reduction dual-responsive prodrug microspheres with high drug content for tumor intracellular triggered release of DOX

Reactive and Functional Polymers 116 (2017) 24–30

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Facile preparation of pH/reduction dual-responsive prodrug microspheres with high drug content for tumor intracellular triggered release of DOX

MARK

Ruinian Zhang, Xu Jia, Mingliang Pei, Peng Liu⁎ State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Prodrug microspheres High drug content pH/reduction dual-responsive Tumor intracellular triggered release Doxorubicin

To integrate the two advantages of upregulated stability during blood circulation and site-specific drug release in cancer cells, pH/reduction dual-responsive prodrug microspheres with high drug content were designed by conjugating doxorubicin (DOX) onto aldehyde-functionalized disulfide-crosslinked copolymer microspheres via acid-labile imine linkage, where the copolymer microspheres were synthesized by facile emulsion copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and 4-formylphenyl acrylate (FPA) with N,N-bis(acryloyl)cystamine (BACy) as crosslinker. Their particle size and average hydrodynamic diameter were 150 nm and 205 nm respectively, with high DOX content of 44.4%. The DOX release ratio reached 73% within 60 h and the prodrug microspheres decrosslinked into water soluble copolymers within 72 h in the simulated tumor microenvironment (pH 5.0 with 10 mM GSH), while only 16% of DOX was released in physiological medium (pH 7.4 with 10 μM GSH), demonstrating their good tumor intracellular triggered release performance. Furthermore, the disintegration of the copolymer microspheres into water soluble copolymers in simulated tumor microenvironment would favor the metabolism of drug carriers. The MTT assay demonstrated that the prodrug microspheres exhibited the enhanced inhibitory efficiency against HepG2 cells in comparison with free DOX, while the bare polymer microspheres were cytocompatible.

1. Introduction Cancer has currently surpassed heart disease as the top killer of human and now chemotherapy becomes as one of the most used clinical approaches to treat cancer, especially after surgical operation. Unfortunately, the cancer patients suffer from severe toxic and side effects of chemotherapeutics due to their non-selectivity [1]. In order to improve the anticancer efficacy, smart drug delivery system (DDS) has been widely studied to reduce the side effects and improve the bioavailability of anticancer chemotherapeutics [2], by means of triggered release anticancer drugs responding to tumor intracellular stimuli, such as pH [3] and reductant level [4]. In such case, the drug release in physiological medium would be suppressed to reduce the toxic and side effects on normal tissues. Compared with the common DDSs in which anticancer drugs are usually loaded or encapsulated via a weak interaction (for examples, electrostatic interaction, hydrogen bond, or hydrophobic interaction), polymer prodrugs, in which one or more drug(s) are covalently attached to the functional groups of the polymer via a weak covalent bond directly or through a spacer, should be a more efficient strategy

⁎

[5]. On the basis of the intracellular differences in the biophysical and biochemical indexes of normal and tumor cells, pH and glutathione (GSH) level have been intensely investigated as switch to trigger the release of anticancer chemotherapeutics from prodrugs in the last decade. For pH-triggered prodrugs, the anticancer drugs are usually conjugated onto the nano-vehicles via acid-labile imine or hydrazone linkage [6], which could be cleaved off to release drugs in tumor intracellular acidic media. As for the reduction-triggering mode [7], the prodrugs could release the derivative of anticancer drugs [8], due to that the drug is conjugated via the bioreducible disulfide bond. Comparatively speaking, the disulfide bond might be more suitable crosslinking structure to control the disintegration of the nano-prodrugs and drug diffusion subsequently. For examples, Zhao and Liu fabricated core-shell-corona micelles by self-assembly of triblock copolymer and shell-crosslinking with disulfide bond as promising tumor microenvironment-responsive nano-vehicles for doxorubicin (DOX) by GSH triggering [9]. Polymer nanoparticles or nanohydrogels have attracted more and more interest owing to their facile preparation in comparison with polymer micelles. Zhou et al. designed monodisperse biodegradable PEGylated pH and reduction dual-stimuli sensitive poly

Corresponding author. E-mail address: [email protected] (P. Liu).

http://dx.doi.org/10.1016/j.reactfunctpolym.2017.05.002 Received 22 November 2016; Received in revised form 29 March 2017; Accepted 4 May 2017 Available online 04 May 2017 1381-5148/ © 2017 Elsevier B.V. All rights reserved.

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purchased from Tianjin Heowns Company. Triethylamine (99%) was purchased from Alfa Aesar and dried by CaH2. Poly(ethylene glycol) methyl ether methacrylate (PEGMA, Mn = 475, 98%) was purchased from Sigma Aldrich and purified with alkaline aluminum oxide column to remove inhibitor before use. Sodium dodecyl sulfate (SDS, CP) was purchased from Shanghai Shiyi chemical reagents Ltd. Ammonium persulphate (APS, 98%) was purchased from Kelong Chemical Reagent Factory. Glutathione (GSH, 97%) and cystamine dihydrochloride (96%) were obtained from Tianjin Heowns Biochemical technology Co, Tianjin, China. Doxorubicin hydrochloride (DOX ⋅ HCl, 99.4%) was obtained from Beijing Huafeng Lianbo Technology Co., Ltd., Beijing, China. Tetrahydrofuran (THF, 99%, Tianjin Kermel Chemical Reagent Co., Ltd) was dried by CaH2 and distilled prior to use. N,NDimethylformamide (DMF), dimethyl sulfoxide (DMSO) and other reagents were achieved from Tianjin Chemical Reagent II Co. and used without further purification. Deionized water was used throughout.

[methacrylic acid-co-poly(ethylene glycol) methyl ether methacrylateco-N,N-bis(acryloyl)cystamine] nanohydrogels via one-step distillation precipitation polymerization as DDS for DOX [10]. Jia et al. synthesized fluorescent poly(methacrylic acid-co-poly(ethylene glycol) methyl ether methacrylate-co-N′-rhodamine B-acrylhydrazine) nanoparticles as potential theranostic nanoplatform via facile distillation precipitation copolymerization with bio-reducible disulfide-containing crosslinker, after DOX loading via electrostatic interaction [11]. Pan et al. fabricated folate-conjugated poly(N-(2-hydroxypropyl)methacrylamide-comethacrylic acid) nanohydrogels for targeted delivery of DOX, via distillation-precipitation polymerization and subsequent folate modification [12]. Most recently, Zhang et al. established pH and reduction dualsensitive prodrug nanogel to integrate the two advantages of upregulated stability during blood circulation and selective release of drug in cancer cells, by simultaneously conjugating DOX via acid sensitive hydrazone bond and cross-linking with reduction responsive disulfide containing linkage in the core through one step “click chemistry” crosslinking of diblock copolymer (methoxy poly(ethylene glycol)-bpoly(γ-propargyl-L-glutamate)) with 2-azidoethyl disulfide into corecrosslinked micelles [13]. In the present work, a facile strategy has been developed for the fabrication of the pH/reduction dual-responsive prodrug microspheres, by conjugating DOX via acid-labile imine linkage onto PEGylated copolymer microspheres prepared by emulsion copolymerization of polyethylene glycol methyl ether methacrylate (PEGMA) and 4-formylphenyl acrylate (FPA) with N,N-bis(acryloyl)cystamine (BACy) as crosslinker (Scheme 1). The in vitro release experiments showed that the accumulative release ratios were 73.3% and 16.6% in the simulated tumor and physiological media respectively, indicating the tumor intracellular triggered release of DOX from the proposed prodrug microspheres.

2.2. Synthesis of PEGylated polymer microspheres 4-Formylphenyl acrylate (FPA) was synthesized by esterification reaction of phenolic hydroxyl with acyl chloride, as reported previously [14]. Typically, a solution of acryloyl chloride (4.5 mL) in 10 mL anhydrous tetrahydrofuran was added dropwise at − 5 °C into a solution of p-hydroxybenzaldehyde (2 g, 0.016 mol) and dried triethylamine (4.5 mL) in 25 mL of anhydrous THF with stirring, the reaction was continued 10 h at room temperature. Then the product was filtered and evaporated under vacuum. The residual organic layer was purified by passing a silica gel column chromatography (eluant: petroleum ether/ethyl acetate = 8:1 v/v). Finally, the product was concentrated and dried in vacuum. (1H NMR spectrum (400 MHz, CDCl3) (Fig. S1): δ 6.08(He,1H), 6.36(Hd,1H), 6.66(Hf,1H), 7.33(Hb,1H), 7.93(Hc,1H), 10.0(−CHO,1H); yield: 75%). BACy was synthesized according to the reported procedure [15,16]. Cystamine dihydrochloride (5.630 g, 0.025 mol) was dissolved in 25 mL deionized water. Then the solution was added into a 250 mL three-necked round bottom flask equipped with a magnetic stirrer. A solution of acryloyl chloride (4.526 g, 0.050 mol) in 5 mL dichloromethane and a NaOH aqueous solution (8.0 g in 10 mL water) were added simultaneously and slowly to the flask within 1 h in ice bath.

2. Experimental 2.1. Materials and reagents p-Hydroxybenzaldehyde (AR, 98%) was purchased from Tianjin Chemical Reagent Co., Tianjin, China. Acryloyl chloride (96%) was

Scheme 1. Schematic illustration of the preparation of prodrug microspheres, their disintegration and DOX release in tumor microenvironment.

25

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Afterward, the reaction mixture was stirred at room temperature for 10 h. The product was filtrated and the filtrate was then extracted with dichloromethane (3 × 50 mL). The organic phase was dried over anhydrous magnesium sulfate and then evaporated under vacuum. The crude product was finally purified by re-crystallization in ethyl acetate. PEGylated polymer microspheres were prepared via an emulsion polymerization approach, as reported previously [17]. Typically, PEGMA (0.12 g, 0.25 mmol), FPA (0.12 g, 0.68 mmol, pre-dissolved in 1 mL DMF) and SDS (0.008 g) were mixed in 36 mL deionized water in a three-necked round bottom flask equipped with a reflux condensing tube and nitrogen gas inlet. After vacuumizing and purging nitrogen constantly for 30 min, the pre-emulsion was heated to 80 °C in an oil bath under magnetic stirring. Then 0.8 mL APS aqueous solution (containing 1 mg APS) was added to initiate the polymerization, followed immediately by adding a solution of BACy (0.012 g BACy in 2 mL DMF). The whole polymerization reaction was conducted under stirring at 80 °C for 8 h. Finally, the PEGylated polymer microspheres were centrifuged at 10000 rpm for 8 min after cooling to room temperature. The obtained product was washed with deionized water three times for purpose of purification. Afterwards, the purified product was dispersed uniformly in 20 mL deionized water. 1.0 mL of the uniform dispersion was taken out to determine its solid content as 8.0 mg/mL, after centrifugation and drying in vacuum. So the yield of the emulsion polymerization could be calculated to be 61%.

where Qt is the amount of drug dissolved in time t, Q0 is the initial amount of drug in the solution (most times, Q0 = 0) and K0 is the zero order release constant expressed in units of concentration/time [20].

Log C = log C0 − K·t 2.303 where C0 is the initial concentration of drug, K is the first order rate constant, and t is the time [20].

Mt = k·t1 2 where Mt is the drug released at time t, and k is the rate constant. Once a plot of Mt/t1/2 is linear with a slope ≥ 1, it is considered to follow the Higuchi drug release kinetics [21].Mt/M∞ = k · tn , Mt/M∞ < 0.6where Mt./M∞ is the drug release fraction at time t; k is a constant; and n is the release exponent. The release model follows Fickian diffusion as n ≤ 0.5, while non-Fickian as 0.5 < n < 1 [22]. 2.5. In vitro cytotoxicity The cytotoxicity of the polymer microspheres and prodrug microspheres was evaluated via MTT assay. First, HepG2 cells were cultivated on 96-well plates (1.0 × 104 cells per well) and the cultures were maintained in an incubator at 37 °C with a standard atmosphere of 5% CO2 for 24 h. Followed by the addition with different concentrations of the polymer microspheres or prodrug microspheres, as well as free DOX. After cultured for 48 h, 20.0 μL of MTT (5.0 mg/mL) was added into each well. After incubation for 4 h, the plates were washed with PBS for three times. Cell viability was measured with a microplate reader at a wavelength of 490 nm.

2.3. Preparation of prodrug microspheres

2.6. Analysis and characterization

Then the prodrug microspheres were prepared by conjugating DOX onto the PEGylated polymer microspheres via acid-labile imine linkage [18]. 6.0 mL of the abovementioned dispersion of the PEGylated polymer microspheres was centrifuged and the solid (48 mg) was redispersed into 1.0 mL DMSO in a 25 mL single neck round bottom flask. 0.074 g DOX·HCl, dissolved in 3 mL of DMSO with an equivalent of triethylamine, was added dropwise to the flask. After stirring for 72 h in the dark at room temperature, the mixture was centrifuged (10,000 rpm for 10 min). The product, prodrug microspheres, was dispersed into deionized water with ultrasonic for 10 min and the supernatant was removed by centrifugation (10,000 rpm for 10 min). This procedure was repeated five times to completely remove DMSO and the excess DOX. The DOX content in the prodrug microspheres was calculated by determining the DOX concentration in the supernatant solution, analyzed with TU-1901 UV–Vis spectrophotometer at its maximum absorbance of 480 nm.

The 1H NMR spectrum of the monomer FPA was characterized by Nuclear Magnetic Resonance instrument (JEOL ECS 400 M) with deuterium chloroform as solvent. FT-IR spectra were recorded on a Nicolet Satellite infrared spectrometer in the range of 400–4000 cm− 1 with a resolution of 4 cm− 1, with KBr pellets. The morphology and size of the microspheres were characterized on a JEM-1200 EX/S transmission electron microscope (TEM) (JEOL, Tokyo, Japan). Samples were dispersed in deionized water in an ultrasonic bath for 10 min, and deposited on a copper grid covered with a carbon film. The hydrodynamic diameters of the microspheres were analyzed by dynamic light scattering (DLS) system constructed on Light Scattering System BI-200SM, Brookhaven Instruments device equipped with the Coherent INOVA 70C argon-ion laser. The scattered laser was collected at a detection angle of 90° during 5 min. DLS analysis had been carried out for more than three times for each samples, with RSD < 5% for the average hydrodynamic diameter (Dh).

2.4. In vitro controlled release The in vitro release profiles of DOX from the prodrug microspheres was investigated at 37 °C under shaking in five different media: (a) acetate buffer (pH 5.0); (b) acetate buffer (pH 6.5); (c) phosphate buffer (pH 7.4); (d) acetate buffer (pH 5.0) with 10 mM GSH; and (e) phosphate buffer (pH 7.4) with 10 μM GSH, respectively. 14.0 mg prodrug microspheres were dispersed into 10 mL of each releasing medium and then immediately transferred to a dialysis tube (molecular weight cut-off of 14,000). The dialysis bag was immersed into 100 mL of corresponding releasing media. At desired time intervals, 5 mL dialysate was taken out and replenished with an equal volume of fresh releasing medium. The amount of the released DOX was determined with TU-1901 UV–Vis spectrophotometer at 480 nm to calculate the accumulative release ratio according to the method reported previously [19]. The zero-order, first-order, Higuchi and Korsmeyer-Peppas release models were used to analyze release data, as below

3. Results and discussion 3.1. Preparation and characterization of the PEGylated polymer microspheres Firstly, 4-formylphenyl acrylate (FPA) was synthesized as a functional hydrophobic monomer for the emulsion copolymerization with PEGMA with BACy as crosslinker, which could introduce aldehyde group in the PEGylated polymer microspheres for conjugating DOX via acid-labile imine linkage (Scheme 1). After the polymerization, the product showed spherical morphology with particle size in 40–100 nm from the TEM analysis (Fig. 1a). Owing to the swelling effect, its average hydrodynamic diameter (Dh) was 150 nm with near monodisperse distribution from the DLS result (Fig. 2a). The characteristic absorbance of aldehyde group at 1698 cm− 1 in FPA units [18], CeOeC stretching band at 1122 cm− 1 in PEGMA units [23], and SeS stretching

Qt = Q0 + K 0·t 26

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Fig. 1. TEM images of the PEGylated microspheres (a) and prodrug microspheres (b).

100

100

(a)

(b)

80

Intensity

Intensity

80 60 40 20

60 40 20

0 100

120

140

160

180

0 120

200

100

100

(c)

80

180

210

240

270

(d)

80

Intensity

Intensity

150

Hydrodynamic diameter (nm)

Hydrodynamic diameter (nm)

60 40 20

60 40 20

0 100

120

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0 80

160

100

90

100 110 120 130 140 150

Hydrodynamic diameter (nm)

Hydrodynamic diameter (nm)

(e)

Intensity

80 60 40 20 0 60

80

100

120

140

Hydrodynamic diameter (nm) Fig. 2. Typical hydrodynamic diameters of the PEGylated microspheres (a), prodrug microspheres (b), and the PEGylated microspheres in 10 mM GSH solution for 12 h (c), 24 h (d), and 48 h (e).

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100

Transmittance (%)

90

Table 1 DOX content and releasing performance of the reported pH/reduction dual-responsive prodrugs.

507

Conjugating bond

PEGylated microspheres

80

1698

Final accumulative release ratio (%)

Refs.

pH 7.4 + μM GSH

pH 5.0 + 10 mM GSH

3–4 15.9 6.2

15 (pH 7.4) 0 (pH 7.4) 35 (pH 7.4)

60, 24 h 56 (with DTT), 48 h 95 (with DTT), 150 h

[26] [27] [28]

39.0 13.0

47.6 (pH 7.4) 30 (pH 7.4)

[29] [30]

22.7 44.4

15.5 16.6

88.4, 72 h 93 pH (5.0 + 10 mM GSH), 72 h 58.4, 58 h 73.3, 60 h

1122 Hydrazone Schiff base cis-Aconityl linkage Hydrazone cis-Aconityl linkage Schiff base Schiff base

Prodrug microspheres

70

1666

60

DOX content (%)

4000 3500 3000 2500 2000 1500 1000 Wavenumber (cm-1 )

[31] The work

500

80

Accumulative release ratio (%)

Fig. 3. FT-IR spectra of the PEGylated microspheres and prodrug microspheres. −1

band at 507 cm in the crosslinker BACy [24] appeared in its FT-IR spectrum (Fig. 3), indicating the successful preparation of the PEGylated polymer microspheres via the emulsion copolymerization of FPA and PEGMA with BACy as crosslinker. To reveal the bioreducible degradability of the PEGylated polymer microspheres, they were treated with pH 5.0 acetate buffer solution containing 10 mM GSH, with concentration of 1.6 mg/mL for different times. From the DLS analysis (Fig. 2c–e), the average hydrodynamic diameter (Dh) decreased as time goes on to approximately 135 nm, 115 nm, and 98 nm within 12 h, 24 h, and 48 h finally. It should be resulted from the dissolution of some de-crosslinked polymers during the bioreducible degradability, due to the cleavage of disulfide crosslinking structure. The result demonstrated that the PEGylated polymer microspheres could disintegrate in presence of reductant, such as GSH. The feature is expected to be beneficial to the diffusion of DOX out of the prodrug microspheres, as well as the metabolism of drug carriers.

70 60 pH 5.0 pH 6.5 pH 7.4 pH 5.0+10mM GSH pH 7.4+10µM GSH

50 40 30 20 10 0 0

10

20

30

40

50

60

Time (h) Fig. 4. Accumulative release of DOX from the prodrug microspheres in different media.

ing the pH triggered release property owing to the acid-labile imine conjugating bond. Furthermore, the accumulative release ratio increased to 73.3% and 16.6% in the media at pH 5.0 with 10 mM GSH and pH 7.4 with 10 μM GSH, mimicking the tumor microenvironment and physiological medium respectively. It indicated that the drug release rate was accelerated in the acidic and reductive media, exhibiting the reduction triggered release characteristics resulted from the acid-labile imine conjugation of DOX and bioreducible disulfide crosslinking structure, which could be cleaved off in acidic media with high GSH level. Therefore, the conjugated DOX was cut off from the carriers and the prodrug microspheres disintegrated, which is favorable to the DOX diffusion out of the carriers. The high drug content and the pH/reduction dual-stimuli triggered release performance of the proposed prodrug microspheres make them potential DDS with reduced drug leakage during blood circulation. Especially in the simulated tumor microenvironment (pH 5.0 with 10 mM GSH), 62% of DOX was released within the first 12 h, and the residual was released in a sustained mode in the last 48 h. The feature might be beneficial to a high intracellular drug concentration, as a result, better anti-cancer efficiency is desired. Since the PEGylated polymer microspheres could not disintegrate completely within 48 h from the DLS analysis (Fig. 2), and the drug release was not complete within 60 h in the releasing medium of pH 5.0 with 10 mM GSH although a sustained release behavior had been found in the last 48 h (Fig. 4), it is speculated that a longer releasing time is needed for the complete disintegration of the polymer microspheres. In order to verify this, 12 mg prodrug microspheres were dispersed in 5.0 mL pH 5.0 acetate buffer solution containing 10 mM GSH mimicking the tumor microenvironment for a longer time. It was found that the

3.2. Preparation and characterization of the prodrug microspheres Then the pH/reduction dual-responsive prodrug microspheres were prepared by conjugating DOX onto the PEGylated polymer microspheres via acid-labile imine linkage. A new signal at 1666 cm− 1, which is assigned to C]N bond as connection between carrier and drug [25], appeared in the FT-IR spectrum of the product, revealing the successful conjugation via imine linkage (Fig. 3). The particle size and average hydrodynamic diameter (Dh) of the prodrug microspheres increased to 121 nm and 205 nm, from the TEM (Fig. 1b) and DLS (Fig. 2b) analysis respectively, owing to the conjugation of the hydrophobic anticancer drug DOX. The DOX content was determined to be 44.4% in the proposed prodrug microspheres. It is the highest value in comparison with the reported pH/reduction dual-responsive nano-prodrugs (Table 1). In the present work, FPA was the main monomer for the PEGylated polymer microspheres, so there were plentiful aldehyde groups in the PEGylated polymer microspheres to achieve such high drug content. With such high DOX content, the dosage of the prodrug could be decreased. So the proposed prodrug microspheres with high drug content are expected to be promising chemotherapeutics for cancer treatment. 3.3. In vitro controlled release performance The in vitro controlled release performance of DOX from the prodrug microspheres was then compared in different simulated media at 37 °C. There was no burst release in all the releasing curves (Fig. 4). The accumulative release ratios were 69%, 26% and 12% at releasing media of pH 5.0, pH 6.5, and pH 7.4 within 60 h, respectively, indicat28

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Fig. 5. Digital photographs of the prodrug microspheres dispersed in simulated tumor microenvironment before (A) and after 72 h (B).

dispersion became transparent solution within 72 h (Fig. 5), meaning the designed prodrug microspheres could disintegrate completely within 72 h. Then the zero-order, first-order, Higuchi and Korsmeyer-Peppas models were used to fit the accumulative release (Figs. S2 and S3), and the correlation coefficients (R2) were used to evaluate the fitting accuracy (Tables 2 and 3). All the values of R2 in the zero-order and first-order models were lower than 0.75, indicating the two models were not applicable to such microgels in the present work [32,33]. For the Higuchi and Korsmeyer-Peppas models (Fig. S3) [34], the linear regression analysis was applied to calculate the release rates of each model and the coefficients of correlation (R2) were used to evaluate the accuracy of the fitting. As listed in Table 3, most of the of R2 data were higher than 0.94, demonstrating the ideal linear coefficients in all cases. In the Higuchi model, the DOX release was diffusion-controlled in acidic releasing media (pH 6.5 or pH 5.0 with or without GSH), while the DOX release in weak basic medium was nondiffusion controlled release with k < 1 [35]. As for the KorsmeyerPeppas model, the release was non-Fickian diffusion in all cases because of the data of n > 0.5 [36].

Table 3 Fitting results with Higuchi and Korsmeyer-Peppas models. Conditions

2

pH 5.0 pH 6.5 pH 7.4 pH 5.0 + 10 mM GSH pH 7.4 + 10 mM GSH

k

R2

n

0.9689 0.9356 0.9983 0.9684 0.9925

2.5217 1.0045 0.4634 2.7288 0.6509

0.9419 0.9083 0.9564 0.9455 0.9530

1.0071 1.4177 1.0750 1.0290 1.1761

DOX Polymer microspheres Prodrug microspheres

Cell Viability (%)

80 60 40 20 0 -5

0

5

10

15

20

25

30

35

Dosage (µg/mL) Fig. 6. Cell viability assay in HepG2 cells of the bare polymer microspheres, prodrug microspheres and free DOX. Cell viability (RSD < 5%, n = 3) was evaluated with MTT assay and normalized against blank samples which were cultured without the microspheres.

Table 2 Fitting results with zero-order, first-order models.

pH 5.0 pH 6.5 pH 7.4 pH 5.0 + 10 mM GSH pH 7.4 + 10 μM GSH

R

100

Considering the possible application of the prodrug microspheres in cancer treatment, cellular toxicity is one of the most important indicators to measure the performances of the DDS, so the in vitro cytotoxicity of the proposed prodrug microspheres was evaluated in HepG2 cells using MTT assays, in comparison with those of the bare

Zero-order

Korsmeyer-Peppas

120

3.4. In vitro cytotoxicity

Conditions

Higuchi

First-order

R2

K0

R2

K

0.2738 0.6462 0.7293 0.6104 0.6956

0.0096 0.0041 0.0019 0.0102 0.0027

0.3203 0.3462 0.5165 0.3169 0.4765

0.0142 0.0141 0.0130 0.0144 0.0154

polymer microspheres and free DOX. As shown in Fig. 6, the bare polymer microspheres possessed very low toxicity, with cell viability of > 95% in the studied dosage range. It demonstrated that the bare polymer microspheres were cytocompatible. To further study the inhibition effect of the prodrug microspheres against cancer cell growth, the cell viability of free DOX and the 29

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prodrug microspheres were compared. The prodrug microspheres displayed obvious inhibition efficacy against HepG2 cells with dosage > 15 μg/mL, with cell viability of 78%. Increasing the dosage of the prodrug microspheres to 30 μg/mL, the cell viability decreased to 57%, while the cell viability was 64% or 39% for the free DOX with the same dosage, respectively. Consideration of the cumulative release of 68% from the prodrug microspheres within 24 h, the prodrug microspheres exhibited a higher inhibition against cancer cell growth than the free DOX in fact. It might be due to the fact that free DOX molecules transported into cells by passive diffusion, whereas the prodrug microspheres can translocate across the biological membrane by endocytosis, which is more efficient and rapid than diffusion [37].

[10]

[11]

[12]

[13]

[14]

4. Conclusions

[15]

In summary, a novel monomer, 4-formylphenyl acrylate (FPA), was synthesized in the present work for novel pH/reduction dual-responsive prodrug microspheres for tumor intracellular triggered DOX release, in which the bioreducible disulfide crosslinked carrier was synthesized by emulsion copolymerization of FPA and PEGMA with BACy as crosslinker, followed by conjugating DOX via acid-labile imine linkage. The prodrug microspheres with high drug content of 44.4% showed average hydrodynamic diameter of 205 nm. The in vitro release profiles demonstrated the excellent good tumor intracellular triggered release performance with reduced drug leakage during blood circulation, and the prodrug microspheres could disintegrate completely within 72 h in the simulated tumor microenvironment (pH 5.0 with 10 mM GSH). The MTT assay demonstrated that bare polymer microspheres were cytocompatible, while the prodrug microspheres exhibited the enhanced inhibitory efficiency against HepG2 cells in comparison with free DOX. These features make the proposed prodrug microspheres potentially promising chemotherapeutics for cancer treatment.

[16]

[17]

[18]

[19]

[20] [21] [22] [23]

[24]

Acknowledgment [25]

This project was granted financial support from the Program for New Century Excellent Talents in University, Ministry of Education of China (Grant No. NCET-09-0441).

[26]

[27]

Appendix A. Supplementary data [28]

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.reactfunctpolym.2017.05.002. [29]

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