0 with 10 mM GSH. The prodrug micelles concentrations were 500 mg L-1. 243x191mm (300 x 300 DPI)



Figure 9. Cell viabilities of (A) HeLa cells and (B) HepG2 cells, treated with SCL-DPD1 prodrug micelles and free DOX with different DOX dosages for 48 h of incubation. 165x246mm (300 x 300 DPI)


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Figure 10. Live cell imaging system images of HeLa cells incubated with (A) SCL-DPD1 micelles and (B) free DOX for different times. The DOX dosage was 4 mg L-1. For each panel, images from left to right show cell nuclei stained by H 33342 (blue), DOX fluorescence in cells (red), and overlays of the blue and red images. The scale bars are 50 µm in all images. 126x143mm (300 x 300 DPI)


Redox Responsive Polymeric Prodrug and

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