Versatile polymeric microspheres with tumor-microenvironment

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Versatile polymeric microspheres with tumor-microenvironment bioreducible degradation, pH-activated surface charge-reversal, pH-triggered “offon” fluorescence and drug release as theranostic nanoplatforms Mingliang Pei, Xu Jia, Guoping Li, and Peng Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00957 • Publication Date (Web): 07 Dec 2018 Downloaded from http://pubs.acs.org on December 9, 2018

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Molecular Pharmaceutics

Versatile polymeric microspheres with tumormicroenvironment bioreducible degradation, pHactivated surface charge-reversal, pH-triggered “offon” fluorescence and drug release as theranostic nanoplatforms Mingliang Pei, Xu Jia, Guoping Li, and Peng Liu *

State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China

ABSTRACT: Facile approach has been developed for the versatile polymeric microspheres with tumor-microenvironment bioreducible degradation, pH-activated surface charge-reversal, pHtriggered “off-on” fluorescence and drug release via emulsion copolymerization of glycidyl methacrylate (GMA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), N-rhodamine 6G-ethyl-acrylamide (Rh6GEAm) with N,N-bis(acyloyl)cystamine) (BACy) as disulfide crosslinker and functionalization. The final PGMA-DMMA microspheres showed excellent

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cytocompatibility, pH-triggered surface charge reversal at pH 5-6, strong fluorescence only in acidic media, and bioreducible degradation with high reductant level, indicating their promising application

as

theranostic

nanoplatforms

for

precise

imaging-guided

diagnosis

and

chemotherapy. The DOX-loaded PGMA-DMMA microspheres with a drug-loading capacity of 18% and particle size of about 150 nm possessed unique pH/reduction dual-responsive controlled release, with a cumulative DOX release of 60.5% within 54 h at the simulated tumor microenvironment but a premature leakage of < 8.0% under the simulated physiological condition. Enhanced inhibition efficacy against HepG2 cells was achieved than the free DOX.

Keywords: theranostic nanoplatforms; polymeric microspheres; tumor-microenvironment bioreducible degradation; pH-activated surface charge-reversal; pH-triggered “off-on” fluorescence; pH-triggered drug release

INTRODUCTION Theranostic nanoplatforms, which are recognized for imaging-guided diagnosis and chemotherapy of cancer, have attracted more and more interest by integrating imaging function into the drug delivery systems (DDSs) in the last decades.1 By now, various imaging reporters, such as optically active small molecules,2 magnetic nanoparticles,3


2

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fluorescent carbon dots,4 or metallic nanoclusters,5 have been intensely investigated for fluorescence imaging, magnetic resonance imaging (MRI), ultrasound imaging, or photoacoustic imaging.6 However, most of them showed imaging function in both the normal physiological medium and the tumor microenvironment, due to their lack of specificity to the tumor-microenvironment. So, only the biodistribution could be achieved with such imaging reporters, while the in-situ real-time fluorescent monitoring of therapeutic response could be realized to a certain extent, by means of tumor-targeting groups.7 The unique “off-on” fluorescent behaviors in normal physiological medium and tumormicroenvironment of the fluorescence imaging groups are expected to provide a precise imaging-guided diagnosis and chemotherapy,4 which could show strong fluorescence only in tumor-microenvironment triggered by the intracellular acidic media8 or high reductant level9 but no fluorescence emission in normal physiological medium, or the fluorescence emission length alters with changing the media pH value.10 Owing to its strong fluorescence only in the acidic media, rhodamine 6G (Rh6G) has been widely used as a pH sensitive fluorochrome in the theranostic nanoplatforms, such as


3


copolymer

micelles,11

double-cross-linked

hyaluronic

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acid

nanoparticles,12

and

multifunctional magnetic nanoparticles.13 These theranostic nanoplatforms exhibit the cancer-associated, stimuli-driven and turn-on features for the precise in-situ real-time fluorescence imaging-guided diagnosis and chemotherapy.14,15 On the other hand, the cellular uptake and tumor-specific intracellular triggered drug release are the main factors affecting the antitumor efficacy. The DDSs are expected to be surface negatively charged to prolong the circulation time, but a weakly positively charged surface is preferable for the cellular internalization. It means a surface-adaptive characteristic is desired for the smart DDSs, mainly via the pH-responsive16,17 or pHactivated18 surface charge-reversal. Furthermore, the pH- triggered drug release responding to the intracellular acidic media could achieve a tumor-targeted drug release with minimized premature leakage during blood circulation.19


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Scheme 1. Schematic illustration of the synthesis of PGMA-DMMA/DOX microspheres.


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In the present work, versatile polymeric microspheres integrating the unique desirable features, tumor-microenvironment bioreducible degradation, pH-activated surface charge-reversal and pH-triggered “off-on” fluorescence and drug release, were designed as theranostic nanoplatforms for precise imaging-guided diagnosis and chemotherapy (Scheme 1). Here, the functional poly(glycidyl methacrylate) (PGMA) microspheres were synthesized via the emulsion copolymerization of glycidyl methacrylate (GMA), poly(ethylene glycol) methyl ether methacrylate (PEGMA), fluorescent monomer N-rhodamine 6G-ethyl-acrylamide (Rh6GEAm) with N,Nbis(acyloyl)cystamine) (BACy) as disulfide crosslinker. PEGMA was used for the PEGylation of the resultant polymer microspheres. The fluorescent monomer Rh6GEAm was selected to endow the microspheres pH-triggered “off-on” fluorescence. The disulfide crosslinkage

with

BACy

could

be

bioreducible

cleaved

off

in

the

tumor-

microenvironment triggered by the high reductant level. GMA was selected to introduce epoxy groups for further functionalization: ring-opening reaction with ethyldiamine (EDA) and amidation with 2,3-dimethylmaleic anhydride (DMMA). Thus, the carboxyl groups in


6

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the final PGMA-DMMA microspheres could be used to load doxorubicin (DOX) via electrostatic interaction for the pH-triggered release and the β-carboxylic amide groups could be hydrolyzed to realize the pH-activated surface negative-to-positive chargereversal.

EXPERIMENTAL SECTION Materials and reagents. Glycidyl methacrylate (GMA, 99%) and poly(ethylene glycol) methylether methacrylate (PEGMA, Mn = 475) were purchased from Sigma Co. Ltd and Shanghai Aladdin Reagent Co., Shanghai, China respectively, and were purified by passing through a basic alumina column before use. Sodium dodecyl sulphate (SDS, C.P.) was bought from Shanghai Shiyi Chemical Reagents Ltd. Aammonium persulphate (APS, A.R. 98%) was purchased from Kelong Chemical Reagent Factory. Rhodamine 6G (Rh6G) was got from Dongsheng Chemical Reagent Company. Acryloyl chloride (96%) was obtained from Tianjin Heowns Company. Ethylenediamine (EDA, 99%) and 2,3-dimethylmaleic anhydride (DMMA, 97%) were achieved from Tianjin Guangfu Fine Chemical Research Institute and San Chemical Technology Co., Ltd., respectively. Doxorubicin hydrochloride (DOX•HCl, 98%) was obtained from Beijing Huafeng Lianbo Technology Co, Ltd. Beijing, China. Glutathione (GSH, 97%) was obtained from Shanghai Aladdin Reagent Co., Shanghai, China. N, N-Dimethylformamide (DMF, A.R. 99.5%) and 1,4-dioxane (99%) were achieved from Tianjin Chemical Reagent II Co. and used without further purification. Other reagents were commercially available and used as received. Double distilled water was used throughout.


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Functional PGMA microspheres. N-Rhodamine 6G-ethyl-acrylamide (Rh6GEAm)11 and N,N-bis(acyloyl)cystamine) (BACy)20 were synthesized with the procedure reported previously. The functional PGMA microspheres were prepared via a facile emulsion polymerization as follows:21 GMA (0.1423 g, 1.0 mmol) and PEGMA (0.1431 g, 0.3 mmol) were dissolved in 1.0 mL of DMF. Then the solution was added into 37 mL of aqueous solution containing SDS (0.0111 g, 0.038 mmol) in a three-necked round bottom flask. After the mixture was heated to 80 ºC with stirring under N2 condition, APS (0.0017 g (0.0074 mmol), 0.0034 g (0.0148 mmol), or 0.0085 g (0.0370 mmol)) pre-dissolved in 0.8 mL of water, and BACy (0.017 g, 0.065 mmol) and GR6GEAm (0.013 g, 0.029 mmol) pre-dissolved in 2.0 mL DMF were added. The polymerization was conducted at 80 ºC with stirring under N2 condition for 8 h. Finally, the functional PGMA microspheres were collected by centrifugation (12000 rpm for 10 min), thoroughly washed with water, and dried under vacuum at 40 ºC. PGMA-DMMA microspheres. The amino groups were introduced into the functional PGMA microspheres with EDA. The PGMA microspheres (0.10 g) was dispersed into 6 mL of 1,4-dioxane, then EDA (5.39 g, 0.09 moL) was added and stirred for 8 h at 80 ºC. The resultant PGMA-EDA microspheres were collected by centrifugation (12000 rpm for 10 min), thoroughly washed with water, and dried under vacuum at 40 ºC. The final PGMA-DMMA microspheres were prepared by reaction of PGMA-EDA microspheres with DMMA. The PGMA-EDA microspheres were dispersed into water with ultrasonication. Then DMMA (0.631g, 5 mmol) was added, and the reaction was continued at room temperature for 48 h. The final PGMA-DMMA microspheres were collected by centrifugation (12000 rpm for 10 min), thoroughly washed with water, and dried under vacuum at 40 ºC.


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DOX-loaded PGMA-DMMA microspheres. Briefly, 10 mg of PGMA-DMMA microspheres were dispersed in 2 mL water, and then 2.0 mL of 2.0 mg/mL DOX aqueous solution was added with stirring. The pH value of the mixture was adjusted to 7.4 with 0.10 mol/mL aqueous NaOH solution, followed by incubating at the room temperature for 48 h in the dark. The PGMADMMA/DOX microspheres were collected by centrifugation (12000 rpm for 10 min) and thoroughly washed with water until no absorbance at 480 nm. The DOX concentration in the fluid supernatant was measured using a UV-vis spectrophotometer at 480 nm, to calculate the drug-loading capacity (DLC), as the mass ratio of the loaded DOX and the carrier microspheres. In vitro triggered drug release profiles. The in vitro DOX release from the PGMADMMA/DOX microspheres were conducted using a dialysis method. Briefly, 10 mL 2.0 mg/mL dispersion of the PGMA-DMMA/DOX microspheres was placed in a dialysis bay with MWCO of 14 kDa, and then it was transferred into a bottle containing 110 mL of release buffer solution to simulate the diverse physiologic environment at 37 ºC with shaking at 120 rpm. At a predetermined time interval, 5.0 mL of dialysate was taken to measure the DOX concentration with UV-Vis spectrophotometer at 480 nm. 5.0 mL of corresponding fresh buffer solution was added to maintain volume constant. Finally, each sample of cumulative release profiles were plotted versus release time. In vitro cellular toxicity. The cytotoxicity of the blank PGMA-DMMA microspheres were evaluated via MTT assay on a normal cell line (293 cells) and a cancer cell line (HepG2 cells) after incubation of 48 h, respectively. The cytotoxicity of the PGMA-DMMA/DOX microspheres were evaluated via MTT assay on three cancer cell lines (HepG2 cells, A549 cells and MCF-7 cells) after incubation of 48 h, respectively. The cells cultivated on 96-well plates (1.0×105 cells per well) in 100 μL DMEM containing 10% FBS and incubated in atmospheric humidity (5% CO2, 95 % air, 37 ºC) for 24 h, and then


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different concentrations of free DOX, blank PGMA-DMMA or PGMA-DMMA/DOX microspheres were added. After cultured for 24 h, 20.0 L of MTT (5.0 mg/mL) was added into each well, incubated for 4 h and washed with PBS for three times. The absorbance of all solution was evaluated at 490 nm on a microplate reader. All cytotoxicity results were obtained from five independent measurements and are expressed as mean value ± SD. In vitro cellular uptake. The cellular uptake of the blank PGMA-DMMA microspheres, the PGMA-DMMA/DOX microspheres and free DOX was exhibited though confocal fluorescence microscope (DMI 4000B, LEICA, Germany) using HepG2 cells after incubation for different times. The cell nuclei were stained with by the DAPI. The location of cellular fluorescence was validated with excitation wavelength of 480 nm (DOX) and 405 nm (DAPI), respectively. Flow cytometric analysis. The flow cytometry was used to monitor cell apoptosis by measuring the cell associated fluorescence stained with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI) by a flow cytometer (BD FACSCalibur), after 5 µg/mL DOX of free DOX, 5 µg/mL DOX of the blank PGMA-DMMA microspheres or the 5 µg/mL DOX equiv/mL of the PGMADMMA/DOX microspheres were added to the HepG2 cell and incubated at 37 °C for 0.5 and 8 h, respectively. Characterization. Fourier transform infrared (FTIR) spectra were recorded by using a Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany). The steady-state emission spectra of the microspheres were measured at different pH media with a Hitachi F-4500 spectrofluorometer. The morphology of the microspheres was examined with a JEM-1200 EX/S transmission electron microscope (TEM) (JEOL, Tokyo, Japan). The samples were dispersed in water with ultrasonication for 3 h at first, drops of the uniform dispersion were deposited on a copper grid covered with a perforated carbon film.


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The mean hydrodynamic diameter and size distribution of the samples were determined by a dynamic light scattering system (BI-200SM, Brookhaven Instruments) using 135 mW intense laser excitation at 514.5 nm at a detection angle of 90° at room temperature. The zeta potentials of the microspheres were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd, UK) in various pH media. The concentration of DOX was measured by a Lambda 35 UV−vis spectrometer at 480 nm at room temperature.

RESULTS AND DISCUSSION

Preparation and characterization of functional PGMA microspheres. After the emulsion copolymerization with different amounts of initiator, the functional PGMA microspheres were obtained. Decreasing the feeding amount of initiator from 0.0085 g to 0.0034 g and 0.0017 g, their average hydrodynamic diameter (Dh) of the resultant microspheres decreased from 430 nm to 283 nm and 210 nm with PDI of 0.230, 0.231 and 0.173, respectively (Figure 1 a-c). So the sample 3, the functional PGMA microspheres prepared with 0.0017 g APS, was selected for further investigation, due to their smaller diameter of 210 nm and narrower distribution.

100

100

(a)

Intensity (%)

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40

80 60 40 20

20 0

(b)

0

100 200 300 400 500 600 Diameter (nm)

0

0

100 200 300 400 500 600 Diameter (nm)


11


(c)

100

80

Intensity (%)

Intensity (%)

100

60 40 20 0

40

0

100

150 300 450 Diameter (nm)

0

100 200 300 400 500 600 Diameter (nm) (d)

80 60 40

0

600

(e)

0

80 60 40 20

150

300 450 Diameter (nm)

600


1000

(f)

100 Intensity (%)

Intensity (%)

40

20

20

80 60 40 20

0

150

300 450 Diameter (%)

0

600

Intensity (%)

80 60 40

0

200

(h)

100

(g)

100

80 60 40 20

20 0

60

100

60

0

80

0

100 200 300 400 500 600 Diameter (nm)

Intensity (%)

Intensity (%)

0

80

0

(d)

20

(c)

100

Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0

2500 5000 7500 10000 12500 Diameter (nm)

0

0

150


750


12

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Figure 1. Typical hydrodynamic diameter distributions of the PGMA-1 (a), PGMA-2 (b), PGMA-3 (c), PGMA-EDA (d), and PGMA-DMMA (e) microspheres, and the PGMADMMA microspheres treated with 10 mM GSH for 4 h (f), 8 h (g), 32 h (h) and 56 h (i), and final PGMA-DMMA/DOX microspheres (j).

In the FT-IR spectrum (Figure 2), the characteristic absorbance of the C=O stretching bond in ester groups of GMA and PEGMA units and the C-N stretching in amide groups of Rh6GEAm and BACy units appeared at 1730 cm-1 and 1520 cm-1, respectively. Furthermore, the C-O-C anti-symmetric stretching band in PEG segment, epoxy group in GMA units, and phenyl band in Rh6GEAm units could be seen at 1149 cm-1, 908 cm 1

and 748 cm

−1,

respectively. The results demonstrated that the functional PGMA

microspheres had been successfully synthesized by the emulsion copolymerization. For further quantitatively estimate the content of fluorescent monomer Rh6GEAm, the content of the fluorescent unit (Rhodamine 6G) in the functional PGMA microspheres was measured to be 1.30% with a UV−vis spectrometer at 527 nm.


13


A

B

C

4000

3200 2400 1600 Wavenumber (nm)

800

Figure 2. FT-IR spectra of the PGMA (A), PGMA-EDA (B), and PGMA-DMMA (C) microspheres.

pH 4 pH 5 pH 6 pH 7 pH 9 pH 11

500 400 Intensity (a.u)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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300 200 100 0 550

575

600 625 Wavelength (nm)

650

Figure 3. Fluorescence emission spectra (λex = 510 nm) of the PGMA microspheres (0.65 mg/mL) as a function of the media pH values. The inset is the image of the microspheres in acidic (pH 5.0) (a) and neutral (pH 7.4) (b) solutions under 365 nm UV light. Then the fluorescent property of the functional PGMA microspheres were investigated, by measuring their dispersions in aqueous solution with different pH values with a solid content of 0.65 mg/mL. As shown in Figure 3, the dispersions in acidic media showed strong fluorescence


14

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emission with an excitation wavelength of 510 nm, while the fluorescent intensity was very low in the neutral and basic media. It demonstrated the pH-modulated fluorescent property of the functional PGMA microspheres, because the nonfluorescent spiro structure transforms reversibly into the fluorescent ring-opened amide in acidic environment.11 The dispersion in pH 5.0 medium showed strong orange fluorescence under 365 nm UV light, distinctly different from the dispersion in pH 7.4 medium (the insert in Figure 3). The results indicated the pH-triggered “offon” cancer-associated, stimuli-driven and turn-on fluorescent property of the functional PGMA microspheres, which are promising for the precise in-situ real-time fluorescence imaging-guided diagnosis and chemotherapy. Preparation and characterization of PGMA-DMMA microspheres. After the ringopening reaction with ethyldiamine (EDA), the characteristic absorbance of the epoxide ring vibration at 908 cm-1 completely disappeared (Figure 2),22 indicating that epoxy group was fully consumed with EDA. The ring-opening reaction would result a more hydrophilic nature of the microspheres, so they should swell better than the functional PGMA microspheres in water, showing a bigger Dh than the later one. However, the DLS analysis showed that the Dh decreased after the ring-opening reaction (Figure 1 c and d). It might be resulted from the inevitable crosslinking reaction of the epoxy groups with EDA. Then the resultant primary amino groups were amidated with 2,3-dimethylmaleic anhydride (DMMA). The PGMA-EDA microspheres showed sphere shaped with particle size of 140 nm from the TEM analysis (Figure 4), and their Dh increased from 183 nm of the functional PGMA microspheres to 266 nm (Figure 1e). The absorbance peaks of the N-H bending and C-N stretching in amide group at 1446 and 1562 cm-1 increased obviously (Figure 2), demonstrating the successful amadiation. Furthermore, the absorbance peak at 3410 cm-1 became broader after the ring-opening reaction, and much broader centered at


15


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3430 cm-1 after the amidation, due to the generation of hydroxyl and amino groups in the ring-opening reaction, and sebsequent carboxyl groups in the amidation with DMMA. These results demonstrated the successful synthesis of the PGMA-DMMA microspheres, as shown in Scheme 1.

(a)

(b)

Figure 4. TEM images of the PGMA-DMMA microspheres (a) and the PGMA-DMMA/DOX microspheres (b).

Then the pH responsive property of the PGMA-DMMA microspheres was investigated with DLS technqiue, after dispersing them into the solution with different pH values for 24 h. The Dh of the PGMA-DMMA microspheres increased from pH 7 to 10 (Figure 5a), due to the deprotonation of the carboxylic acid groups into carboxylate ion. While decreasing the medium pH to 6, the Dh decreased distinctly. The Dh remained constant in pH 5-6 and increased at pH 4. It should be resulted from the hydrolysis of the amide groups, in which DMMA molecules shedded and the regained amino groups protonated at the acidic media. Considering the effect of ion strength on the Dh of the microspheres in the dispersions, the


16

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DLS analysis results could only reveal the pH responsive property of the PGMA-DMMA microspheres, but not the direct evidence of the pH-activated surface charge-reversal.

a

Zeta potential (mV)

400 320 Dh (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


240 160 80 4

5

6

7 pH

8

9

10

20

b

10 0 -10 -20

3

4

5

6 pH

7

8

9

Figure 5. (a) Variation of hydrodynamic diameter of PGMA-DMMA microspheres (0.01 mg/mL) in pH value. Mean ± S.D. (n=3), and (b) Zeta potential of the PGMA-DMMA microspheres at various pH value conditions. Mean ± S.D. (n=5).

The zeta potentials of the PGMA-DMMA microspheres increased from –(15.8±0.7) mV to 20.7±1.0 mV as the media pH value decreased from to 9.0 to 3.0 (Figure 5b), with surface charge-reversal in the range of pH 5-6. The result indicated that the PGMA-DMMA microspheres possessed the pH-activated surface negative-to-positive charge-reversal feature via the acid-triggered hydrolysis of the β-carboxylic amide groups in slightly acidic media, i.e. the tumor extracellular environments (pH ~ 6),23 which may synergistically enhance cellular internalization via electrostatic interaction with cell membrane.16 Then the bioreducible cleavage of the disulfide crosslinkage in the PGMA-DMMA microspheres was investigated by treating them with 10 mM GSH for different times. As shown in Figure 1 e-i, the hydrodynamic diameter increased in the first 8 h, because of the bioreducible cleavage of the disulfide crosslinkage which resulted a lower and lower crosslinking degree in the microspheres. Then the hydrodynamic diameter showed two peaks


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centered at 124 nm and 506 nm after 32 h (Figure 1 h), indicating the fracture of the microspheres. Further increasing the treating time to 56 h, the hydrodynamic diameter showed three peaks centered at 0.7 nm, 450 nm and 6910 nm (Figure 1 i). The nanoscale fragments should be the water-soluble polymers generated from the bioreducible degradation of the PGMA-DMMA microspheres, while the microscale ones should be the degraded products with very low crosslinking degree, maybe the crosslinkage formed by the ringopening reaction of two epoxy groups in different polymer chains with one EDA molecule. Such reduction-triggered de-crosslinking is desirable for the tumor-specific intracellular drug delivery with low premature drug leakage during blood circulation.24 DOX-loading and in vitro controlled release performance. Owing to the excellent features such as tumor-microenvironment bioreducible degradation, pH-activated surface charge-reversal and pH-triggered “off-on” fluorescence, the PGMA-DMMA microspheres are expected as promising theranostic nanoplatforms for tumor treatment. After DOX-loading via the electrostatic interaction, the particle size and Dh of the PGMA-DMMA/DOX microspheres increased to 144 nm (Figure 4) and 334 nm (Figure 1 j) with a DLC of 18%. Then the in vitro controlled release of the PGMA-DMMA/DOX microspheres was evaluated under different conditions: phosphate buffered saline (PBS) at pH 7.4, acetate buffered solution (ABS) at pH 5.0, PBS at pH 7.4 + 10 M GSH mimicking the normal physiological medium and ABS at pH 5.0 + 10 mM GSH mimicking the tumor microenvironment. No obviously burst release could be seen for all profiles, as shown in Figure 6. At pH 7.4, the cumulative release increased to 5.3% within the first 12 h, and almost remained in further 42 h. It should be the release of the DOX molecules loaded in the skin layer of the microspheres. At the simulated normal physiological medium, the cumulative release increased slowly in the whole process and finally reached 8.0%, due to the reduction-triggered de-crosslinking to a certain extent with low


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reductant level. The severe toxic and side effects of the chemotherapeutic drug DOX on the normal cells and tissues could be avoided efficiently with the proposed theranostic nanoplatform owing to the low premature drug leakage.

Cumulative release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 40

pH 5.0 + 10 mM GSH pH 5.0 pH 7.4 + 10 uM GSH pH7.4

20 0

0

15

30 Time (h)

45

60

Figure 6. Cumulative drug release profiles of DOX from the PGMA-DMMA/DOX microspheres in the presence and absence of GSH under different pH media. Mean ± SD (n=3).

As for the pH 5.0 releasing medium, the cumulative release increased to ~15% within the first 12 h, and increased slowly and finally reached 16.2% in further 42 h. The drug releasing rate was faster than that at pH 7.4, showing pH-responsive property due to the two factors: i) DOX could be protonated at such acidic media and the protonated DOX has higher solubility;25 ii) the pHactivated surface charge-reversal in which the DMMA structure, the bridged linkage between the drug and the carrier, was cleaved off. In the simulated tumor microenvironment, a sustained DOX release was achieved with a cumulative release of >60% in 54 h. In such case, the cumulative release was much higher than that at pH 5.0 without GSH, due to the efficiently decrosslinking of the microspheres triggered by the high GSH level, besides the acidity effects


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abovementioned. Such pH/reduction dual-responsive tumor-specific intracellular triggered drug release property is expected for a high antitumor efficacy. To further investigate the release mechanism, the in vitro controlled release profiles in different media were fitted with the Higuchi and Korsmeyer-Peppas equations (Figure S1). The values of the regression coefficient (R2), kinetic constant (k), and release exponent (n) are summarized in Table 1. All the correlation coefficients from the Higuchi model were higher than those from the Korsmeyer-Peppas model, demonstrating the DOX release was mainly controlled by diffusion.26 As for the Korsmeyer-Peppas model, all the values of the release exponent (n) were higher than 0.85 in all three releasing media, demonstrating the non-Fickian diffusion mechanism, such as Super case II transport involving erosion of the matrices.27

Table 1. Fitted DOX release parameters with Higuchi and Korsmeyer-Peppas models. KorsmeyerHiguchi Peppas

Conditions R2

k

R2

n

pH 7.4

0.9691 0.285

0.8740

1.508

pH 7.4 + 10 μM GSH

0.9366 0.179

0.9075

0.952

pH 5.0

0.9411 0.804

0.9145

1.001

pH 5.0 + 10 mM

0.9715 0.9948 1.432

0.903

GSH

In vitro cellular toxicity. Considering the further applications of the PGMA-DMMA


20

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microspheres in biomedical fields, cellular toxicity is one of the most important indicators to measure the performances of the drug delivery systems, so the in vitro cytotoxicity of the blank PGMA-DMMA microspheres was evaluated on a normal cell line (293 cells) and a cancer cell line (HepG2 cells) after incubation of 48 h using MTT assays, respectively. As shown in Figure 7a, the PGMA-DMMA microspheres possessed excellent cytocompatibility, with a cell viability > 95% at a high concentration of 20 μg/mL for both cell lines. Contrastly, the PGMA-DMMA/DOX microspheres showed obvious cytotoxicity, and the cell viability decreased to 53% with increasing their concentration to 20 μg/mL (Figure 7b). The PGMA-DMMA/DOX microspheres displayed a lower inhibition efficacy against HepG2 cells than the free DOX with same concentration. Consideration of the cumulative release of < 44% from the PGMA-DMMA/DOX microspheres within 48 h and the drug content of 15%, the actually final DOX concentration gradiently released from the PGMADMMA/DOX microspheres was only 1.32 μg/mL. At such low actual DOX concentration, the cell viability was near to that with 2.5 μg/mL free DOX. The results demonstrated an enhanced antitumor efficacy with the proposed versatile theranostic nanoplatforms than the free DOX.25,28 To further study the inhibition of the PGMA-DMMA/DOX microspheres against cancer cell growth, the cell viability of the other two cancer cell lines (A549 cells and MCF-7 cells) after incubation of 48 h, respectively. As shown in Figure 7c, the PGMA-DMMA/DOX microspheres possessed efficient inhibition of cancer cell growth on the two cancer cell lines, similar as on the HepG2 cells. The results indicated that the proposed microspheres could be used as broad-spectrum anti-cancer formulations for cancer chemotherapy.


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HepG2 cells 293 cells

(a)

100 75 50 25 0

125 Cell viability (%)

Cell viability (%)

0

2.5 5 10 20 Concentration (g/mL)

120 Cell viability (%)

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PGMA-DMMA/DOX DOX

(b)

100 75 50 25 0

(c)

0


A549 cell MCF-7 cell

100 80 60 40 20 0

0


Figure 7. (a) Cell viability assay of the PGMA-DMMA microspheres for 293 cells and HepG2 cells after incubation for 48 h, Mean ± S.D. (n=5), (b) Cell viability assay in HepG2 cell of the PGMA-DMMA/DOX microspheres and free DOX after incubation for 48 h, Mean ± S.D. (n=5), and (c) Cell viability assay in A549 cells and MCF-7 cells of the PGMA-DMMA/DOX microspheres after incubation for 48 h, Mean ± S.D. (n=5). Cell viability (%) was evaluated with MTT assay and normalized against blank samples which were cultured without the microspheres.

Cellular uptake and flow cytometry analysis. The cellular uptake of the PGMA-DMMA/DOX theranostic nanoplatforms was also explored in HepG2 cells with the confocal fluorescence


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technique after different incubation times, in comparison with the blank PGMA-DMMA microspheres and free DOX. As shown in Figure 8a, the red fluorescence could be seen in the HepG2 cells after incubation with the blank PGMA-DMMA microspheres for 8 h, indicating the unique cellular uptake due to the pH-activated surface negative-to-positive charge-reversal and pH-triggered fluorescent emission of the polymeric microspheres. Furthermore, it could be clearly seen in the merged image that the red fluorescence of the Rh6G groups surround the blue fluorescence of the nuclei stained with DAPI, demonstrating the blank PGMA-DMMA microspheres or their degraded products were only accumulated in the cytoplasm of HepG2 cells.

(a)


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(b)

(c) Figure 8. Confocal laser scanning microscopy (CLSM) images of HepG2 cells upon incubating


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with the blank PGMA-DMMA microspheres (20 μg/mL) for 8 h (a), free DOX (5 μg/mL) (b), and the DOX-loaded PGMA-DMMA microspheres (5 μg DOX equiv/mL) (c) for varying time intervals (such as 0.5, 2.0 ,8.0 h), respectively. Each set of images, from left to represented cell nuclei in bright flied, stained by the DAPI, DOX fluorescence, the merged image, respectively, scale bar: 50 μm.

As for the free DOX (Figure 8b), the red fluorescence of DOX became stronger and stronger with prolonging the incubation time from 0.5 h to 8.0 h, indicating the DOX concentration increased with the incubation time. Meantime, the red fluorescence of DOX overlapped completely with the blue fluorescence of the cell nuclei stained with DAPI in the different stages. It demonstrated that the free DOX was uptaken and mainly accumulated in the cell nuclei.28 After the incubation of HepG2 cells with the DOX-loaded PGMA-DMMA microspheres with 5 μg DOX equiv/mL, the red fluorescence in both nuclei (DOX) and cytoplasm (Rh6G group) of HepG2 cells became stronger and stronger with prolonging the incubation time from 0.5 h to 8.0 h. It demonstrated that more DOX-loaded PGMA-DMMA microspheres had been uptaken by the HepG2 cells and the more DOX had been released and accumulated in the cell nuclei, with increasing the incubation time. However, the red fluorescence of DOX in nuclei was weaker than the red fluorescence of Rh6G group in cytoplasm in the investigated incubation time range. The phenomena demonstrated the potential fluorescent imaging performance of the proposed theranostic nanoplatforms owing to their pH-triggered “off-on” fluorescence, although the fluorescence of DOX overlaps with the fluorescence of the Rh6G groups.


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Figure 9. Cell death analysis of HepG2 cells by flow cytometry with v-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining after treating for 0.5 and 8 h, respectively, from the left to represented control, free DOX (5 µg/mL), PGMA-DMMA (5 µg/mL) and the PGMA-DMMA/DOX microspheres at DOX-equivalent concentration of 5 µg/mL.

HepG2 cell apoptosis was evaluated by FITC/PI double staining with via flow cytometry to assess the cell death phenotype. The results are presented in Figure 9. The total apoptotic ratios were calculated as the sum of the late apoptotic ratio and the early apoptotic ratio. As for the cells incubated with free DOX with a concentration of 5 µg/mL for 0.5 h, the survival of cells was 94.7% while the total apoptotic ratio was about 5%. Prolonging the incubation time to 8 h, the survival decreased to less than 0.1% with


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a total apoptotic ratio of 96.7%. The results indicated the DOX-induced cell apoptosis.29 In the case of the PGMA-DMMA/DOX microspheres at DOX-equivalent concentration of 5 µg/mL, the cell survival was 1.33% while the total apoptotic ratio was about 6.26% after incubation for 0.5 h. Then the cell survival decreased to 0 with a total apoptotic ratio of 12.9% in 8 h. The cell death level with the PGMA-DMMA/DOX microspheres was much higher than that with free DOX, indicating the proposed theranostic nanoplatforms possessed a higher efficiency to induce cell death than the free DOX,30 consistent with the MTT results.

CONCLUSIONS

In summary, versatile polymeric microspheres integrating the unique desirable features, tumormicroenvironment bioreducible degradation, pH-activated surface charge-reversal, pH-triggered “off-on” fluorescence and drug release, were designed via emulsion polymerization and postpolymerization modification, as theranostic nanoplatforms for precise imaging-guided diagnosis and chemotherapy. The proposed PGMA-DMMA microspheres possessed excellent cytocompatibility, while an enhanced inhibition efficacy against HepG2 cells was achieved than the free DOX after DOX-loading, owing to the facts that the pH-triggered surface negative-topositive charge reversal at pH 5-6 favored the cellular uptake and internalization and the


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pH/reduction dual-responsive controlled release endowing the tumor-specific intracellular triggered drug delivery with low premature leakage. The versatile polymeric microspheres emitted strong fluorescence only in acidic media such as the tumor microenvironment. Such features indicated their promising application as theranostic nanoplatforms for precise imagingguided diagnosis and chemotherapy in tumor treatment.

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

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

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For Table of Contents Use Only

Versatile

polymeric

nanoparticles

with

tumor-microenvironment

bioreducible

degradation, pH-activated surface charge-reversal, pH-triggered “off-on” fluorescence and drug release as theranostic nanoplatforms Mingliang Pei, Xu Jia, Guoping Li, and Peng Liu*


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Versatile polymeric microspheres with tumor-microenvironment

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