Chitosan-Based Nanocarriers with pH and Light Dual Response for

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Chitosan-Based Nanocarriers with pH and Light Dual Response for Anticancer Drug Delivery Lili Meng,† Wei Huang,*,† Dali Wang,† Xiaohua Huang,‡ Xinyuan Zhu,† and Deyue Yan*,† †

School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China ‡ Key Laboratory of New Processing Technology for Nonferrous Metal & Materials Ministry of Education and School of Material Science and Engineering, Guilin University of Technology, Guilin 541004, PR China S Supporting Information *

ABSTRACT: Currently, the major challenge for cancer treatment is to develop new types of smart nanocarriers that can efficiently retain the encapsulated drug during blood circulation and quickly release the drug in tumor cells under stimulation. In this study, the dual pH-/light-responsive crosslinked polymeric micelles (CPM) were successfully prepared by the self-assembly of amphiphilic glycol chitosan−onitrobenzyl succinate conjugates (GC-NBSCs) and then cross-linking with glutaraldehyde (GA), which was synthesized by grafting hydrophobic light-sensitive o-nitrobenzyl succinate (NBS) onto the main chain of hydrophilic glycol chitosan (GC). The GC-NBSC CPMs exhibited good biocompatibility according to the MTT assay against NIH/3T3 cells. The cell viability was maintained higher than 75% after 24 h incubation with CPMs even at a high concentration of 1.0 mg mL−1. The hydrophobic anticancer drug camptothecin (CPT) was selected as a model drug and loaded into GC-NBSC CPMs. The results of in vitro evaluation showed that the encapsulated CPT could be quickly released at low pH with the light irradiation. Meanwhile, the CPT-loaded CPMs displayed a better cytotoxicity against MCF-7 cancer cells under UV irradiation, and IC50 of the loaded CPT was as low as 2.3 μg mL−1, which was close to that of the free CPT (1.5 μg mL−1). Furthermore, the flow cytometry and confocal laser scanning microscopy (CLSM) measurements confirmed that the CPT-loaded CPMs could be internalized by MCF-7 cells efficiently and release CPT inside the tumor cells to enhance the inhibition of cell proliferation. Thereby, such excellent GC-NBSC CPMs provide a favorable platform to construct smart drug delivery systems (DDS) for cancer therapy.

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INTRODUCTION In the past few decades, nanosized polymeric micelles have emerged as promising carriers for anticancer drug delivery due to their outstanding characteristics, such as high drug loading capacity, long circulation in the bloodstream, and passive targeting capability based on the enhanced permeability and retention (EPR) effect.1−4 However, polymeric micelles formed by supramolecular self-assembly of amphiphilic copolymers are always too fragile to protect the encapsulated drugs while circulating in blood due to the dynamic nature and also inefficient in achieving controlled release.5 Therefore, there is growing interest in designing intelligent micelles that are stable under physiological conditions but can fall apart in a controlled fashion upon stimulation to release the payloads. Up to now, various stimuli-responsive CPMs have been developed for this purpose.6−17 Among them, the CPMs with acid-cleavable crosslinkers have been intensively investigated owing to the low pH (about 5.0−6.5) in tumor tissues.18−22 For example, Jeong et al. prepared a kind of shell cross-linked micelles with a pH-labile ketal cross-linker based on the self-assembly of poly(ethylene © XXXX American Chemical Society

glycol)−poly(L-aspartic acid)−poly(L-phenylalanine) which showed a rapid Dox release triggered by endosomal pH.19 Wang and co-workers displayed another kind of boronate cross-linked micelles made from telodendrimer pair (PEG5K(nitroboronic acid/catechol)4-CA8), and the stability of such CPMs was strongly interrupted by lowering the pH value from 7.4 to 5.0, leading to release of the encapsulated paclitaxel.22 Besides these single pH-responsive CPMs, more and more CPMs with dual stimuli-responsive properties have been fabricated recently to meet the conflicting requirements, i.e., the extracellular stability and intracellular release of drug.23−27 McCormick et al. manufactured imine shell cross-linked micelles starting from a temperature-responsive triblock copolymer for the first time and investigated the pH-triggered release of loaded hydrophobic drug.25 Liu and co-workers also developed novel thiol and pH dual-responsive CPMs based on Received: March 30, 2013 Revised: July 1, 2013

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and deuterated trifluoroacetic acid (CF3COOD) as solvents. Fouriertransform infrared (FT-IR) spectra were recorded on a PerkinElmer Paragon 1000 spectrometer by the KBr sample holder method. Spectra were obtained by collecting and averaging 16 scans at frequencies ranging from 500 to 4000 cm−1 at room temperature. The thermogravimetric analysis (TGA) was measured on a PE TGA-7 thermogravimetric analyzer from 100 to 800 °C at a rate of 20 °C/min under a nitrogen atmosphere. Dynamic light scattering (DLS) measurements were performed with a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at wavelength of 633 nm. All samples were measured at a scattering angle of 173°. Every data point was detected in triplicate and averaged. Transmission electron microscopy (TEM) measurements were carried out on a JEOL TEM-100CX-II instrument operating at a voltage of 200 kV. Samples were prepared by dropping the aqueous solutions of micelles or CPMs onto carbon-coated copper grids and then air-drying at room temperature before measurements. Fluorescent spectra were recorded using a PerkinElmer LS 50B luminescence spectrometer at room temperature. The fluorescence measurements were taken at an excitation wavelength of 550 nm, and the emission was monitored from 570 to 750 nm. For the UV light irradiation, the CPMs solution was placed vertically under a high-pressure mercury lamp (365 nm, 150 W) at a distance of 10 cm. In the cell experiments, an ultraviolet lamp (365 nm, 25 W) was used. Synthesis of o-Nitrobenzyl Succinate. o-Nitrobenzyl succinate (NBS) was synthesized according to the previous report.46 Typically, o-nitrobenzyl alcohol (4.0 g, 26.12 mmol), succinic anhydride (5.23 g, 52.24 mmol), and DMAP (1.60 g, 13.06 mmol) were dissolved completely in dried chloroform (86 mL) and refluxed under a nitrogen atmosphere for 24 h. After removing partial chloroform under reduced pressure, the mixture was washed three times with 10% HCl and then extracted with saturated NaHCO3 solution. The basic aqueous phase was washed with ether and acidified to pH 5.0 with 10% HCl. The white solid precipitate was collected and dried in vacuo at 40 °C overnight to give NBS (6.08 g, 92%). 1H NMR (CDCl3): δ (ppm) 8.10−8.12 (d, 1H, ArH), 7.47−7.67 (m, 3H, ArH), 5.56 (s, 2H, ArCH2), 2.75 (s, 4H, CH2CH2). Synthesis of Glycol Chitosan−o-Nitrobenzyl Succinate Conjugates. GC-NBSCs with different degree of substitution (DS) were synthesized by grafting different amount of NBS onto the main chain of GC according to the method in previous literature.42 Typically, GC (250.0 mg, 0.98 mmol glucosamine residues) was dissolved completely in the mixed solvent of methanol and deionized water (1:1 v/v, 60 mL). After that, NBS (56.7 mg, 0.22 mmol), EDC (63.3 mg, 0.33 mmol), and NHS (38.0 mg, 0.33 mmol) were added into the above mixture in sequence, and the reaction mixture was stirred at ambient temperature for 24 h. The resulting mixture was dialyzed in the mixed solvent of methanol/deionized water (4:1 v/v) for 2 days and deionized water for another 2 days (MWCO 14 kDa) and followed by lyophilization to produce pure GC-NBSC with the DS of 19.3 (GC-NBSC19.3). Other samples with the DS of 10.0 and 4.8 were synthesized by the same procedure. The DS was determined by measuring the concentration of unreacted NBS in the reaction mixture through UV absorbance at 260 nm and calculated according to the equation

an amphiphilic diblock copolymer (PCL-b-P(OEGMA-coMAEBA)), which could largely minimize the premature leakage of drug under physiological conditions and exhibit accelerated drug release under mildly acidic pH and/or reductive microenvironment.26 Despite their potential for drug delivery, smart CPMs still face some inevitable problems, such as achieving time and sitecontrolled drug delivery only by virtue of the internal biological stimuli related to the targeted diseased sites. Nowadays, multiple external stimuli, such as light,28−31 heat,32,33 electric/ magnetic fields,34,35 and ultrasound,36,37 are being explored to fabricate DDS which may show unprecedented control over drug delivery.38,39 Light as a trigger for drug release has drawn a great deal of attention since it provides a possibility for realizing remote and spatiotemporal drug release by tuning the wavelength, energy, and site of irradiation.31,40 Among the numerous chromophores applied in light-responsive materials, o-nitrobenzyl is of particular importance since it can undergo a photolysis reaction under either UV light or near-infrared light, disrupting the hydrophilic−hydrophobic balance of the onitrobenzyl-containing polymer and thus the integrity of assemblies.31,41 Until now, only a few CPMs are reported with both internal and external stimuli response.42 On the other hand, as an important hydrophilic derivative of chitosan, GC has been widely used in biomedical fields due to its favorable advantages, including abundant reactive groups for facile modification, good biocompatibility and biodegradability, and enhanced cell permeability with less toxicity.43−45 In this work, we attempted to synthesize amphiphilic GCNBSCs by covalently conjugating hydrophobic light-sensitive NBS onto the main chains of GC. Then, the nanosized micelles were prepared by the self-assembly of GC-NBSCs in water and further stabilized by cross-linking their GC shells with GA to form GC-NBSC CPMs. Finally, the hydrophobic anticancer drug CPT was encapsulated into the CPMs, and their performance as smart DDS was also investigated in terms of the stabilization under physiological conditions, in vitro drug release under pH-/light stimuli, anticancer activity in vitro, and cellular internalization behavior.

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EXPERIMENTAL SECTION

Materials. Glycol chitosan (degree of polymerization = 2800, degree of deacetylation = 82.7%) was purchased from Sigma-Aldrich (St. Louis, MO), which was purified by filtration and dialysis in deionized water before use. Succinic anhydride, o-nitrobenzyl alcohol, 4-(dimethylamino)pyridine (DMAP), N-hydroxysuccinimide (NHS), glutaraldehyde (GA) (25% in water), and Nile red were purchased from Acros Organics and used without further purification. 1-Ethyl-3(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was obtained from Titan Chemical Corp. Propidium iodide (PI) and 3(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) were from Sigma-Aldrich. Camptothecin was purchased from Shaanxi Sciphar. Biotechnology Co. Chloroform, methanol, and dimethyl sulfoxide (DMSO) were supplied by Sinopharm Chemical Reagent Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and phosphate buffered solution (PBS) were purchased from PAA Laboratories GmbH. Chloroform was dried over calcium hydride (CaH2) and distilled just before use. Dialysis tube (MWCO, 14 and 3.5 kDa) was obtained from Shanghai Lvniao Technology Corp. All the other chemicals were purchased from domestic suppliers and used as received. Clear polystyrene tissue culture treated 6-well and 96-well plates were obtained from Corning Costar. Methods. 1H NMR spectroscopy was performed on a Varian Mercury-400 spectrometer with deuterated chloroform (CDCl3), deuterated oxide (D2O), deuterated dimethyl sulfoxide (DMSO-d6),

DS =

M total − M unreacted × 100 Mglucosamine

(1)

where Mtotal, Munreacted, and Mglucosamine are the amount (in moles) of NBS in feed, NBS unreacted in reaction mixture, and glucosamine unit in GC. Critical Micelle Concentration (CMC) Measurement. The CMC value of GC-NBSCs was determined by the fluorescence probe technique. Here, Nile red was used as the fluorescence probe. Typically, the solid GC-NBSC19.3 was dissolved in deionized water to prepare the aqueous solutions with various concentration of GCNBSC19.3 (from 0.023 to 0.248 mg mL−1). Then, 25 μL of Nile red acetone solution (1.6 × 10−4 M) was added into 4.0 mL of GCNBSC19.3 micelles solution and evaporated overnight to remove the B

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Scheme 1. Synthesis of NBS (A) and GC-NBSC (B)

acetone. The final concentration of Nile red in the solution was 1.0 × 10−6 M. Finally, the fluorescence emission spectra of the solutions were recorded in the range from 570 to 750 nm on a fluorescence spectrometer with the exciting wavelength of 550 nm. Preparation of GC-NBSC CPMs. GC-NBSC19.3 (50.0 mg) was dissolved in 20 mL of phosphate buffer saline (PBS, pH 7.4) under mild stirring until the micelles formed. Then, 1 mL of GA aqueous solution (1.22 × 10−2 mmol mL−1) was added dropwise into the above micelle solution over 4 h under vigorous stirring at room temperature. After that, the resulting mixture was kept stirring for another 4 h and further purified by dialysis (MWCO 3500 Da) in PBS solution for 24 h, during which the PBS solution was replaced by fresh one every 4 h. Finally, the mixture was filtrated through 0.45 μm Millipore filter, and the pure GC-NBSC19.3 CPMs were obtained after lyophilizing the filtrate with a yield of 80%. Dual Stimuli Response of GC-NBSC CPMs. Typically, 3 mL of aqueous solution of GC-NBSC19.3 CPMs purified by dialysis was incubated in 0.1 M PBS solution (pH 7.4) or acetic buffer solution (pH 5.0) at 37 °C for 5 h and then irradiated with UV light (365 nm, 150 W) for 10 min. As a control, the samples without UV light irradiation were also prepared. After that, the size and morphology of samples were determined by DLS and TEM measurement. Preparation of CPT-Loaded GC-NBSC CPMs. In brief, GCNBSC19.3 (50.0 mg) and CPT (3.0 mg) were simultaneously added into DMSO (20 mL) under the vigorous stirring until both of them dissolved completely. Then, the mixture was transferred into a dialysis bag (MWCO 3500 Da) and dialyzed in PBS solution for 2 days, during which the PBS solution was renewed every 4 h. During this process, CPT-loaded micelles were formed. After that, 1 mL of GA aqueous solution (1.22 × 10−2 mmol mL−1) was added dropwise into the above micelle solution over 4 h under vigorous stirring at room temperature. The resulting mixture was kept stirring for another 4 h and further purified by dialysis (MWCO 3500 Da) in PBS solution for 24 h, during which the PBS solution was replaced by fresh one every 4 h. Finally, the mixture was filtrated through 0.45 μm Millipore filter to remove the free CPT, and the CPT-loaded CPMs were obtained by lyophilizing the filtrate. To determine the drug loading content (DLC) and drug loading efficacy (DLE), the CPT-loaded CPMs were incubated in PBS solution (pH = 5.0) for 24 h, then lyophilized, and dissolved in DMSO again. The drug concentration was determined by measuring the fluorescence intensity of CPT (excitated at 340 nm). DLC and DLE were calculated according to the following equations:

DLE (%) = Wloaded /Wtotal × 100%

(2)

DLC (%) = Wloaded /(Wpolymer + Wloaded) × 100%

(3)

immersed in 50 mL of PBS solution (pH 7.4) or acetate buffer solution (pH 5.0) in a shaking water bath at 37 °C to acquire sink conditions. Meanwhile, the samples without UV light irradiation were also prepared as a control. At the predetermined time interval, the external buffer solution (3 mL) was taken out and replenished with an equal volume of fresh media. The amount of released CPT was determined by fluorescence measurement. All the drug release experiments were performed in triplicate, and the results were the average data. Cell Culture. NIH/3T3 normal cells (a mouse embryonic fibroblast cell line) and MCF-7 cancer cells (a human breast cancer cell line) were cultivated in DMEM supplied with 10% FBS (fetal bovine serum) and antibiotics (50 U mL−1 penicillin and 50 U mL−1 streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. In Vitro Cytotoxicity Measurements. The relative cytotoxicity of GC-NBSC19.3 CPMs was estimated by a MTT viability assay against NIH/3T3 normal cells. In the MTT assay, NIH/3T3 cells were seeded into 96-well plates with a density of 8 × 103 cell per well in 200 μL of medium. After 24 h incubation, the culture medium was carefully removed and replaced with 200 μL of fresh medium containing serial dilutions of GC-NBSC19.3 CPMs. The cells were grown for another 24 h. Then, 25 μL of MTT assay stock solution (5 mg mL−1) in PBS was added into each well. After incubation for an additional 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL of DMSO per well, and the absorbance were measured in a BioTek SynergyH4 at a wavelength of 490 nm. Activity Analysis. The cytotoxicity of CPT-loaded CPMs of GCNBSC19.3 against MCF-7 cancer cells was examined in vitro by the MTT assay. MCF-7 cells were seeded into 96-well plates at an initial density of 8 × 103 cells per well in 200 μL of medium. After incubation for 24 h, the culture medium was replaced with fresh one, and 50 μL of PBS solution containing serial dilutions of CPT-loaded CPMs was added into each well. The cells were irradiated under UV light at 365 nm (25 W) for 30 min after incubation with CPT-loaded CPMs for 3 h and cultured for another 24 h. Meanwhile, cells without UV light irradiation were also set as a control. The cytotoxicity of blank GCGC-NBSC19.3 CPMs with or without UV light irradiation was also investigated as controls. Then, 25 μL of 5 mg mL−1 MTT assays stock solution in PBS was added into each well, and the cells were further grown for 4 h, followed by removing the medium carefully. The obtained blue formazan crystals were dissolved in 200 μL of DMSO per well, and the absorbance was measured in a BioTek SynergyH4 at a wavelength of 490 nm. Cellular Internalization of CPT-Loaded GC-NBSC CPMs. The cellular internalization experiments of CPT-loaded CPMs of GCNBSC19.3 were performed by flow cytometry and CLSM measurements. Flow Cytometry. MCF-7 cells were seeded into six-well plates at 5 × 105 cells per well in 1 mL complete DMEM and cultured for 24 h. Then, the medium containing CPT-loaded CPMs at a final CPT concentration of 3.3 μg/mL was added into different wells, and the

where Wtotal, Wloaded, and Wpolymer are the weight of total CPT used, the loaded CPT, and GC-NBSC19.3 CPMs. In Vitro Drug Release. For in vitro drug release study, the lyophilized CPT-loaded CPMs of GC-NBSC19.3 (10 mg) were dispersed in 4 mL of PBS solution (pH 7.4) and then transferred into a dialysis bag (MWCO 3500 Da). After being irradiated under UV light at 365 nm (150 W) for 10 min, the mixture in dialysis bag was C

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cells were incubated at 37 °C for 5, 60, and 180 min. Thereafter, the samples were prepared for flow cytometry analysis by removing the cell culture medium, rinsing with cold PBS, and treating with trypsin. Data for 1.0 × 104 gated events were collected and analyzed by means of a BDFACS Calibur flow cytometer and CELL Quest software. CLSM Study. For the CLSM study, MCF-7 cells were seeded into six-well plates at 2 × 105 cell per well in 1 mL of complete DMEM and cultured for 24 h, followed by removing the culture medium and adding 1 mL of medium containing CPT-loaded CPMs at a final CPT concentration of 3.3 μg/mL into different wells. The cells were incubated at 37 °C for predetermined intervals. Then, the culture medium was removed, and cells were washed with PBS three times. Subsequently, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and the slides were rinsed with cold PBS three times. Finally, the slides were mounted and observed with a LSM510 META.

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Figure 2. 1H NMR spectra of GC in D2O (a), GC-NBSC19.3 in D2O (b), and GC-NBSC19.3 in the mixed solvent of DMSO-d6/CF3COOD (1:1, v/v) (c).

RESULTS AND DISCUSSION Synthesis and Characterization of GC-NBSC. Scheme 1 gives the synthesis route of GC-NBSC. First, NBS was

process of coupling reaction. The carboxyl acid group of NBS was first activated by NHS and then coupled with the primary amino groups in GC to form an amide linkage. The DS is a key parameter to determine the hydrophobic/hydrophilic property of GC-NBSC and finally affects the size of self-assembled aggregates.43 Here the DS is defined as the number of NBS groups per 100 glucosamine units of GC and can be easily controlled by adjusting the feed ratio of NBS to GC. Therefore, three samples of GC-NBSC with different DS were prepared at various feed molar ratio of NBS to glucosamine units of GC (0.230, 0.115, and 0.058) and fully characterized by FTIR and TGA measurements. The FTIR spectra of GC-NBSC and GC are shown in Figure 1. Compared with GC (a), a new shoulder peak assigned to the carbonyl of ester group is observed at 1726 cm−1 in the spectra of GC-NBSC (b−d) and is gradually intensified while increasing the feed ratio. Besides, the characteristic peaks at 1654 and 1564 cm−1 observably increase after conjugation, which are attributed to the carbonyl of amide I band and N −H bending of amide II band, respectively. All the above results confirm the formation of amide linkage between the carboxyl group of NBS and the amino groups of GC. The DS value of GC-NBSC was determined by the UV absorbance-based quantification according to eq 1, and the results are shown in Table 1.44 Apparently, it increases with increasing the feed ratio of NBS and GC. This result is also confirmed by the TGA measurement. The TGA curves (Figure S2 ) show an increased weight loss percentage of GC-NBSC in the first stage with the increase of feed ratio. Self-Assembly Behavior of GC-NBSC. Similar to other polymeric amphiphiles, GC-NBSC might also be able to selfassemble into nanosized micelles because of its amphiphilic structure comprised of hydrophobic NBS groups and hydrophilic GC backbone. The self-assembly behavior of GC-NBSC was well investigated by means of 1H NMR, fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). First, taking GC-NBSC19.3 as an example, 1H NMR was used to study the self-assembly behavior, and the results are shown in Figure 2. Compared with the spectrum of GC in D2O (a), only some weak signals ascribed to the protons of appended NBS groups are observed at 2.46, 5.4, and 7.4−8.0 ppm in the spectrum of GC-NBSC19.3 in D2O (b). Especially those signals at 7.4−8.0 ppm assignable to the aromatic protons are barely visible, indicating that GC-NBSC19.3 are self-assembled into

Figure 1. FTIR spectra of GC (a), GC-NBSC4.8 (b), GC-NBSC10.0 (c), and GC-NBSC19.3 (d).

Table 1. General Properties of GC-NBSC with Different DS GC-NBSC4.8 GC-NBSC10.0 GC-NBSC19.3

feed ratioa

DSb

CMC (mg mL−1)

size (nm)

0.06 0.12 0.23

4.8 10.0 19.3

0.29 0.18 0.08

513.0 378.3 58.8

a Molar ratio of NBS to glucosamine units of GC. bDS measured by the UV absorbance-based quantification.

synthesized from o-nitrobenzyl alcohol by the carboxylation of succinic anhydride under the catalysis of DMAP in CHCl3 at 60 °C (Scheme 1A). The results show that the hydroxyl groups of o-nitrobenzyl alcohol are able to react with succinic anhydrides quantitatively to produce NBS with a high yield of 92%. The chemical structure of NBS is confirmed by 1H NMR measurement (Supporting Information Figure S1), which clearly shows the signals of aromatic protons at 7.47− 8.12 ppm (4H, ArH), the benzyl methylene protons at 5.56 ppm (2H, CH2), and the succinic methylene protons at 2.75 ppm (4H, CH2CH2). Then, NBS was further coupled with the primary amine groups in GC to form GC-NBSC in the presence of EDC and NHS (Scheme 1B). Because of the dramatically different water solubility of NBS and GC, a mixed solvent of CH3OH/deionized water (1:1, v/v) was used in the D

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Figure 3. (A) Emission spectra of Nile red (λex = 550 nm) at various concentration of GC-NBSC19.3. (B) Emission intensity of Nile red versus the concentration of GC-NBSC19.3.

Scheme 2. Preparation of CPT-Loaded GC-NBSC CPMs and Intracellular Drug Release Triggered by pH and UV Light

Furthermore, the micellar self-assembly of GC-NBSC was monitored by the fluorescence spectroscopy using Nile red as a probe. As shown in Figure 3A, the fluorescence emission intensity of Nile red increases slowly as the concentration of GC-NBSC19.3 is low. However, it increases dramatically once the concentration exceeds a certain value, implying the formation of micelles and encapsulation of Nile red into the hydrophobic cores. This concentration can be defined as the critical micelle concentration (CMC). It is suggested in Figure 3B that the CMC of GC-NBSC19.3 in water is about 0.08 mg mL−1. The CMC of other two conjugates with different DS were also determined by the same method and listed in Table 1. Clearly, it decreases from 0.29 to 0.08 mg mL−1 with increasing the DS, due to the enhanced hydrophobicity of the conjugates. After that, the size of GC-NBSC micelles was evaluated by DLS. Not surprisingly, it exhibits the same change with the DS, since the enhanced hydrophobicity induces the formation of more dense hydrophobic cores.43 It is well-known that the CMC of amphiphilic polymers and the size of their self-assemblies are two important parameters for DDS. Low CMC and small size makes micelles very stable in vivo and circulate longer in the blood. Therefore, GC-NBSC19.3 was selected as a drug carrier and used to prepare the cross-linked micelles as a potential DDS. Preparation of Cross-Linked Micelles and Their Dual Stimuli-Responsive Property. Generally, upon intravenous injection, micelles can quickly be diluted to a concentration

Figure 4. 1H NMR spectra of the cross-linking reaction between GA and GC-NBSC19.3 micelles in D2O at pH 7.4.

core−shell structures in aqueous environment, and the hydrophobic NBS groups are confined within the core domain shielded by hydrophilic GC corona, thus leading to weak signals of NBS protons. However, when GC-NBSC19.3 is dissolved in the mixed solvent of DMSO-d6/CF3COOD (1:1, v/v) (c), the self-assemblies are disrupted, causing the increased signals for NBS at 2.48, 5.48, and 7.35−7.90 ppm. E

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Figure 5. TEM photographs (A) and DLS results (B) of GC-NBSC19.3 micelles (a), GC-NBSC19.3 CPMs at pH 7.4 (b), and GC-NBSC19.3 at pH 5.0 after UV irradiation (c).

shell of GC-NBSC19.3 micelles in PBS solution (pH 7.4) because GA can easily react with amino groups of GC-NBSC19.3 to form acid-labile imine bonds which induces the formation of dual stimuli-responsive GC-NBSC19.3 CPMs (Scheme 2). 1H NMR spectroscopy was adopted to track the cross-linking reaction, and the results are shown in Figure 4. The signal of aldehyde protons in GA at 9.60 ppm weakens gradually with increasing the reaction time and almost disappears after 4 h. Unfortunately, the signal ascribed to the protons of imine bonds is not found clearly because it is overlapped with the signals assigned to the aromatic protons of NBS at 7.54−8.10 ppm. On the other hand, the signals of GC backbones (3.64 ppm) and methylene protons from GA (1.22−1.68 ppm) also decrease with the reaction time, due to the increased viscosity of the reaction mixture induced by the interparticle crosslinking which is difficult to avoid when the reaction was conducted in a magnetic tube without stirring. Besides, the ATR-FTIR spectra of GC-NBSC19.3 and its cross-linked product was measured and shown in Figure S3. The characteristic peak of imine bonds cannot be observed due to overlapping with the absorbance of amide I band. However, the peak of N−H bending at 1525 cm−1 becomes very weak in the spectrum of GC-NBSC19.3 CPMs, which indicates the successful cross-linking reaction. TEM and DLS measurements were also taken to evaluate the size and morphology change of micelles after cross-linking. As shown in Figure 5A-b, the morphology of CPMs is similar to that of non-cross-linked micelles, while the diameter decreases to 30.0 nm after crosslinking, indicating the formation of more compact structures. The DLS result reveals a decreased average diameter of 37.8 nm for CPMs (Figure 5B-b), which is basically consistent with that determined by TEM. Thereafter, dual pH-/light-responsive properties of GCNBSC19.3 CPMs were studied through DLS and TEM. As mentioned above, the average diameter of CPMs is about 37.8 nm by DLS at pH 7.4 (Table 2). When the pH was adjusted to 5.0 and kept for 5 h, the size of CPMs increases to 71.2 nm, attributing to the cleavage of cross-linked imine bonds in the shell. Besides, amino groups in GC are protonated at such a low pH which greatly enhances the hydrophilicity of CPMs, leading to the expanded volume. When this acid CPMs solution is further irradiated under UV light for 10 min, the size measured

Table 2. Dual Stimuli-Responsive Properties of GCNBSC19.3 CPMs condition

pH 7.4

pH 5.0

size (nm)

37.8 ± 3.0

71.2 ± 9.9

pH 7.4 with UV 52.4 ± 11.5

pH 5.0 with UV 14.6 ± 3.0

Figure 6. In vitro drug release behavior of CPT-loaded GC-NBSC19.3 CPMs under different stimulus conditions.

Figure 7. In vitro cytotoxicity of GC-NBSC19.3 CPMs against NIH/ 3T3 cells after 24 h incubation.

below CMC and disaggregate, resulting to the drug leakage before reaching the diseased site. Therefore, cross-linking of the core or shell of micelles is a feasible and effective method to improve the stability. Here we selected GA to cross-link the F

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Figure 8. (A) Cytotoxicity of free CPT and CPT-loaded GC-NBSC19.3 CPMs with or without UV irradiation against MCF-7 cells after 24 h incubation. (B) Cytotoxicity of blank GC-NBSC19.3 CPMs with or without UV irradiation against MCF-7 cells after 24 h incubation.

irradiation time increasing and levels off after 10 min, indicating that the photocleavable reaction proceed completely in a short time. In Vitro Drug Release Study. In order to evaluate the potential of this dual pH-/light-responsive GC-NBSC19.3 CPMs as a smart DDS, the anticancer drug CPT was selected and capsulated into the CPMs. We first evaluated the photostability of CPT by using the fluorescence spectroscopy. The fluorescence excitation and emission spectra of CPT in Figure S8 are nearly the same before and after the UV irradiation, confirming the high photostability of CPT for UV light irradiation. Then the dual-triggered CPT release behavior was investigated in vitro by the dialysis method. The DLE and DLC of the CPT-loaded CPMs are found to be 5.0% and 5.93%, respectively, on the basis of the standard curve of CPT. Different pH- and UV light-triggered drug release curves of CPT-loaded CPMs are presented in Figure 6. At pH 7.4 without UV irradiation, only 18.35% of the loaded CPT is released from CPMs within 12 h. However, at pH 7.4 with UV irradiation, approximately 31.96% of the loaded CPT is released within the same period. It indicates that the photolysis of NBS side groups occurs in the core of CPMs, but the core−shell structure of CPMs is still preserved due to the cross-linking effect. Similarly, at pH 5.0 without UV irradiation, the crosslinked imine bonds in the CPMs shell are destroyed, but the core−shell structure is still integrated because of the amphiphilic nature of GC-NBSC19.3. Thus, only about 49.08% of the loaded CPT is released out within 12 h under this condition. However, when the low pH and UV irradiation are simultaneously exerted on the CPMs, a burst release of CPT (45.30%) is observed during the initial first hour because of the quick disaggregation of CPMs, and the cumulative amount of released drug is up to 81.68% after 12 h. This is consistent with the results of acid-stimuli hydrolysis of imine linkage and photolysis of NBS. Overall, these results show that this smart GC-NBSC19.3 CPMs are able to realize a quick release of the encapsulated drugs only when it is synchronously triggered by pH and UV light. On the contrary, the encapsulated drug release would be dramatically suppressed with a sole stimulation. In Vitro Cytotoxicity of GC-NBSC CPMs and Anticancer Activity of CPT-Loaded CPMs. As drug delivery system materials, the biocompatibility or cytotoxicity of the carrier is a key index for its biomedical application. Thus, the cytotoxicity of GC-NBSC19.3 CPMs in vitro was evaluated by the MTT method against a mouse embryonic fibroblast NIH/ 3T3 cell line. Figure 7 shows the cell viability after 24 h

Figure 9. Cellular uptake of CPT-loaded GC-NBSC19.3 CPMs by MCF-7 cells versus the incubation time by flow cytometry analysis. Inset is the representative flow cytometry histogram profiles of MCF-7 cells incubated with CPT-loaded CPMs for 3 h.

by DLS sharply decreases to about 14.6 nm (Figure 5B-c). At the same time, no regular spherical micelles are observed except some irregular tiny fragments in the TEM photograph (Figure 5A-c). This implies that light-sensitive side NBS groups are cleaved from GC chains under UV irradiation, causing the complete disaggregation of CPMs into the analogue of glycol chitosan (Scheme S1). In order to confirm the above speculation, the size and morphology of GC were also investigated by DLS and TEM. As shown in Figure S4, some similar small irregular particles with a diameter of less than 20 nm are found and verify the complete disaggregation of CPMs into the analogue of glycol chitosan. On the other hand, if the CPMs solution at pH 7.4 is irradiated by UV light for 10 min, only small size change of CPMs is observed, increasing from 37.8 to 52.4 nm, suggesting that the core−shell structures are still maintained even if the photolysis of NBS core occurs. The acid-cleavable process of imine linkage was also investigated by 1 H NMR spectroscopy, and the results are shown in Figure S5. When GC-NBSC19.3 CPMs were incubated in D2O at pH = 5.0 and 37 °C for different times, the signal of aldehyde was monitored and quantified by comparing with the signal of CHCl3 as an external standard. Then the hydrolysis ratio of GC-NBSC19.3 CPMs was calculated and displayed in Figure S6. Obviously, half of the cross-linkage is broken in about 2 h, and a complete hydrolysis of the cross-linkage is achieved within 5 h. Besides, the photolysis of NBS in GC-NBSC was also evaluated by UV−vis spectroscopy by using the aqueous solution of GC-NBSC19.3 micelles as a model (Figure S7). The characteristic peak of NBS at 260 nm decreases with the G

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Figure 10. CLSM images of MCF-7 cells incubated with the CPT-loaded GC-NBSC19.3 CPMs for 5 min (A), 30 min (B), and 1 h (C). Cell nuclei were stained with PI.

same dose, probably due to the time-consuming drug release from the CPMs and thus delayed nuclear uptake.48 Additionally, in order to eliminate the possibility that the increased anticancer efficacy of CPT-loaded CPMs after light irradiation comes from the cell death under UV light or the cytotoxicity of irradiated products, the in vitro cytotoxicity of blank CPMs against MCF-7 cells with or without light irradiation was also conducted by the MTT method. It is exhibited in Figure 8B that when the CPMs concentration is up to 1.0 mg mL−1, the cell viability without light irradiation still retains about 75%, and only a slight decrease, 3%, is found after irradiation. Cell Internalization Studies. It is an important factor for therapeutic efficacy of anticancer drugs that whether they can be quickly transported into cancer cells. Therefore, the flow cytometry analysis was performed to measure the cellular uptake of CPT-loaded GC-NBSC19.3 CPMs by the MCF-7 cell line. The CPT-loaded CPMs solution with a CPT concentration of 3.3 μg mL−1 was added into the culture medium, and the cells were incubated in this culture medium at 37 °C for the predetermined time intervals. As a control, the MCF-7 cells were also incubated in the culture medium without the CPTloaded CPMs solution. As shown in Figure 9, the fluorescence intensity of MCF-7 cells is found within 5 min, and its intensity increases greatly with prolonging the incubation time. After 1 h incubation, the relative geometrical mean fluorescence intensity

incubation with CPMs at the concentration varying from 8 to 1000 μg mL−1. Obviously, the cell viability remains above 75.0% even when the concentration of CPMs increases to 1000 μg mL−1. Consequently, GC-NBSC19.3 CPMs have good biocompatibility and are a suitable material for drug delivery system. Besides, the anticancer activity of CPT-loaded GC-NBSC19.3 CPMs with or without UV irradiation was also investigated by MTT assay against MCF-7 cancer cells, and the free CPT was used as a control. Figure 8A shows that the dose of the capsulated CPT required for 50% cellular growth inhibition (IC50) with light irradiation is about 2.3 μg mL−1, while the one without irradiation is about 5.0 μg mL−1. It is well reported that tumor extracellular environment is acidic (pH 6.5), and the pH values in endosome and lysosome are even lower (pH 5.0− 5.5).47 Therefore, after the CPMs are internalized by cancer cells through an endocytosis process, the imine linkages are quickly cleaved in the acidic intracellular compartments and the amino groups of GC are protonated to facilitate the endosome/ lysosome escape. Combining the light stimulation, those CPMs disassemble completely and release the drug intracellularly to achieve high anticancer activities. As a comparison, the IC50 of free CPT is 1.4 μg mL−1, suggesting a slightly better anticancer effect than loaded CPT. The free drug often shows higher activity than the loaded drug in polymeric nanoparticles at the H

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of the CPT-loaded CPMs pretreated cells is about 1.2-fold of that of the nonpretreated cells and further increases rapidly to 4-fold in a period of 3 h. The rapid enhancement of fluorescence intensity indicates that the CPT-loaded CPMs can be effectively internalized by MCF-7 cells. Moreover, the cellular uptake behavior was further evaluated by CLSM. By taking advantage of the red fluorescence from propidium iodide (PI) and blue fluorescence from CPT, the intracellular location of drug was studied. After CPT-loaded GC-NBSC19.3 CPMs with a CPT concentration of 3.3 μg mL−1 were added into the culture medium, MCF-7 cells were incubated at 37 °C for 5 min, 30 min, and 1 h. Thereafter, the cell nuclei were stained with propidium iodide (PI), and the treated samples were observed directly using CLSM. As shown in Figure 10A, the cells pretreated with CPT-loaded CPMs for 5 min display weak blue fluorescence in cytoplasm. When the incubation time was extended to 30 min or 1 h, the strong fluorescence is observed mainly in cytoplasm while a small amount in nuclei due to a small drug release from the CPTloaded CPMs (Figure 10B,C). Therefore, these results also confirm the effective internalization of CPT-loaded CPMs by cancer cells.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (W.H.); [email protected] (D.Y.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program (No. 2013CB834506, 2012CB821500, 2009CB930400) and the National Natural Science Foundation of China (No. 21204048, 91127047, 21174086, 21074069).

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CONCLUSIONS A novel dual pH-/light-responsive nanocarrier was successfully prepared by the self-assembly of amphiphilic GC-NBSC, which was synthesized by grafting hydrophobic NBS onto the hydrophilic GC and then cross-linking with GA. The results of in vitro drug release evaluation showed that the release of loaded CPT from CPMs was greatly inhibited in a neutral environment like normal tissue or blood circulation, whereas a rapid release was observed under the acidic environment with UV light irradiation. Only a small part of the capsulated CPT could be released when it was triggered either by the low pH or UV light. The results of MTT assay against the NIH/3T3 cell line confirmed the good biocompatibility of blank CPMs, where cell viability remained higher than 75% after incubation for 24 h. Furthermore, CPT-loaded CPMs under UV irradiation showed a better cytotoxicity against MCF-7 cancer cells than that of the nonirradiated ones, the IC50 of which was 2.3 μg mL−1 and close to that of the free CPT (1.5 μg mL−1). The flow cytometry and CLSM measurement indicated that these CPT-loaded CPMs could be effectively internalized by MCF-7 cells to facilitate an efficient anticancer activity of CPT. In a conclusion, this new type of dual responsive nanocarriers holds great potential for targeted and spatiotemporal drug delivery.

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ASSOCIATED CONTENT

S Supporting Information *

1 H NMR spectrum of NBS, TGA curves of GC, GC-NBSC4.8, GC-NBSC10.0, and GC-NBSC19.3, ATR-FTIR spectra of GCNBSC19.3 before and after cross-linking, TEM photograph and DLS result of glycol chitosan, 1H NMR spectra of GCNBSC19.3 CPMs incubated in D2O at pH = 5.0 and 37 °C for different time, hydrolysis ratio of imine bonds of GC-NBSC19.3 CPMs incubated in D2O at pH = 5.0 and 37 °C for different time, UV−vis spectra of GC-NBSC19.3 micelles in aqueous solution under UV irradiation at 365 nm for different time, fluorescence excitation and emission spectra of CPT before and after under UV irradiation, and chemical structures of GCNBSC CPMs and the final products after light and pH triggers. This material is available free of charge via the Internet at http://pubs.acs.org.

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