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pH-sensitive amphiphilic chitosan-quercetin conjugate for intracellular delivery of doxorubicin enhancement
Carbohydrate Polymers 223 (2019) 115072
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
pH-sensitive amphiphilic chitosan-quercetin conjugate for intracellular delivery of doxorubicin enhancement
T
Yuzhi Mua, Guangsheng Wub, Chang Sua, Yao Donga, Kaichao Zhanga, Jing Lia, Xiaojie Suna, ⁎ Yang Lia, Xiguang Chena,c, Chao Fenga, a
College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, Shandong Province, China Navy Qingdao First Sanatorium of PLA, No. 27 West Hong Kong Road, Qingdao 266071, Shandong Province, China c Qingdao National Laboratory for Marine Science and Technology, 1# Wenhai Road, Qingdao 266000, Shandong Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Quercetin Drug conjugate Intracellular delivery Multidrug resistance effect P-glycoprotein
A novel pH-responsive nanomicelle (QT-CA-CS) based on Chitosan, Quercetin and Citraconic anhydride was reported in this study. The QT-CA-CS could self-assemble into nanomicelles for encapsulating anticancer drug doxorubicin (DOX) by ultrasound. The novel nanomicelles had P-gp inhibition and pH responsiveness, which was capable of inhibiting drug efflux and responding to an endo/lysosomal acidic environment. The drug loaded nanomicelles had high encapsulation rate (more than 80%), small particle size (133.52 ± 4.13 nm) and positive zeta potential (+13.5 mV). The release rate of doxorubicin and quercetin in pH 4.5 was faster than that in pH 7.4. QT-CA-CS-DOX nanomicelles could promote cellular uptake of doxorubicin by drug resistance cell line (MCF-7/ADR), which was 8.62 folds higher than that of free doxorubicin. Most importantly, QT-CA-CS-DOX nanomicelles could escape from lysosomes and rapidly release doxorubicin and quercetin in the cytoplasm, which had an enhanced inhibitory effect on tumor cells, especially for MCF-7/ADR. The above results proved that the high potential of QT-CA-CS-DOX nanomicelles for multidrug resistance related tumor therapy.
1. Introduction Intracellular delivery of anticancer drug in cancer cells is primary for chemotherapy, which determines the efficacy and side effect of anticancer agent (Kim, Faix, & Schnitzer, 2017; Rao, Ko, Lee, & Park, 2018; Tashima, 2018). This process comprises series of successive events that can be summarized as drug intracellular accumulation and drug efflux mitigation. On the one hand, anticancer drug should pass through cell recognition, cell uptake, endo-lysosomal escape, and cytoplasmic localization, to achieve effective accumulation in cancer cell. On the other hand, it must get rid of drug efflux resulted from multidrug resistance effect (MDR) of cancer cells, to maintain high level drug concentration in cancer cell. The strategy for improving intracellular drug accumulation and reducing MDR is urgently required (Tuguntaev et al., 2017). Stimuli responsive nano-carrier provides a smart platform for responding specific physiological tumor microenvironment, such as acidic pH, redox property, certain enzymes or glucose concentration or their combinations (Aji Alex et al., 2017; Tayo, 2017; Yang, Feng, & Liu, 2016; Zuo, Kong, Mu, Feng, & Chen, 2017). Due to the particular acidic pH around tumor tissue (pH 6.0) and endo/ environment (pH 4.5), ⁎
various pH-sensitive nanocarriers have been designed to realize triggered drug release in cytoplasm (Turk & Turk, 2009). One strategy is the introduction of acid sensitive group, such as amido, carboxyl or imidazolyl in polymer chain, that release drug by pH-induced disassembly or swelling behavior. The other one is the incorporation an acid labile linkage between polymer chain and drug molecule that is cleaved in acidic environment to bring drug release (Feng, Zhang, Zhi, Gao, & Nakanishi, 2018; Li et al., 2015; Liu et al., 2014; Zhang, Zhao, Guo, Lin, & Guo, 2017, 2014). A previous research proved that the two approaches above had shown excellent intracellular drug accumulation in cancer cell. However, these stimuli responsive nano-carrier rarely involved reversal of MDR of cancer cells, which restricted their therapeutic efficacy. MDR, derived from recurrent chemotherapy, is important cause of chemotherapy failure. MDR mainly associate with the overexpression of multidrug-resistant proteins and membrane P-glycoprotein (P-gp) transporters, which actively increase drug efflux to limit the efficiency of anticancer agents (Mohana, Ganesan, Rajendra Prasad, Ananthakrishnan, & Velmurugan, 2018; Mollazadeh, Sahebkar, Hadizadeh, Behravan, & Arabzadeh, 2018; WT, 1996). It has been suggested that the MDR of cancer cells to chemotherapeutics can be
Corresponding author. E-mail addresses: [email protected], [email protected] (C. Feng).
https://doi.org/10.1016/j.carbpol.2019.115072 Received 10 December 2018; Received in revised form 2 July 2019; Accepted 7 July 2019 Available online 08 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy succinimide were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) and RPMI-1640 were purchased from Hyclone (Logan, UT, USA). All other chemicals were of analytical grade and were used without further purification. MCF-7 cells and L929 cells were kindly provided by Cell Bank, Chinese Academy of Sciences. MCF-7/ADR cells were purchased from Shanghai Yu Bo Biotech Co., Ltd.
suppressed by combine anticancer agents with P-gp inhibitor, such as cyclosporine A, verapamil and so on (Tuguntaev et al., 2017). Compare to various synthetic P-gp inhibitor, quercetin (QT), a polyphenolic flavonoid compound derived from tea, apple, onions, broccoli and berries, display promising P-gp inhibition activities and minimal side effects. Importantly, QT also exhibit favorable competitively inhibition against the most MDR protein family, such as MRP1 and BCRP, as well as CYP3A subfamily of the cytochrome P-450, which have been verified on various drug-resistant cancer cell lines and animal models (Borska et al., 2012; Wang et al., 2014; Xia, Cole, Cai, & Cai, 2017). Chitosan (CS) is a natural high molecular polysaccharide that is hydrophilic, cationic, biocompatible and biodegradable. In an acidic solution, the amino group of CS is protonated to carry a positive charge. CS has significant advantages in blood circulation retention and negligible accumulation in body tissues, and thus CS is currently a suitable excellent material for pharmaceutical carriers (Kean & Thanou, 2010; Mu et al., 2019). To combat MDR and improve accumulation of anticancer drug in tumor cells, a pH-responsive amphiphilic chitosan-quercetin conjugate was synthesized using an acid labile linkage (citraconoyl, CA) between QT and CS (QT-CA-CS) in this study. The amide bond formed by the reaction of citraconic anhydride with an amino group could be destroyed under acidic conditions, thereby generating an acid-responsive behavior (Cao et al., 2014; Prata et al., 2004; Yuan et al., 2012). It was expected that the QT-CA-CS nanomicelles could disassembly responding the acid pH of endo/lysosome environment by breakage of linkage, thus bring out rapid release of DOX and QT to achieve highefficiency tumor inhibition (Fig. 1). This study devoted to concentrate the intracellular drug accumulation and the MDR mitigation in QT-CACS nano-micelles for developing an effective delivery platform, particularly for MDR cancer cells. The self-assemble behavior, drug encapsulation and pH-responsive release pattern of QT-CA-CS nano-micelles were investigated in vitro. Furthermore, the cellular uptake, endo/lysosome escape, intracellular drug release and antitumor activity were comparatively investigated in drug-sensitive and resistant cancer cells.
2.2. Synthesis of quercetin-chitosan conjugates (QT-CA-CS and QT-CS) The pH-sensitive QT-CA-CS was synthetized using carbodiimide reaction as follow (Mu et al., 2019). QT was firstly treated with citraconic anhydride (citraconic anhydride: QT = 1.3:1, at molar ratio) and magnetic stirred overnight at 40 °C in DMSO. The product was precipitated with deionized water. After centrifugation (RCF 900g), the precipitation was redissolved in DMSO. EDC·HCl and NHS mixture, which molar ratio was EDC: NHS = 1:1.5 in 1 ml of DMSO was added under stirring 30 min at 25 °C. 100 mg low molecular weight water soluble chitosan dissolved in 5 ml deionized water and the chitosan solution was dropped into the mixture. After overnight reaction, the mixture was dialyzed using dialysis tube with molecular weight cut-off of 3500 DA in distilled water for 3 days, then, freeze-dried (SIM, FD-5, USA). The preparation process of non-pH sensitive QT-CS was similar to that of QT-CA-CS, in which citraconic anhydride was replaced by succinic anhydride.
2.3. Preparation of nano- micelles QT-CA-CS and QT-CS nanomicelles were prepared by Ultrasound method according to previously report (Pang, Lu, Du, Yang, & Zhai, 2014). Briefly, QT-CA-CS or QT-CS was dissolved in DI water and dispersed using probe-type ultrasonic in ice bath for 6 min. DOX loaded QT-CA-CS-DOX and QT-CS-DOX nanomicelles were prepared as follow. QT-CA-CS or QT-CS conjugate with DOX were dissolved in 10 ml H2O-DMSO mixture (1:1, v/v). The mixture was stirred for 30 min and ultrasonicated for 6 min in ice bath by an ultrasonicator. Then, the mixture was put into a dialysis bag (MWCO 3500 DA) and dialyzed using deionized water for 12 h to remove the unentrapped DOX. The solution was lyophilized to obtain the QT-CSDOX nano-micelles after centrifugation (RCF 13700 g).
2. Materials and methods 2.1. Materials Low molecular weight water soluble chitosan, whose deacetylation degree and molecular weight are 84.2% and 11.7 kDa, respectively, was made by our laboratory as previous report (Mu et al., 2019). Cell Navigator Lysosome Staining Kit *Green* AAT bioquest 22656 was purchased from AAT Bioquest Inc. Quercetin and Dimethyl sulfoxide were
Fig. 1. Scheme of the high-efficiency MDR tumor inhibition of QT-CA-CS-DOX Micelles. 2
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Fig. 2. Synthesis route of the QT-CA-CS and QT-CS conjugates (A), 1H-NMR spectra of QT, CS and QT-CA-CS (B), FT-IR spectra of QT, CS and QT-CA-CS (C).
to determine the chemical structure of QT-CA-CS and QT-CS. For FTIR, the sample was dried in the oven at 80 °C and analyzed by FTIR with a disk of KBr. For 1H NMR spectroscopy, 5 mg sample was dissolved in 400 μL D2O and analyzed by 1H NMR The degree of substitution of QT on chitosan (DS) was calculated by UV spectrometry according our previously described method (Mu et al., 2019) using the equation:
Table 1 Degree of substitution of QT-CS and QT-CA-CS (by UV analysis). Polymer
CS glucosamine unit: QT (M/M)
DS of QT (%)
QT-CA-CS QT-CS
1:1 1:1
4.32 ± 0.43 4.67 ± 0.51
2.4. Characterization of conjugates and nanomicelles
DS (mol%) = Mcs × mQT/ (mQT-CS − mQT) MQT × 100%
FT-IR (FTIR, Nicolet, 5DX/550II, USA) and 1H NMR (Bruker ARX400 MHz spectrometer, Germany) spectroscopy were carrying out
Where mQT was the weight of QT (mg), mQT-CS was the weight of QTCA-CS or QT-CS (mg), MQT was the molecular weight of QT, MCS was 3
(1)
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Fig. 3. TEM images of QT-CA-CS and QT-CA-CS-DOX micelles (A), TEM images of QT-CA-CS, QT-CS, QT-CA-CS-DOX and QT-CS-DOX micelles at different pH (B), the Zeta potential and particle size of nanomicelles at different pH (C).
4
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Table 2 Nanomicelles' characteristics (pH 7.4). QT-CA-CS:DOX weight ratio (W/W)
Size (nm)
10:0 10:0.5 10:1 10:2
112.26 118.76 133.52 456.70
± ± ± ±
PDI 1.35 5.26 4.13 28.71
0.217 0.255 0.335 0.643
± ± ± ±
0.023 0.073 0.045 0.135
EE (%)
LC (%)
Zeta potential (mV)
– 89.79 ± 2.14 88.61 ± 1.57 –
– 4.72 ± 0.53 8.23 ± 0.82 –
+11.21 +12.21 +12.84 +13.56
± ± ± ±
1.25 1.65 1.66 1.19
Fig. 4. The release profiles of DOX and QT from QT-CS-CA-DOX micelles (A) and QT-CS- DOX micelles (B) in different pH.
Where DOXt was the total amount of DOX added; DOXf was the content of encapsulated DOX; Nanomicellest was the weight of nanomicelles.
the molecular weight of CS unit. The critical micelle concentration (CMC) of QT-CA-CS was determined by fluorescence measurement used pyrene as a fluorescence probe. Briefly, 10 μL of 1 × 10−5 M pyrene acetone solution was added to empty 1.5 ml tubes. After the acetone was removed, the solutions of QT-CA-CS with concentrations varying from 1.5625 μg/mL to 100 μg/ mL were added. After equilibrated 4 h at room temperature in dark, the different samples were measured using a spectrophotometer (F-4500 FL, Hitachi Co., Japan). The detection wavelength was 330 nm and emission wavelength in the range 350–500 nm was collected. The intensity ratio I373/I384 was plotted against the logarithm of conjugate concentration. The CMC value of QT-CA-CS conjugate was determined as the intersection point of two lines obtained by linear regression. 2 mg of the lyophilized sample was weighed and dissolved in deionized water or buffer. The nanomicelles solution was obtained after ultrasonication. The average size, polydispersity index (PDI) and zeta potential of the nanomicelles were measured by nano ZS90 Zetasizer (Malvern Instruments, Malvern, United Kingdom). The detector angle was 90◦ and the detect wave was 670 nm. The morphology of nano-micelles was observed by a transmission electron microscopy (TEM, JEM-2010, Japan). The copper mesh for transmission electron microscopy was carefully immersed in the diluted freshly prepared micelle suspension. After 3 min, the copper mesh was carefully removed and the liquid on the surface of the copper mesh was blotted with filter paper. The copper mesh was immersed in 1% phosphotungstic acid, stained for 1 min, dried and observed under a transmission electron microscope.
2.6. In vitro pH-responsive release of DOX The drug release profile of QT-CA-CS-DOX or QT-CS-DOX nanomicelles was examined using a dialysis method at different pH simulating physiological environment (pH 7.4), tumor environment (pH 6.0) and Lysosomal environment (pH 4.5) (Ray et al., 2018). Briefly, 5 mg QTCA-CS-DOX or QT-CS-DOX nanomicelles was dispersed in PBS buffer (pH 7.4), MES buffer (pH 6.2) or citric acid sodium citrate buffer (pH 4.5) buffers (1 mL) dialyzed (MWCO 3500) in the same buffers (4 mL) at 37 ℃. At fixed time intervals, the release buffer was taken out and replenished with the equal volume of fresh buffer. A Microplate Reader (PerkinElmer, Boston, MA, USA) was used to determine the release amount of DOX (480 nm) and QT (340 nm) by absorbance measurement of release buffer. 2.7. Cytotoxicity assay The cytotoxicity of MCF-7 or MCF-7/ADR cells incubated with QTCA-CS-DOX or QT-CS-DOX nanomicelles was evaluated by MTT assay. After 12 h incubation, the culture medium was replaced by RPMI-1640 without serum containing blank nanomicelles (QT-CA-CS or QT-CS), free DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX nanomicelles whose concentrations were equal to free DOX (50 μg/mL). The untreated cells were used as control. After incubating for another 24 h or 48 h, 20 μL, 5 mg/mL, MTT was added into wells and incubated for 4 h. Then, the media was replaced with 150 μL DMSO, shacked 15 min at 37 °C, and then, measured with Microplate Reader at 560 nm (PerkinElmer, Boston, MA, USA).
2.5. Drug encapsulation efficiency and loading efficiency QT-CA-CS-DOX or QT-CS-DOX nanomicelles was dispersed in DMSO, and then ultrasonicated for 20 min on ice bath. The structure of nano-micelles was destroyed and DOX was dissolved into DMSO. After centrifugation (RCF 13700 g), the content of encapsulated DOX was determined by a Microplate Reader (PerkinElmer, Boston, MA, USA, detection wavelength 480 nm). The drug loading capacity (LC%) and encapsulation efficiency (EE%) were calculated using Eqs. (2) and (3), respectively: EE (%) = (DOXt− DOXf) / DOXt × 100%
(2)
LC (%) = (DOXt− DOXf) / Nanomicellest × 100%
(3)
2.8. Intracellular delivery of DOX 2.8.1. Cellular uptake MCF-7 or MCF-7/ADR cells were grown in complete RPMI-1640. The cells at a logarithm phase were transferred to CLSM (confocal laser scanning microscopy) dishes at cell density of 2 × 105 cells/mL and incubated for 12 h. Then the culture medium was replaced with serumfree RPMI-1640 to equilibrium. After 30 min, the culture medium was replaced by 2 ml RPMI-1640 containing free DOX, 0.6 mg/ml QT-CA5
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Fig. 5. MCF-7 and MCF-7/ADR cells viability after treatment with QT-CS and QT- CA-CS after 24 h and 48 h (A), MCF-7 and MCF-7/ADR cells viability after treatment with free DOX, QT-CA-CS-DOX micelles, QT-CS-DOX micelles after 24 h (B), 48 h (C). The cell viability was relative to untreated control.
DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX at equal DOX concentration (50 μg/mL) for incubation 1 h. The cells were washed twice with PBS (pH 7.4), and treated by Trypsin-EDTA solution (2.5%), then collected by centrifugation (1000 RPM). Finally, the cells were resuspended in PBS and subjected to flow cytometry (BD Calibur, USA).
CS-DOX or 0.5 mg/ml QT-CS-DOX nanomicelles at the same DOX concentration (50 μg/mL). After incubated at 37 °C for another 1 h, the cells were rinsed with PBS (pH 7.4) and added 2 ml 4% formaldehyde was to fix cells. T Then, the nuclei was stained with 4', 6-diamidino-2-phenylindole (DAPI, 100 ng/ml, in PBS) for 10 min and rinsed three times with PBS. The cells were observed by confocal laser scanning microscopy.
2.8.3. Intracellular tracking assay MCF-7/ADR cells (2 × 105 cells/mL) were cultured on CLSM dishes (35 mm) in complete RPMI-1640 for 12 h. The culture medium was replaced with serum-free RPMI-1640 to equilibrium for 30 min. Then, the culture medium was replaced by 2 ml RPMI-1640 containing free DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX
2.8.2. Flow cytometric analysis MCF-7 or MCF-7/ADR cells (2 × 105 cells/mL) were culture on sixwell culture in complete RPMI-1640 for 12 h. Then the culture medium was replaced with serum-free RPMI-1640 to equilibrium. After 30 min, the culture medium was replaced by 2 ml RPMI-1640 containing free 6
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Fig. 6. Flow cytometry analysis results of fluorescence signals and CLSM images of the internalization of QT-CA-CS-DOX micelles, QT-CS-DOX micelles and free DOX in MCF-7 (A) and MCF-7/ADR cells (B). Cells were counter-stained with DAPI (blue) for nuclei.
nanomicelles at the same DOX concentration (50 μg/mL). After 4 h and 6 h incubation, the cells were washed twice with PBS (pH 7.4) and stained by Cell Navigator Lysosome Staining Kit *Green* bioquest AAT 22,656. The cells were washed twice with PBS again and 2 ml 4% formaldehyde was added to fix cells for 30 min. The nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI, 100 ng/ml, in PBS) for 10 min and washed triple times with PBS and observed by CLSM.
2.9. Statistical analysis The data was expressed as ‘mean ± SD’ and statistically analyzed by one way analysis of variance with Sigma Plot, version 11.0 (Systat Software Inc., US). The differences were considered to be statistically significant when the p value was less than 0.05. 3. Results and discussion
2.8.4. P-gp efflux inhibition The P-gp efflux inhibition of QT-CA-CS-DOX or QT-CS-DOX nanomicelles was evaluated using CLSM as our previous report (Mu et al., 2019). The verapamil was used as the positive control. MCF-7/ADR cells (2 × 105 cells/mL) were cultured on CLSM dishes (35 mm) in complete RPMI-1640 for 12 h. Then the culture medium was replaced by 2 ml RPMI-1640 containing verapamil (100 μM), QT(0.045 mg/ml), QT-CA-CS(1 mg/ml), or QT-CS(1 mg/ml) for 12 h. The RPMI-1640 without drug was used as negative control. Then, the culture medium was replaced by RPMI-1640 containing 5 μg/mL DOX and incubated for 6 h, Then washed triple times with PBS and observed using CLSM.
3.1. Synthesis and characterization of quercetin-chitosan conjugates with/ without acid labile linkage (QT-CA-CS and QT-CS) The pH-responsive QT-CA-CS conjugate was synthesized using citraconic anhydride to link QT and CS, which involved two-steps: modification of quercetin with citraconic anhydride to prepare QT-CA acid ester and carbodiimide reaction between amino groups of CS and carboxyl group of QT-CA acid ester to prepare QT-CA-CS conjugate (Fig. 2A). Under acidic conditions, the double bond in the citraconic acid amide promoted the internal attack of the amide carbonyl group by 7
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Fig. 7. CLSM images of the internalization of QT-CA-CS-DOX micelles, QT-CS-DOX micelles and free DOX in MCF-7/ADR cells after 4 h (A) or 12 h (B) incubation. Cells were counter-stained with DAPI (blue) for nuclei and Cell Navigator™ Lysosomal Staining Kit (green) for lysosomes.
characteristic peaks of benzene ring of QT (b 6.47 ppm, d 7.33 ppm, g 9.28 ppm), double bond (r 5.8 ppm) and eCH3 (q 2.52 ppm) of citraconic acid ester, which further confirmed the successful formation of QT-CA-CS conjugate (Fig. 2B). The DS of QT on CS molecular chain was evaluated by ultraviolet spectroscopy as our previous report. The DS of QT on two conjugates was similar (QT-CA-CS: 4.32 ± 0.43%, QT-CS: 4.67 ± 0.51%), when the molar ratio of the carboxyl group on QT to the amino group on chitosan was 1:1. (Table 1) In NMR data, the proton absorption peak area is proportional to the protons involved. The ratio of the proton peak areas of the two groups is the same as the molar ratio between the two groups (Holzgrabe, 2010). The method of substitution degree detection refers to the report of Wang et al. (Wang, Jiang, Chen, & Bai, 2016). The degree of substitution could calculate from the ratio of protons on the benzene ring of quercetin (Id 7.33 ppm) to the area under
the carboxylate, thereby accelerating the cleavage of the amide bond (Kang et al., 2014). Succinic anhydride was structurally similar to citraconic anhydride, which lacked double bonds compared to citraconic anhydride. Succinamide did not produce an intramolecular catalytic effect under acidic conditions and was more stable than citraconamide. Therefore, the no pH-responsive QT-CS conjugate was synthesized using succinic anhydride instead of citraconic anhydride to link QT and CS as similar process. The structure of QT-CA-CS conjugate was characterized by FTIR. The characteristic peaks of γ CeH (703 cm−1 and 802 cm−1) in QT were observed in QT-CA-CS conjugate spectrum (721 cm−1 and 824 cm−1). The characteristic peak of δ NeH (1649 cm−1) on CS was strengthened and changed to 1641 cm−1 on QT-CA-CS, which attributed to the formation of amido link between -NH3 on CS and –COOH on QT-CA (Fig. 2C). 1H-NMR spectrum of QT-CA-CS exhibited the 8
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Fig. 8. CLSM images of the internalization of DOX in MCF-7/ADR cells pretreat with verapamil, free QT, blank QT-CS nanomicelles or blank QT-CA-CS nanomicelles. Cells were counter-stained with DAPI (blue) for nuclei.
in aqueous solution via hydrophobic interactions. In general, nanomicelles have significantly lower CMC than low molecular weight surfactants, thus providing greater resistance to dissociation upon dilution. The CMC value of QT-CA-CS conjugate was 0.034 mg/mL. QT-CA-CS nano-micelles showed spherical in shape with smooth surface (Fig. 3A), positive surface charge (Zeta potential: +11.21 ± 1.25 mV to +13.56 ± 1.19 mV), favorable dispersibility (PDI: 0.217 ± 0.023 to 0.335 ± 0.045) and high cargo capacity for DOX. The size distribution histograms of QT-CA-CS and QT-CA-CS-DOX were shown in Fig. S1. As the pH decreased, the structure of the pH-sensitive QT-CA-CS or QT-CACS-DOX micelles gradually disintegrated and the particle size increased.
the H2-H6 proton signal (I l, m, n, o, p 2.96–3.73 ppm) on the chitosan unit. DS = (5 Id /I l, m, n, o, p) ×100%. The DS of QT-CA-CS and QT-CS detected by 1H-NMR spectrum was 2.06% and 2.23% respectively, which was significant less than those of UV-results. This phenomenon was consistent with previous reports, which might attribute to inadequate dissolution of polymer conjugates (Wang et al., 2014). 3.2. Preparation and characterization of nano-micelles QT-CA-CS conjugate was able to self-assemble to form nano-micelles 9
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pH-responsive QT-CA-CS-DOX nano-micelles exhibited the highest cytotoxicity during 48 h incubation, especially for MCF-7/ADR. The MCF7/ADR cell viability treated by 50 μg/mL QT-CA-CS-DOX reduced to less than 25% after 24 h incubation, even less than 4% after 48 h incubation, which was about 9% and 7% of free DOX and QT-CS-DOX nano-micelles treated groups, respectively. In our previous study, QTCS-DOX with no pH-responsive nano-micelles showed favorable P-gp inhibition against MDR, however, in this study, QT-CS-DOX nano-micelles displayed slight inhibitory effect for both of MCF-7 and MCF-7/ ADR at all doses during 48 h incubation, even weaker than that of free DOX treated groups. The low cytotoxicity of QT-CS-DOX compared to free DOX may be due to the difficulty in releasing DOX from the nanomicelles in the cells, so that DOX could not exert its effects in the nucleus. Based on the profiles of in vitro drug release (Fig. 4), the above results could be concluded that the improved anti-cancer effect of QTCA-CS-DOX nano-micelles was attributed its pH-responsive drug release behavior. The acid labile linkage between QT and CS in QT-CA-CS-DOX nanomicelles was broken at endo/lysosome environment, leading to rapidly release of DOX and QT, thus generating high efficiency inhibitory effect against tumor cells. We studied the cytotoxicity of our nanomicelles on healthy cells, the L929 cell line. We found that nanomicelles without DOX showed no cytotoxicity, DOX and DOX-containing nanomicelles were less toxic within 24 h. The possible reason is due to the low sensitivity of the L929 cell line to DOX (Fig. S2).
As a comparison, the structure of QT-CS or QT-CS-DOX did not change significantly (Fig. 3B, C). This may be due to the fact that the citraconic amide in QT-CA-CS breaks under acidic conditions, causing the structure of the micelle to be destroyed. The succinamide in QT-CS is stable under acidic conditions, so that the micelle structure remains intact. The maximum EE and LE was 88.61 ± 1.57% and 8.23 ± 0.82%, respectively, when weight ratio of QT-CA-CS to DOX was 10:1. With increase of weight ratio of QT-CA-CS to DOX, the particle size increase from 112.26 ± 1.35 nm to 133.52 ± 4.13 nm, which indirectly proved the successful encapsulation of QT-CA-CS conjugate for DOX. Compared to QT-CS nanomicelles in our previously reported, QT-CA-CS nanomicelles had a larger particle size and a lower surface charge. This might be due to the reaction of residual free citraconic acid with chitosan. The carboxyl group in citraconic acid reduced the surface charge of the QT-CA-CS nanomicelles, and the hydrophilicity of the surface of the micelles increases, and the hydration radius of the micelles increases. For QT-CS conjugate, the same ratio of QT-CS to DOX (10:1) bring out similar EE (89.31 ± 1.67%) and LE (8.76 ± 0.24%), which was used as control for subsequent studies (Table 2). 3.3. In vitro release of DOX In vitro drug release experiments were carried out at different pH for simulating corresponding physiological environment (pH 7.4: blood circulation environment, pH 6.0: tumor tissue environment, pH 4.5: endo/lysosome environment). For pH-responsive QT-CA-CS-DOX nanomicelles, the drug release rate was significantly increased with decrease of pH value (Fig. 4A). At pH 7.4, QT-CA-CS-DOX exhibited slowly sustained release of DOX, reached to 36.34% within 48 h. On contrast, no significant QT release (less than 4%) was observed in QT-CA-CSDOX, indicated that the acid labile linkage between CS and QT was stable at blood circulation environment. At pH 6.0, the cumulative release rate of DOX and QT from QT-CA-CS-DOX was raised to 45.32% and 16.22%, respectively, which were slight higher than that of QT-CSDOX nano-micelles (Fig. 4B). At pH 4.5, QT-CA-CS-DOX nano-micelles could disassembly by breakage of acid labile linkage to achieve rapid release of DOX and QT, on the other hand, the doxorubicin was able to protonate in acid medium, which changed it from hydrophobic to hydrophilic and promoted the diffusion capacity of DOX. The cumulative drug release rate was obvious faster than that in pH 6.0 and pH 7.4, giving 80.75% for DOX and 42.34% for QT within 48 h. For no pHresponsive QT-CS-DOX nano-micelles, no significant QT release (less than 5%) was detected at each pH within 48 h, due to the stable covalent linkage between CS and QT. Meanwhile, the similar DOX release profiles (no more than 40%) were found at different pH value within 48 h, demonstrated that the structural integrity of QT-CS-DOX nano-micelles hampered the drug release at physiological environment (Fig. 4B). The QT and DOX release of QT-CS-DOX at pH 7.4 were much lower than the previously reported release rate of QT-CS-DOX. In our previous study, we used simulated intestinal fluid at pH 7.4 as the drug release medium. The simulated intestinal fluid contains trypsin, which may react with chitosan to destroy the structure of the micelle and cause drug release. This may be why QT-CS-DOX has different release behaviors in this study.
3.5. Intracellular delivery of DOX 3.5.1. Cellular uptake The cellular uptake of free DOX, QT-CA-CS-DOX and QT-CS-DOX by MCF-7 and MCF-7/ADR cell line was studied using CLSM and flow cytometry after 4 h incubation (Fig. 6). The overlap of red (DOX) and blue (DAPI) fluorescence signal performed purple indicated the location of DOX in nucleus. For MCF-7 cell line, QT-CS-DOX and QT-CA-CS-DOX treated groups exhibited stronger DOX signal compared to that of free DOX treated group, which was in accord with results of flow cytometry (Fig. 6A). However, the purple signal of QT-CS-DOX and QT-CA-CSDOX treated groups was weaker than that of free DOX treated group, which attributed to the relative drug release rate of nanomicells. For MCF-7/ADR, weak DOX signal was detected in free DOX treated group, verified the drug resistance of MCF-7/ADR. QT-CS-DOX and QT-CA-CSDOX obviously promoted DOX cellular uptake by MCF-7/ADR, which were about eight-fold higher than that of free DOX. This phenomenon indicated that the QT component on QT-CS-DOX and QT-CA-CS-DOX was able to mitigate drug efflux effect of MCF-7/ADR. QT-CA-CS-DOX treated group displayed the strongest purple signal compared to free DOX and QT-CS-DOX treated groups, which means more internalized DOX entered into MCF-7/ADR nucleus. Based on the drug release profile and cytotoxicity assay results, we speculated that the rapid DOX release of QT-CA-CS-DOX in acidic environment provide more released DOX in cytoplasm, thus, actively promoted accumulation of DOX in MCF-7/ADR nucleus, leading to high efficiency inhibitory effect against tumor cells. 3.5.2. Intracellular tracking assay To confirm our hypothesis, intracellular tracking assay was carried out in kinetics of incubation time exposure MCF-7/ADR. The endo/lysosomes were stained by Cell Navigator™Lysosomal Staining Kit with green fluorescence. After 4 h incubation, nearly all of DOX signal in cells treated with free DOX was overlapped with lysosomes green signal. For QT-CS-DOX treated group, a few scattering dots like DOX signal was disperse in tumor nucleus and most of that still located in lysosomes. In contrast, as for QT-CA-CS-DOX treated group, extensive DOX signal were diffused in nucleus, which rarely overlapped with green fluorescence of lysosomes (Fig. 7A). After 12 h incubation, strong DOX signal was observed in tumor nuclear regions treated with QT-CA-
3.4. Cytotoxicity assay Human breast cancer cell line with and without DOX resistance (MCF-7, MCF-7/ADR) were used as models to evaluate the cytotoxicity of drug loaded and blank nano-micelles (Fig. 5). No significant cytotoxicity was observed in blank QT-CS or QT-CA-CS nano-micelles treated MCF-7 and MCF-7/ADR groups, indicating that the favorable cytocompatibility of QT-CA-CS nano-micelles (Fig. 5A). Concentrationdependent cell viability, which decreased with the increase of DOX concentration, was observed in the cells treated with free DOX, QT-CACS-DOX and QT-CS-DOX nano-micelle (Fig. 5B and C). Among them, 10
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References
CS-DOX, and its intensity significantly higher than that treated with free DOX and QT-CS-DOX (Fig. 7B). Under endo/lysosomal acidic environment (pH 4.5–5.5), the broken of acid labile linkage between QT and CS in QT-CA-CS-DOX bring out the rapid release of DOX and QT. On the other hand, the“proton sponge” effect resulting from the protonation of amino group in CS component of QT-CA-CS-DOX promoted the disruption of endo/lysosomes and facilitate the released DOX and QT escape from endo/lysosome (Richard, Thibault, De Crescenzo, Buschmann, & Lavertu, 2013). Compared with QT-CA-CS-DOX, no pHsensitive QT-CS-DOX was difficult to release the loaded DOX and trapped in endo/lysosome because of its stable structure in endo/lysosomal acidic environment. The red fluorescence of DOX signal in QTCS-DOX treated group was weak and similar with that of free DOX treated group. These results was well verified our hypothesis and illuminated the intracellular delivery kinetics of QT-CA-CS-DOX.
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Effective cancer therapy based on selective drug delivery into cells across their membrane using receptor-mediated endocytosis. Bioorganic & Medicinal Chemistry Letters, 28(18), 3015–3024. Tayo, L. L. (2017). Stimuli-responsive nanocarriers for intracellular delivery. Biophysical Reviews, 9(6), 931–940. Tuguntaev, R. G., Chen, S., Eltahan, A. S., Mozhi, A., Jin, S., Zhang, J., ... Liang, X. J. (2017). P-gp inhibition and mitochondrial impairment by dual-functional nanostructure based on vitamin e derivatives to overcome multidrug resistance. ACS Applied Materials & Interfaces, 9(20), 16900–16912. Turk, B., & Turk, V. (2009). Lysosomes as “suicide bags” in cell death: Myth or reality? The Journal of Biological Chemistry, 284(33), 21783–21787. Wang, J., Jiang, J. Z., Chen, W., & Bai, Z. W. (2016). Data of (1)H/(13)C NMR spectra and degree of substitution for chitosan alkyl urea. Data in Brief, 7, 1228–1236. Wang, X., Chen, Y., Dahmani, F. Z., Yin, L., Zhou, J., & Yao, J. (2014). Amphiphilic carboxymethyl chitosan-quercetin conjugate with P-gp inhibitory properties for oral delivery of paclitaxel. Biomaterials, 35(26), 7654–7665. WT, B. (1996). P-glycoproteins and multidrug resistance. Annual Review of Pharmacology and Toxicology, 1996(36), 161–183. Xia, X., Cole, S. P. C., Cai, T., & Cai, Y. (2017). Effect of traditional Chinese medicine components on multidrug resistance in tumors mediated by P-glycoprotein. Oncology Letters, 13(6), 3989–3996. Yang, K., Feng, L., & Liu, Z. (2016). Stimuli responsive drug delivery systems based on nano-graphene for cancer therapy. Advanced Drug Delivery Reviews, 105(Pt. B), 228–241. Yang, X., Ding, Y., Xiao, M., Liu, X., Ruan, J., & Xue, P. (2017). Anti-tumor compound RY10-4 suppresses multidrug resistance in MCF-7/ADR cells by inhibiting PI3K/Akt/ NF-kappaB signaling. Chemico-Biological Interactions, 278, 22–31. Yuan, Y. Y., Mao, C. Q., Du, X. J., Du, J. Z., Wang, F., & Wang, J. (2012). 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3.5.3. P-gp efflux inhibition P-gp was closely associated with MDR development and highly expressed in MCF-7/ADR cell (Yang et al., 2017). To further confirm P-gp efflux inhibition of nano-micellar, the MCF-7/ADR were respectively pretreated with commercialize P-gp inhibitor verapamil, free QT, blank QT-CS nano-micelles or blank QT-CA-CS nano-micelles, then, incubated with DOX and observed its fluorescence intensity using CLSM (Fig. 8). Compared with control group directly treated with DOX, the verapamil, QT, QT-CS and QT-CA-CS pretreated group exhibited strong DOX signal, reflected their high efficiency of P-gp efflux. There was no significant difference in DOX signal intensity among QT and QT-CA-CS pretreated group, which was apparently higher than that pretreated with QT-CS. The difference in the efficiency of P-gp efflux was due to the different utilization of the QT loaded on the carrier. Unlike QT-CS nano-micellar whose P-gp efflux inhibition rooted in QT component in conjugate, the P-gp efflux inhibition of QT-CA-CS derived from the free QT that released by the broken of acid labile linkage between QT and CS in QT-CA-CS. The above data proves that QT-CA-CS-DOX nano-micellar could prevent the drug from being discharged by P-gp to maintain the concentration of intracellular drugs during the treatment within 12 h. Flow cytometry analysis results were shown in supporting information (Fig. S4). 4. Conclusions In conclusion, a pH-sensitive QT-CA-CS conjugate was prepared using an acid labile linkage between QT and CS for intracellular delivery of DOX. QT-CA-CS conjugate could self-assemble to nano-micelle by ultrasound method and provide high encapsulation efficiency for DOX. The pH-sensitivity of QT-CA-CS-DOX gave it efficient drug intracellular accumulation and drug efflux mitigation, performed as cell uptake promotion, escape and rapid drug release response to endo/lysosome acidic pH, along with high DOX level accumulation in tumor nucleus, generating high efficiency inhibitory effect against MCF-7 with/without MDR. This study provided an efficient nano-carrier platform for delivery of DOX against MDR cancer cells. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81801846, U1706212 and 81671828), Natural Science Foundation of Shandong Province (ZR2019QD005), Applied Basic Research Plan of Qingdao (No.16-5-1-70-jch) and the Taishan Scholar Program. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115072. 11
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and mechanism underlying endolysosomal escape for intracellular delivery. Biomacromolecules, 15(11), 4032–4045. Zuo, Y., Kong, M., Mu, Y., Feng, C., & Chen, X. (2017). Chitosan based nanogels stepwise response to intracellular delivery kinetics for enhanced delivery of doxorubicin. International Journal of Biological Macromolecules, 104(Pt. A), 157–164.
Zhang, C., Zhao, X., Guo, S., Lin, T., & Guo, H. (2017). Highly effective photothermal chemotherapy with pH-responsive polymer-coated drug-loaded melanin-like nanoparticles. International Journal of Nanomedicine, 12, 1827–1840. Zhang, X., Chen, D., Ba, S., Zhu, J., Zhang, J., Hong, W., ... Qiao, M. (2014). Poly(lhistidine) based triblock copolymers: pH induced reassembly of copolymer micelles
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
pH-sensitive amphiphilic chitosan-quercetin conjugate for intracellular delivery of doxorubicin enhancement
T
Yuzhi Mua, Guangsheng Wub, Chang Sua, Yao Donga, Kaichao Zhanga, Jing Lia, Xiaojie Suna, ⁎ Yang Lia, Xiguang Chena,c, Chao Fenga, a
College of Marine Life Science, Ocean University of China, 5# Yushan Road, Qingdao 266003, Shandong Province, China Navy Qingdao First Sanatorium of PLA, No. 27 West Hong Kong Road, Qingdao 266071, Shandong Province, China c Qingdao National Laboratory for Marine Science and Technology, 1# Wenhai Road, Qingdao 266000, Shandong Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Quercetin Drug conjugate Intracellular delivery Multidrug resistance effect P-glycoprotein
A novel pH-responsive nanomicelle (QT-CA-CS) based on Chitosan, Quercetin and Citraconic anhydride was reported in this study. The QT-CA-CS could self-assemble into nanomicelles for encapsulating anticancer drug doxorubicin (DOX) by ultrasound. The novel nanomicelles had P-gp inhibition and pH responsiveness, which was capable of inhibiting drug efflux and responding to an endo/lysosomal acidic environment. The drug loaded nanomicelles had high encapsulation rate (more than 80%), small particle size (133.52 ± 4.13 nm) and positive zeta potential (+13.5 mV). The release rate of doxorubicin and quercetin in pH 4.5 was faster than that in pH 7.4. QT-CA-CS-DOX nanomicelles could promote cellular uptake of doxorubicin by drug resistance cell line (MCF-7/ADR), which was 8.62 folds higher than that of free doxorubicin. Most importantly, QT-CA-CS-DOX nanomicelles could escape from lysosomes and rapidly release doxorubicin and quercetin in the cytoplasm, which had an enhanced inhibitory effect on tumor cells, especially for MCF-7/ADR. The above results proved that the high potential of QT-CA-CS-DOX nanomicelles for multidrug resistance related tumor therapy.
1. Introduction Intracellular delivery of anticancer drug in cancer cells is primary for chemotherapy, which determines the efficacy and side effect of anticancer agent (Kim, Faix, & Schnitzer, 2017; Rao, Ko, Lee, & Park, 2018; Tashima, 2018). This process comprises series of successive events that can be summarized as drug intracellular accumulation and drug efflux mitigation. On the one hand, anticancer drug should pass through cell recognition, cell uptake, endo-lysosomal escape, and cytoplasmic localization, to achieve effective accumulation in cancer cell. On the other hand, it must get rid of drug efflux resulted from multidrug resistance effect (MDR) of cancer cells, to maintain high level drug concentration in cancer cell. The strategy for improving intracellular drug accumulation and reducing MDR is urgently required (Tuguntaev et al., 2017). Stimuli responsive nano-carrier provides a smart platform for responding specific physiological tumor microenvironment, such as acidic pH, redox property, certain enzymes or glucose concentration or their combinations (Aji Alex et al., 2017; Tayo, 2017; Yang, Feng, & Liu, 2016; Zuo, Kong, Mu, Feng, & Chen, 2017). Due to the particular acidic pH around tumor tissue (pH 6.0) and endo/ environment (pH 4.5), ⁎
various pH-sensitive nanocarriers have been designed to realize triggered drug release in cytoplasm (Turk & Turk, 2009). One strategy is the introduction of acid sensitive group, such as amido, carboxyl or imidazolyl in polymer chain, that release drug by pH-induced disassembly or swelling behavior. The other one is the incorporation an acid labile linkage between polymer chain and drug molecule that is cleaved in acidic environment to bring drug release (Feng, Zhang, Zhi, Gao, & Nakanishi, 2018; Li et al., 2015; Liu et al., 2014; Zhang, Zhao, Guo, Lin, & Guo, 2017, 2014). A previous research proved that the two approaches above had shown excellent intracellular drug accumulation in cancer cell. However, these stimuli responsive nano-carrier rarely involved reversal of MDR of cancer cells, which restricted their therapeutic efficacy. MDR, derived from recurrent chemotherapy, is important cause of chemotherapy failure. MDR mainly associate with the overexpression of multidrug-resistant proteins and membrane P-glycoprotein (P-gp) transporters, which actively increase drug efflux to limit the efficiency of anticancer agents (Mohana, Ganesan, Rajendra Prasad, Ananthakrishnan, & Velmurugan, 2018; Mollazadeh, Sahebkar, Hadizadeh, Behravan, & Arabzadeh, 2018; WT, 1996). It has been suggested that the MDR of cancer cells to chemotherapeutics can be
Corresponding author. E-mail addresses: [email protected], [email protected] (C. Feng).
https://doi.org/10.1016/j.carbpol.2019.115072 Received 10 December 2018; Received in revised form 2 July 2019; Accepted 7 July 2019 Available online 08 July 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy succinimide were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) and RPMI-1640 were purchased from Hyclone (Logan, UT, USA). All other chemicals were of analytical grade and were used without further purification. MCF-7 cells and L929 cells were kindly provided by Cell Bank, Chinese Academy of Sciences. MCF-7/ADR cells were purchased from Shanghai Yu Bo Biotech Co., Ltd.
suppressed by combine anticancer agents with P-gp inhibitor, such as cyclosporine A, verapamil and so on (Tuguntaev et al., 2017). Compare to various synthetic P-gp inhibitor, quercetin (QT), a polyphenolic flavonoid compound derived from tea, apple, onions, broccoli and berries, display promising P-gp inhibition activities and minimal side effects. Importantly, QT also exhibit favorable competitively inhibition against the most MDR protein family, such as MRP1 and BCRP, as well as CYP3A subfamily of the cytochrome P-450, which have been verified on various drug-resistant cancer cell lines and animal models (Borska et al., 2012; Wang et al., 2014; Xia, Cole, Cai, & Cai, 2017). Chitosan (CS) is a natural high molecular polysaccharide that is hydrophilic, cationic, biocompatible and biodegradable. In an acidic solution, the amino group of CS is protonated to carry a positive charge. CS has significant advantages in blood circulation retention and negligible accumulation in body tissues, and thus CS is currently a suitable excellent material for pharmaceutical carriers (Kean & Thanou, 2010; Mu et al., 2019). To combat MDR and improve accumulation of anticancer drug in tumor cells, a pH-responsive amphiphilic chitosan-quercetin conjugate was synthesized using an acid labile linkage (citraconoyl, CA) between QT and CS (QT-CA-CS) in this study. The amide bond formed by the reaction of citraconic anhydride with an amino group could be destroyed under acidic conditions, thereby generating an acid-responsive behavior (Cao et al., 2014; Prata et al., 2004; Yuan et al., 2012). It was expected that the QT-CA-CS nanomicelles could disassembly responding the acid pH of endo/lysosome environment by breakage of linkage, thus bring out rapid release of DOX and QT to achieve highefficiency tumor inhibition (Fig. 1). This study devoted to concentrate the intracellular drug accumulation and the MDR mitigation in QT-CACS nano-micelles for developing an effective delivery platform, particularly for MDR cancer cells. The self-assemble behavior, drug encapsulation and pH-responsive release pattern of QT-CA-CS nano-micelles were investigated in vitro. Furthermore, the cellular uptake, endo/lysosome escape, intracellular drug release and antitumor activity were comparatively investigated in drug-sensitive and resistant cancer cells.
2.2. Synthesis of quercetin-chitosan conjugates (QT-CA-CS and QT-CS) The pH-sensitive QT-CA-CS was synthetized using carbodiimide reaction as follow (Mu et al., 2019). QT was firstly treated with citraconic anhydride (citraconic anhydride: QT = 1.3:1, at molar ratio) and magnetic stirred overnight at 40 °C in DMSO. The product was precipitated with deionized water. After centrifugation (RCF 900g), the precipitation was redissolved in DMSO. EDC·HCl and NHS mixture, which molar ratio was EDC: NHS = 1:1.5 in 1 ml of DMSO was added under stirring 30 min at 25 °C. 100 mg low molecular weight water soluble chitosan dissolved in 5 ml deionized water and the chitosan solution was dropped into the mixture. After overnight reaction, the mixture was dialyzed using dialysis tube with molecular weight cut-off of 3500 DA in distilled water for 3 days, then, freeze-dried (SIM, FD-5, USA). The preparation process of non-pH sensitive QT-CS was similar to that of QT-CA-CS, in which citraconic anhydride was replaced by succinic anhydride.
2.3. Preparation of nano- micelles QT-CA-CS and QT-CS nanomicelles were prepared by Ultrasound method according to previously report (Pang, Lu, Du, Yang, & Zhai, 2014). Briefly, QT-CA-CS or QT-CS was dissolved in DI water and dispersed using probe-type ultrasonic in ice bath for 6 min. DOX loaded QT-CA-CS-DOX and QT-CS-DOX nanomicelles were prepared as follow. QT-CA-CS or QT-CS conjugate with DOX were dissolved in 10 ml H2O-DMSO mixture (1:1, v/v). The mixture was stirred for 30 min and ultrasonicated for 6 min in ice bath by an ultrasonicator. Then, the mixture was put into a dialysis bag (MWCO 3500 DA) and dialyzed using deionized water for 12 h to remove the unentrapped DOX. The solution was lyophilized to obtain the QT-CSDOX nano-micelles after centrifugation (RCF 13700 g).
2. Materials and methods 2.1. Materials Low molecular weight water soluble chitosan, whose deacetylation degree and molecular weight are 84.2% and 11.7 kDa, respectively, was made by our laboratory as previous report (Mu et al., 2019). Cell Navigator Lysosome Staining Kit *Green* AAT bioquest 22656 was purchased from AAT Bioquest Inc. Quercetin and Dimethyl sulfoxide were
Fig. 1. Scheme of the high-efficiency MDR tumor inhibition of QT-CA-CS-DOX Micelles. 2
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Fig. 2. Synthesis route of the QT-CA-CS and QT-CS conjugates (A), 1H-NMR spectra of QT, CS and QT-CA-CS (B), FT-IR spectra of QT, CS and QT-CA-CS (C).
to determine the chemical structure of QT-CA-CS and QT-CS. For FTIR, the sample was dried in the oven at 80 °C and analyzed by FTIR with a disk of KBr. For 1H NMR spectroscopy, 5 mg sample was dissolved in 400 μL D2O and analyzed by 1H NMR The degree of substitution of QT on chitosan (DS) was calculated by UV spectrometry according our previously described method (Mu et al., 2019) using the equation:
Table 1 Degree of substitution of QT-CS and QT-CA-CS (by UV analysis). Polymer
CS glucosamine unit: QT (M/M)
DS of QT (%)
QT-CA-CS QT-CS
1:1 1:1
4.32 ± 0.43 4.67 ± 0.51
2.4. Characterization of conjugates and nanomicelles
DS (mol%) = Mcs × mQT/ (mQT-CS − mQT) MQT × 100%
FT-IR (FTIR, Nicolet, 5DX/550II, USA) and 1H NMR (Bruker ARX400 MHz spectrometer, Germany) spectroscopy were carrying out
Where mQT was the weight of QT (mg), mQT-CS was the weight of QTCA-CS or QT-CS (mg), MQT was the molecular weight of QT, MCS was 3
(1)
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Fig. 3. TEM images of QT-CA-CS and QT-CA-CS-DOX micelles (A), TEM images of QT-CA-CS, QT-CS, QT-CA-CS-DOX and QT-CS-DOX micelles at different pH (B), the Zeta potential and particle size of nanomicelles at different pH (C).
4
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Table 2 Nanomicelles' characteristics (pH 7.4). QT-CA-CS:DOX weight ratio (W/W)
Size (nm)
10:0 10:0.5 10:1 10:2
112.26 118.76 133.52 456.70
± ± ± ±
PDI 1.35 5.26 4.13 28.71
0.217 0.255 0.335 0.643
± ± ± ±
0.023 0.073 0.045 0.135
EE (%)
LC (%)
Zeta potential (mV)
– 89.79 ± 2.14 88.61 ± 1.57 –
– 4.72 ± 0.53 8.23 ± 0.82 –
+11.21 +12.21 +12.84 +13.56
± ± ± ±
1.25 1.65 1.66 1.19
Fig. 4. The release profiles of DOX and QT from QT-CS-CA-DOX micelles (A) and QT-CS- DOX micelles (B) in different pH.
Where DOXt was the total amount of DOX added; DOXf was the content of encapsulated DOX; Nanomicellest was the weight of nanomicelles.
the molecular weight of CS unit. The critical micelle concentration (CMC) of QT-CA-CS was determined by fluorescence measurement used pyrene as a fluorescence probe. Briefly, 10 μL of 1 × 10−5 M pyrene acetone solution was added to empty 1.5 ml tubes. After the acetone was removed, the solutions of QT-CA-CS with concentrations varying from 1.5625 μg/mL to 100 μg/ mL were added. After equilibrated 4 h at room temperature in dark, the different samples were measured using a spectrophotometer (F-4500 FL, Hitachi Co., Japan). The detection wavelength was 330 nm and emission wavelength in the range 350–500 nm was collected. The intensity ratio I373/I384 was plotted against the logarithm of conjugate concentration. The CMC value of QT-CA-CS conjugate was determined as the intersection point of two lines obtained by linear regression. 2 mg of the lyophilized sample was weighed and dissolved in deionized water or buffer. The nanomicelles solution was obtained after ultrasonication. The average size, polydispersity index (PDI) and zeta potential of the nanomicelles were measured by nano ZS90 Zetasizer (Malvern Instruments, Malvern, United Kingdom). The detector angle was 90◦ and the detect wave was 670 nm. The morphology of nano-micelles was observed by a transmission electron microscopy (TEM, JEM-2010, Japan). The copper mesh for transmission electron microscopy was carefully immersed in the diluted freshly prepared micelle suspension. After 3 min, the copper mesh was carefully removed and the liquid on the surface of the copper mesh was blotted with filter paper. The copper mesh was immersed in 1% phosphotungstic acid, stained for 1 min, dried and observed under a transmission electron microscope.
2.6. In vitro pH-responsive release of DOX The drug release profile of QT-CA-CS-DOX or QT-CS-DOX nanomicelles was examined using a dialysis method at different pH simulating physiological environment (pH 7.4), tumor environment (pH 6.0) and Lysosomal environment (pH 4.5) (Ray et al., 2018). Briefly, 5 mg QTCA-CS-DOX or QT-CS-DOX nanomicelles was dispersed in PBS buffer (pH 7.4), MES buffer (pH 6.2) or citric acid sodium citrate buffer (pH 4.5) buffers (1 mL) dialyzed (MWCO 3500) in the same buffers (4 mL) at 37 ℃. At fixed time intervals, the release buffer was taken out and replenished with the equal volume of fresh buffer. A Microplate Reader (PerkinElmer, Boston, MA, USA) was used to determine the release amount of DOX (480 nm) and QT (340 nm) by absorbance measurement of release buffer. 2.7. Cytotoxicity assay The cytotoxicity of MCF-7 or MCF-7/ADR cells incubated with QTCA-CS-DOX or QT-CS-DOX nanomicelles was evaluated by MTT assay. After 12 h incubation, the culture medium was replaced by RPMI-1640 without serum containing blank nanomicelles (QT-CA-CS or QT-CS), free DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX nanomicelles whose concentrations were equal to free DOX (50 μg/mL). The untreated cells were used as control. After incubating for another 24 h or 48 h, 20 μL, 5 mg/mL, MTT was added into wells and incubated for 4 h. Then, the media was replaced with 150 μL DMSO, shacked 15 min at 37 °C, and then, measured with Microplate Reader at 560 nm (PerkinElmer, Boston, MA, USA).
2.5. Drug encapsulation efficiency and loading efficiency QT-CA-CS-DOX or QT-CS-DOX nanomicelles was dispersed in DMSO, and then ultrasonicated for 20 min on ice bath. The structure of nano-micelles was destroyed and DOX was dissolved into DMSO. After centrifugation (RCF 13700 g), the content of encapsulated DOX was determined by a Microplate Reader (PerkinElmer, Boston, MA, USA, detection wavelength 480 nm). The drug loading capacity (LC%) and encapsulation efficiency (EE%) were calculated using Eqs. (2) and (3), respectively: EE (%) = (DOXt− DOXf) / DOXt × 100%
(2)
LC (%) = (DOXt− DOXf) / Nanomicellest × 100%
(3)
2.8. Intracellular delivery of DOX 2.8.1. Cellular uptake MCF-7 or MCF-7/ADR cells were grown in complete RPMI-1640. The cells at a logarithm phase were transferred to CLSM (confocal laser scanning microscopy) dishes at cell density of 2 × 105 cells/mL and incubated for 12 h. Then the culture medium was replaced with serumfree RPMI-1640 to equilibrium. After 30 min, the culture medium was replaced by 2 ml RPMI-1640 containing free DOX, 0.6 mg/ml QT-CA5
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Fig. 5. MCF-7 and MCF-7/ADR cells viability after treatment with QT-CS and QT- CA-CS after 24 h and 48 h (A), MCF-7 and MCF-7/ADR cells viability after treatment with free DOX, QT-CA-CS-DOX micelles, QT-CS-DOX micelles after 24 h (B), 48 h (C). The cell viability was relative to untreated control.
DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX at equal DOX concentration (50 μg/mL) for incubation 1 h. The cells were washed twice with PBS (pH 7.4), and treated by Trypsin-EDTA solution (2.5%), then collected by centrifugation (1000 RPM). Finally, the cells were resuspended in PBS and subjected to flow cytometry (BD Calibur, USA).
CS-DOX or 0.5 mg/ml QT-CS-DOX nanomicelles at the same DOX concentration (50 μg/mL). After incubated at 37 °C for another 1 h, the cells were rinsed with PBS (pH 7.4) and added 2 ml 4% formaldehyde was to fix cells. T Then, the nuclei was stained with 4', 6-diamidino-2-phenylindole (DAPI, 100 ng/ml, in PBS) for 10 min and rinsed three times with PBS. The cells were observed by confocal laser scanning microscopy.
2.8.3. Intracellular tracking assay MCF-7/ADR cells (2 × 105 cells/mL) were cultured on CLSM dishes (35 mm) in complete RPMI-1640 for 12 h. The culture medium was replaced with serum-free RPMI-1640 to equilibrium for 30 min. Then, the culture medium was replaced by 2 ml RPMI-1640 containing free DOX, 0.6 mg/ml QT-CA-CS-DOX or 0.5 mg/ml QT-CS-DOX
2.8.2. Flow cytometric analysis MCF-7 or MCF-7/ADR cells (2 × 105 cells/mL) were culture on sixwell culture in complete RPMI-1640 for 12 h. Then the culture medium was replaced with serum-free RPMI-1640 to equilibrium. After 30 min, the culture medium was replaced by 2 ml RPMI-1640 containing free 6
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Fig. 6. Flow cytometry analysis results of fluorescence signals and CLSM images of the internalization of QT-CA-CS-DOX micelles, QT-CS-DOX micelles and free DOX in MCF-7 (A) and MCF-7/ADR cells (B). Cells were counter-stained with DAPI (blue) for nuclei.
nanomicelles at the same DOX concentration (50 μg/mL). After 4 h and 6 h incubation, the cells were washed twice with PBS (pH 7.4) and stained by Cell Navigator Lysosome Staining Kit *Green* bioquest AAT 22,656. The cells were washed twice with PBS again and 2 ml 4% formaldehyde was added to fix cells for 30 min. The nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI, 100 ng/ml, in PBS) for 10 min and washed triple times with PBS and observed by CLSM.
2.9. Statistical analysis The data was expressed as ‘mean ± SD’ and statistically analyzed by one way analysis of variance with Sigma Plot, version 11.0 (Systat Software Inc., US). The differences were considered to be statistically significant when the p value was less than 0.05. 3. Results and discussion
2.8.4. P-gp efflux inhibition The P-gp efflux inhibition of QT-CA-CS-DOX or QT-CS-DOX nanomicelles was evaluated using CLSM as our previous report (Mu et al., 2019). The verapamil was used as the positive control. MCF-7/ADR cells (2 × 105 cells/mL) were cultured on CLSM dishes (35 mm) in complete RPMI-1640 for 12 h. Then the culture medium was replaced by 2 ml RPMI-1640 containing verapamil (100 μM), QT(0.045 mg/ml), QT-CA-CS(1 mg/ml), or QT-CS(1 mg/ml) for 12 h. The RPMI-1640 without drug was used as negative control. Then, the culture medium was replaced by RPMI-1640 containing 5 μg/mL DOX and incubated for 6 h, Then washed triple times with PBS and observed using CLSM.
3.1. Synthesis and characterization of quercetin-chitosan conjugates with/ without acid labile linkage (QT-CA-CS and QT-CS) The pH-responsive QT-CA-CS conjugate was synthesized using citraconic anhydride to link QT and CS, which involved two-steps: modification of quercetin with citraconic anhydride to prepare QT-CA acid ester and carbodiimide reaction between amino groups of CS and carboxyl group of QT-CA acid ester to prepare QT-CA-CS conjugate (Fig. 2A). Under acidic conditions, the double bond in the citraconic acid amide promoted the internal attack of the amide carbonyl group by 7
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Fig. 7. CLSM images of the internalization of QT-CA-CS-DOX micelles, QT-CS-DOX micelles and free DOX in MCF-7/ADR cells after 4 h (A) or 12 h (B) incubation. Cells were counter-stained with DAPI (blue) for nuclei and Cell Navigator™ Lysosomal Staining Kit (green) for lysosomes.
characteristic peaks of benzene ring of QT (b 6.47 ppm, d 7.33 ppm, g 9.28 ppm), double bond (r 5.8 ppm) and eCH3 (q 2.52 ppm) of citraconic acid ester, which further confirmed the successful formation of QT-CA-CS conjugate (Fig. 2B). The DS of QT on CS molecular chain was evaluated by ultraviolet spectroscopy as our previous report. The DS of QT on two conjugates was similar (QT-CA-CS: 4.32 ± 0.43%, QT-CS: 4.67 ± 0.51%), when the molar ratio of the carboxyl group on QT to the amino group on chitosan was 1:1. (Table 1) In NMR data, the proton absorption peak area is proportional to the protons involved. The ratio of the proton peak areas of the two groups is the same as the molar ratio between the two groups (Holzgrabe, 2010). The method of substitution degree detection refers to the report of Wang et al. (Wang, Jiang, Chen, & Bai, 2016). The degree of substitution could calculate from the ratio of protons on the benzene ring of quercetin (Id 7.33 ppm) to the area under
the carboxylate, thereby accelerating the cleavage of the amide bond (Kang et al., 2014). Succinic anhydride was structurally similar to citraconic anhydride, which lacked double bonds compared to citraconic anhydride. Succinamide did not produce an intramolecular catalytic effect under acidic conditions and was more stable than citraconamide. Therefore, the no pH-responsive QT-CS conjugate was synthesized using succinic anhydride instead of citraconic anhydride to link QT and CS as similar process. The structure of QT-CA-CS conjugate was characterized by FTIR. The characteristic peaks of γ CeH (703 cm−1 and 802 cm−1) in QT were observed in QT-CA-CS conjugate spectrum (721 cm−1 and 824 cm−1). The characteristic peak of δ NeH (1649 cm−1) on CS was strengthened and changed to 1641 cm−1 on QT-CA-CS, which attributed to the formation of amido link between -NH3 on CS and –COOH on QT-CA (Fig. 2C). 1H-NMR spectrum of QT-CA-CS exhibited the 8
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Fig. 8. CLSM images of the internalization of DOX in MCF-7/ADR cells pretreat with verapamil, free QT, blank QT-CS nanomicelles or blank QT-CA-CS nanomicelles. Cells were counter-stained with DAPI (blue) for nuclei.
in aqueous solution via hydrophobic interactions. In general, nanomicelles have significantly lower CMC than low molecular weight surfactants, thus providing greater resistance to dissociation upon dilution. The CMC value of QT-CA-CS conjugate was 0.034 mg/mL. QT-CA-CS nano-micelles showed spherical in shape with smooth surface (Fig. 3A), positive surface charge (Zeta potential: +11.21 ± 1.25 mV to +13.56 ± 1.19 mV), favorable dispersibility (PDI: 0.217 ± 0.023 to 0.335 ± 0.045) and high cargo capacity for DOX. The size distribution histograms of QT-CA-CS and QT-CA-CS-DOX were shown in Fig. S1. As the pH decreased, the structure of the pH-sensitive QT-CA-CS or QT-CACS-DOX micelles gradually disintegrated and the particle size increased.
the H2-H6 proton signal (I l, m, n, o, p 2.96–3.73 ppm) on the chitosan unit. DS = (5 Id /I l, m, n, o, p) ×100%. The DS of QT-CA-CS and QT-CS detected by 1H-NMR spectrum was 2.06% and 2.23% respectively, which was significant less than those of UV-results. This phenomenon was consistent with previous reports, which might attribute to inadequate dissolution of polymer conjugates (Wang et al., 2014). 3.2. Preparation and characterization of nano-micelles QT-CA-CS conjugate was able to self-assemble to form nano-micelles 9
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pH-responsive QT-CA-CS-DOX nano-micelles exhibited the highest cytotoxicity during 48 h incubation, especially for MCF-7/ADR. The MCF7/ADR cell viability treated by 50 μg/mL QT-CA-CS-DOX reduced to less than 25% after 24 h incubation, even less than 4% after 48 h incubation, which was about 9% and 7% of free DOX and QT-CS-DOX nano-micelles treated groups, respectively. In our previous study, QTCS-DOX with no pH-responsive nano-micelles showed favorable P-gp inhibition against MDR, however, in this study, QT-CS-DOX nano-micelles displayed slight inhibitory effect for both of MCF-7 and MCF-7/ ADR at all doses during 48 h incubation, even weaker than that of free DOX treated groups. The low cytotoxicity of QT-CS-DOX compared to free DOX may be due to the difficulty in releasing DOX from the nanomicelles in the cells, so that DOX could not exert its effects in the nucleus. Based on the profiles of in vitro drug release (Fig. 4), the above results could be concluded that the improved anti-cancer effect of QTCA-CS-DOX nano-micelles was attributed its pH-responsive drug release behavior. The acid labile linkage between QT and CS in QT-CA-CS-DOX nanomicelles was broken at endo/lysosome environment, leading to rapidly release of DOX and QT, thus generating high efficiency inhibitory effect against tumor cells. We studied the cytotoxicity of our nanomicelles on healthy cells, the L929 cell line. We found that nanomicelles without DOX showed no cytotoxicity, DOX and DOX-containing nanomicelles were less toxic within 24 h. The possible reason is due to the low sensitivity of the L929 cell line to DOX (Fig. S2).
As a comparison, the structure of QT-CS or QT-CS-DOX did not change significantly (Fig. 3B, C). This may be due to the fact that the citraconic amide in QT-CA-CS breaks under acidic conditions, causing the structure of the micelle to be destroyed. The succinamide in QT-CS is stable under acidic conditions, so that the micelle structure remains intact. The maximum EE and LE was 88.61 ± 1.57% and 8.23 ± 0.82%, respectively, when weight ratio of QT-CA-CS to DOX was 10:1. With increase of weight ratio of QT-CA-CS to DOX, the particle size increase from 112.26 ± 1.35 nm to 133.52 ± 4.13 nm, which indirectly proved the successful encapsulation of QT-CA-CS conjugate for DOX. Compared to QT-CS nanomicelles in our previously reported, QT-CA-CS nanomicelles had a larger particle size and a lower surface charge. This might be due to the reaction of residual free citraconic acid with chitosan. The carboxyl group in citraconic acid reduced the surface charge of the QT-CA-CS nanomicelles, and the hydrophilicity of the surface of the micelles increases, and the hydration radius of the micelles increases. For QT-CS conjugate, the same ratio of QT-CS to DOX (10:1) bring out similar EE (89.31 ± 1.67%) and LE (8.76 ± 0.24%), which was used as control for subsequent studies (Table 2). 3.3. In vitro release of DOX In vitro drug release experiments were carried out at different pH for simulating corresponding physiological environment (pH 7.4: blood circulation environment, pH 6.0: tumor tissue environment, pH 4.5: endo/lysosome environment). For pH-responsive QT-CA-CS-DOX nanomicelles, the drug release rate was significantly increased with decrease of pH value (Fig. 4A). At pH 7.4, QT-CA-CS-DOX exhibited slowly sustained release of DOX, reached to 36.34% within 48 h. On contrast, no significant QT release (less than 4%) was observed in QT-CA-CSDOX, indicated that the acid labile linkage between CS and QT was stable at blood circulation environment. At pH 6.0, the cumulative release rate of DOX and QT from QT-CA-CS-DOX was raised to 45.32% and 16.22%, respectively, which were slight higher than that of QT-CSDOX nano-micelles (Fig. 4B). At pH 4.5, QT-CA-CS-DOX nano-micelles could disassembly by breakage of acid labile linkage to achieve rapid release of DOX and QT, on the other hand, the doxorubicin was able to protonate in acid medium, which changed it from hydrophobic to hydrophilic and promoted the diffusion capacity of DOX. The cumulative drug release rate was obvious faster than that in pH 6.0 and pH 7.4, giving 80.75% for DOX and 42.34% for QT within 48 h. For no pHresponsive QT-CS-DOX nano-micelles, no significant QT release (less than 5%) was detected at each pH within 48 h, due to the stable covalent linkage between CS and QT. Meanwhile, the similar DOX release profiles (no more than 40%) were found at different pH value within 48 h, demonstrated that the structural integrity of QT-CS-DOX nano-micelles hampered the drug release at physiological environment (Fig. 4B). The QT and DOX release of QT-CS-DOX at pH 7.4 were much lower than the previously reported release rate of QT-CS-DOX. In our previous study, we used simulated intestinal fluid at pH 7.4 as the drug release medium. The simulated intestinal fluid contains trypsin, which may react with chitosan to destroy the structure of the micelle and cause drug release. This may be why QT-CS-DOX has different release behaviors in this study.
3.5. Intracellular delivery of DOX 3.5.1. Cellular uptake The cellular uptake of free DOX, QT-CA-CS-DOX and QT-CS-DOX by MCF-7 and MCF-7/ADR cell line was studied using CLSM and flow cytometry after 4 h incubation (Fig. 6). The overlap of red (DOX) and blue (DAPI) fluorescence signal performed purple indicated the location of DOX in nucleus. For MCF-7 cell line, QT-CS-DOX and QT-CA-CS-DOX treated groups exhibited stronger DOX signal compared to that of free DOX treated group, which was in accord with results of flow cytometry (Fig. 6A). However, the purple signal of QT-CS-DOX and QT-CA-CSDOX treated groups was weaker than that of free DOX treated group, which attributed to the relative drug release rate of nanomicells. For MCF-7/ADR, weak DOX signal was detected in free DOX treated group, verified the drug resistance of MCF-7/ADR. QT-CS-DOX and QT-CA-CSDOX obviously promoted DOX cellular uptake by MCF-7/ADR, which were about eight-fold higher than that of free DOX. This phenomenon indicated that the QT component on QT-CS-DOX and QT-CA-CS-DOX was able to mitigate drug efflux effect of MCF-7/ADR. QT-CA-CS-DOX treated group displayed the strongest purple signal compared to free DOX and QT-CS-DOX treated groups, which means more internalized DOX entered into MCF-7/ADR nucleus. Based on the drug release profile and cytotoxicity assay results, we speculated that the rapid DOX release of QT-CA-CS-DOX in acidic environment provide more released DOX in cytoplasm, thus, actively promoted accumulation of DOX in MCF-7/ADR nucleus, leading to high efficiency inhibitory effect against tumor cells. 3.5.2. Intracellular tracking assay To confirm our hypothesis, intracellular tracking assay was carried out in kinetics of incubation time exposure MCF-7/ADR. The endo/lysosomes were stained by Cell Navigator™Lysosomal Staining Kit with green fluorescence. After 4 h incubation, nearly all of DOX signal in cells treated with free DOX was overlapped with lysosomes green signal. For QT-CS-DOX treated group, a few scattering dots like DOX signal was disperse in tumor nucleus and most of that still located in lysosomes. In contrast, as for QT-CA-CS-DOX treated group, extensive DOX signal were diffused in nucleus, which rarely overlapped with green fluorescence of lysosomes (Fig. 7A). After 12 h incubation, strong DOX signal was observed in tumor nuclear regions treated with QT-CA-
3.4. Cytotoxicity assay Human breast cancer cell line with and without DOX resistance (MCF-7, MCF-7/ADR) were used as models to evaluate the cytotoxicity of drug loaded and blank nano-micelles (Fig. 5). No significant cytotoxicity was observed in blank QT-CS or QT-CA-CS nano-micelles treated MCF-7 and MCF-7/ADR groups, indicating that the favorable cytocompatibility of QT-CA-CS nano-micelles (Fig. 5A). Concentrationdependent cell viability, which decreased with the increase of DOX concentration, was observed in the cells treated with free DOX, QT-CACS-DOX and QT-CS-DOX nano-micelle (Fig. 5B and C). Among them, 10
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References
CS-DOX, and its intensity significantly higher than that treated with free DOX and QT-CS-DOX (Fig. 7B). Under endo/lysosomal acidic environment (pH 4.5–5.5), the broken of acid labile linkage between QT and CS in QT-CA-CS-DOX bring out the rapid release of DOX and QT. On the other hand, the“proton sponge” effect resulting from the protonation of amino group in CS component of QT-CA-CS-DOX promoted the disruption of endo/lysosomes and facilitate the released DOX and QT escape from endo/lysosome (Richard, Thibault, De Crescenzo, Buschmann, & Lavertu, 2013). Compared with QT-CA-CS-DOX, no pHsensitive QT-CS-DOX was difficult to release the loaded DOX and trapped in endo/lysosome because of its stable structure in endo/lysosomal acidic environment. The red fluorescence of DOX signal in QTCS-DOX treated group was weak and similar with that of free DOX treated group. These results was well verified our hypothesis and illuminated the intracellular delivery kinetics of QT-CA-CS-DOX.
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3.5.3. P-gp efflux inhibition P-gp was closely associated with MDR development and highly expressed in MCF-7/ADR cell (Yang et al., 2017). To further confirm P-gp efflux inhibition of nano-micellar, the MCF-7/ADR were respectively pretreated with commercialize P-gp inhibitor verapamil, free QT, blank QT-CS nano-micelles or blank QT-CA-CS nano-micelles, then, incubated with DOX and observed its fluorescence intensity using CLSM (Fig. 8). Compared with control group directly treated with DOX, the verapamil, QT, QT-CS and QT-CA-CS pretreated group exhibited strong DOX signal, reflected their high efficiency of P-gp efflux. There was no significant difference in DOX signal intensity among QT and QT-CA-CS pretreated group, which was apparently higher than that pretreated with QT-CS. The difference in the efficiency of P-gp efflux was due to the different utilization of the QT loaded on the carrier. Unlike QT-CS nano-micellar whose P-gp efflux inhibition rooted in QT component in conjugate, the P-gp efflux inhibition of QT-CA-CS derived from the free QT that released by the broken of acid labile linkage between QT and CS in QT-CA-CS. The above data proves that QT-CA-CS-DOX nano-micellar could prevent the drug from being discharged by P-gp to maintain the concentration of intracellular drugs during the treatment within 12 h. Flow cytometry analysis results were shown in supporting information (Fig. S4). 4. Conclusions In conclusion, a pH-sensitive QT-CA-CS conjugate was prepared using an acid labile linkage between QT and CS for intracellular delivery of DOX. QT-CA-CS conjugate could self-assemble to nano-micelle by ultrasound method and provide high encapsulation efficiency for DOX. The pH-sensitivity of QT-CA-CS-DOX gave it efficient drug intracellular accumulation and drug efflux mitigation, performed as cell uptake promotion, escape and rapid drug release response to endo/lysosome acidic pH, along with high DOX level accumulation in tumor nucleus, generating high efficiency inhibitory effect against MCF-7 with/without MDR. This study provided an efficient nano-carrier platform for delivery of DOX against MDR cancer cells. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81801846, U1706212 and 81671828), Natural Science Foundation of Shandong Province (ZR2019QD005), Applied Basic Research Plan of Qingdao (No.16-5-1-70-jch) and the Taishan Scholar Program. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115072. 11
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