Polydopamine coated hollow mesoporous silica nanoparticles as pH-sensitive nanocarriers for overcoming multidrug resistance

Colloids and Surfaces B: Biointerfaces 183 (2019) 110427

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Polydopamine coated hollow mesoporous silica nanoparticles as pHsensitive nanocarriers for overcoming multidrug resistance

T

Mei Shaoa,1, Cong Changb,1, Zuhao Liub, Kai Chena, Yimin Zhoua, Guohua Zhengb, Zhijun Huanga, ⁎ Haixing Xua, Peihu Xua, Bo Lua, a b

School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology, Wuhan 430070, PR China College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Polydopamine Hollow mesoporous silica nanoparticles Multidrug resistance Stimuli response Drug delivery system

A nanocarrier system of methoxypolyethylene glycol amine (mPEG-NH2) functionalized polydopamine (PDA) coated hollow mesoporous silica nanoparticles (HMSNs-PDA-PEG) was developed with pH-responsive, which combined doxorubicin hydrochloride (DOX) and quercetin (QUR) to reverse multidrug resistance (MDR) and improved anticancer effects on taxol (TAX) and DOX double resistant human colorectal cancer cell line HCT-8 (HCT-8/TAX cells). Well-dispersed nanoparticles (HMSNs-PDA-PEG) were prepared with a dimension of around 170 nm. The surface morphology and chemical properties of HMSNs-PDA-PEG were also successfully characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), thermal gravimetric analysis (TGA), X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET) method, Fourier transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS). Drug release experiments results indicated that DOX and QUR (QD) loaded nanoparticles (HMSNs-PDA-PEG@QD) had similar release kinetic profiles of each drug, which all exhibited highly sensitive to pH value due to the surface PDA coating. Additionally, the HCT-8 cells or HCT-8/TAX cells were employed to assess the cellular uptake and cytotoxicity of various drug-free or drug-loaded HMSNs samples. Meanwhile, a series of biological evaluations demonstrated that the HMSNs-PDAPEG@QD exhibited remarkable ability to overcome MDR compared with free DOX and HMSNs-PDA-PEG@DOX. Taken together, these results revealed that HMSNs-PDA-PEG@QD was suitable as a prospective and efficient drug delivery nanosystem for overcoming multidrug resistance.

1. Introduction Multidrug resistance (MDR) is one of the major obstacles for cancer effective chemotherapeutic [1,2]. The main causes of MDR can be summarized into two general mechanisms: the drug efflux pump mechanism which mainly refers to the overexpression of P-glycoprotein (P-gp) and the activated or overexpressed antiapoptotic mechanism [3,4]. To solve this problem, multidrug combination therapy has emerged, which can not only effectively improve therapeutic effects, decrease side effects and reverse MDR [5,6], but also produce synergistic or additive effects according to different cell signaling pathways to satisfy the needs of diverse systems [7]. Doxorubicin hydrochloride (DOX), an anthraquinone drug can kill cancer cells in types of growth cycles through inhibiting RNA and DNA synthesis [8,9], are widely used in treating multiple tumors such as bladder, breast, lung and so on [10]. However, long-term use of DOX

could lead to MDR and serious side effects like cardiotoxicity, nausea, vomit, etc. [11]. Quercetin (QUR) as a natural flavonoid has a wide range of biological activities, such as antitumor, anti-inflammatory, anti-oxidation and hyperlipidemia [12,13]. It has been reported that QUR can inhibit the expression of P-gp and mutant p53, which promote cell apoptosis as a potential chemosensitizer [14]. As a result, a synergistic effects could be produced when QUR is combined with DOX [15,16]. Mesoporous silica nanoparticles (MSNs) have become ideal nanocarriers for intelligent drug delivery systems due to their good biocompatibility, easy functionalized surface, adjustable pore size and good specific surface area [17,18]. The surface of MSNs contains a large number of hydroxyl groups, which can be easily modified with ligand molecules, such as β-cyclodextrine (β-CD) [19], hyaluronic acid (HA) [20], peptides [21] and so forth. At the same time, different responsive systems based on MSNs can be designed according to the specific

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Corresponding author. E-mail address: [email protected] (B. Lu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.colsurfb.2019.110427 Received 23 June 2019; Received in revised form 31 July 2019; Accepted 5 August 2019 Available online 06 August 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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8 (DOX sensitive) and HCT-8/TAX (TAX and DOX double resistant) were selected as in vitro models. After the system (HMSNs-PDA-PEG@ QD) reached the cancer cell site through the EPR effects [40], the two drugs were released when exposed to acidic microenvironment, and the QUR enhanced the anti-cancer activity of DOX by inhibiting the expression of P-gp and mutant p53. We anticipate that this nanocarriers can provide a viable strategy to efficiently combat multidrug-resistant cancers.

environment of the cancer site, including pH response [22], redox response [23], enzyme response [24] and others. Although traditional MSNs have made great progress as nanocarriers, their low drug loading capacity, which is generally less than 10%, has limited their applications [25,26]. Based on this, hollow mesoporous silica nanoparticles (HMSNs) have been developed. Compared with MSNs, the hollow structure of HMSNs has greatly improved drug loading capacity, which can reduce the accumulation of foreign materials in vivo and enhance the safety of cancer treatment [27]. Therefore, reports about HMSNs as nanocarriers for intelligent drug delivery systems have been published increasingly [28,29]. Polydopamine (PDA) is a melanin-like mussel adhesion protein mimic [30], which can form an adhesive layer on almost any kind of material surface with good biocompatibility [31]. Besides, dopamine (DA) can oxidize and self-polymerize in the presence of weak alkaline (pH˜8.5) to furnish water-insoluble PDA films. These unique properties make PDA an ideal coating material [32,33]. It is noteworthy that PDA coating is very sensitive to pH value, which can be stable coating material under weak alkaline or neutral conditions, and easy to fall off under acidic conditions [34]. At the same time, quinone groups on the surface of PDA can easily react with amino- and thiol-terminated molecules by Schiff base or Michael addition reaction, which will facilitate the modification of different functional ligands onto the surface of PDA [35]. For instance, Wang’s group [36] coated polydopamine on NaGdF4 nanoparticles and externally attached polyethylene glycol (PEG) to achieve anatomic and functional imaging of the tumor site simultaneously. Rahoui’s group [32] developed novel nanoparticles (MSNsPDA-AuNPs) by coating gold labelled polydopamine on mesoporous silica to construct a dual-responsive system of pH and NIR photothermal effects for cancer therapy. Additionally, Mei’s group [37] modified D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS)functionalized polydopamine on the surface of mesoporous silica, which was both a pH-sensitive goalkeeper and used to combat multidrug resistance in lung cancer chemotherapy. Herein, a pH-responsive drug delivery system for multidrug-resistant cancer therapy was designed (Scheme 1a). We first encapsulated DOX and QUR into HMSNs by physical embedding method. Then, PDA film was introduced into the system as a pH sensitive encapsulating material, which could exist stably under normal physiological conditions and degrade in acidic environment of cancer cells. Meanwhile, the protective layer of mPEG-NH2 was modified on the surface of PDA by Michael addition reaction, which could effectively inhibit non-specific protein adsorption and enhance the stability of blood circulation. Most importantly, mPEG-NH2 in the system could hinder the interaction between nanoparticles and cells, thus reducing intracellular differentiation [38,39] (Scheme 1b). Human colorectal cancer cell line HCT-

2. Experimental section 2.1. Materials Tetraethylorthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), ammonia solution, methanol, ethanol, hydrochloric acid (HCl) and sodium carbonate (Na2CO3) were purchased from Sinopharm chemical reagent Co., Ltd (Shanghai, China). Tris (hydroxymethyl) methyl aminomethane was purchased from Macklin (Shanghai, China). Dopamine hydrochloride, methoxypolyethylene glycol amine (mPEGNH2, M.W. 2000), doxorubicin hydrochloride (DOX) and quercetin (QUR) were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Human colorectal cancer cell line HCT-8 (DOX sensitive) and HCT-8/TAX (TAX and DOX double resistant). Gibco Roswell Park Memorial Institute (RPMI) medium 1640 basic, Hoechst 33342, fetal bovine serum (FBS), 5-diphenyl tetrazolium bromide (MTT) were obtained from Gibco Invitrogen Corp. (Carlsbad, CA, USA). All the involved reagents were analytical grade and were used without further purification.

2.2. Preparation of HMSNs-PDA-PEG@QD and drug loading For the prepared of HMSNs, the procedures were furnished via a selective etching method based on previous reports with slight modifications [27,41]. DOX and QUR were loaded into HMSNs through diffusion, which were designated as HMSNs@QD. Then the dopamine (DA) through oxidize and self-polymerize furnished water-insoluble PDA films on surface of HMSNs [42,43]. The products were designated as HMSNs-PDA@QD. The mPEG-NH2 was bound to the surface of PDAcoated HMSNs through Michael addition reaction [38], which were designated as HMSNs-PDA-PEG@QD and the blank samples (drug-free HMSNs-PDA-PEG) and DOX loaded samples (HMSNs-PDA-PEG@DOX) were also prepared by the same method. The preparation of HMSNsPDA-PEG@QD and drug loading detailed synthesis steps can be found in the supporting information.

Scheme 1. (a) Schematic illustration of HMSNs-PDA-PEG@QD. (b) Synthesis of PEG-conjugated polydopamine film through Oxidative Polymerization and Michael Addition Reaction. 2

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cells were treated with different samples in a different concentration culture medium and incubated for 24 h and 48 h. Then, the cells were washed by PBS and all the medium were replaced with 20 μL of MTT (5 mg /mL) for another 4 h. Finally, the culture medium was discarded and 150 μL of DMSO was used to dissolve crystalline formazan and then the optical density (OD) value of the solution was measured at 492 nm using a microplate reader (BIO-RAD 550) to obtain the cell viability, which could be calculated as follows:

2.3. Characterization of nanoparticles The morphologies of different HMSNs images were acquired on the transmission electron microscopy (TEM, JEM-2100 F). The zeta potential and the hydrodynamic size of the nanoparticles were measured by Malvern Zetasizer NanoZS instrument. For thermal gravimetric analysis (TGA), the thermogravimetric analyzer (STA449F3) was used to determine the weight loss of the different HMSNs samples and all of the measurements were performed within an O2 atmosphere from 50 to 1000 °C at a temperature rate of 10 °C/min. The Brunauer–Emmett–Teller (BET) measurement was employed to determine the specific surface areas of different HMSNs samples and the Barrett–Joyner–Halenda (BJH) (ASAP 2020, Micromeritics) analysis was used to determine their corresponding pore characteristics. Before measurement, all the samples were pretreated at 110 °C for 24 h under N2 atmosphere. After modification with PDA and mPEG-NH2, the changes in the surfaces chemical composition of the HMSNs samples were analyzed by Fourier transform infrared spectroscopy using a PerkinElmer infrared spectrophotometer with a pellet of powdered KBr. Small-angle X-ray diffraction was performed on a Rotation Anode High Power X-ray Diffractormeter (RU-200B/D/MAX-RB). XPS data were characterized on X-ray photoelectron spectroscopy (XPS) (ESCALAB 250Xi) using a monochromatic Al Ka X-ray source. To charge correction, the C1s peak was performed at 284.8 eV and binding energies were according to the Fermi edge.

Cell viability = (OD sample / OD control) × 100% Where the OD sample and control were measured from cells corresponding to the presence and absence of samples respectively. 2.8. Western blot assay Proteins were extracted from HCT-8/TAX cells which were pre-incubated with different HMSNs samples at 37 °C for 24 h and antiGAPDH antibody chosen as loading control. The cells were lysed at 4 °C for 20 min in a cell lysis buffer supplemented with PMSF after washing with cold PBS. The lysates were then collected by centrifugation and stored at −20 °C. The proteins (25 mg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then electro transferred onto the polyvinylidene fluoride (PVDF) membrane. Subsequently, the transferred blots were blocked by TBST, which containing 5% skim milk solution and incubated with primary antibody under mild stirring overnight at 4 °C. After that, the membrane was incubated with secondary antibody under shaking gently at 37 °C for 1 h. Finally, enhanced chemiluminescence detection kit was used to visualize specific bands.

2.4. In vitro drug release In order to evaluate the in vitro DOX and QUR release profile of HMSNs-PDA-PEG@QD samples, drug release experiments were carried out at different pH values. 5 mg accurately weighed of HMSNs-PDAPEG@QD were dispersed in 3 mL of phosphate buffered saline (PBS) and then transferred into a dialysis bag (MW. 14 kDa) outer filled with 10 mL of PBS (pH 5.0, 6.0 or 7.4) in centrifuge tubes shaken in constanttemperature shaker at 37 °C. At appropriate time intervals, 3 mL of the outside release solution was taken out and added with same volumes of fresh solution and the whole released DOX and QUR was measured using RPHPLC.

3. Results and discussion 3.1. Characterization of nanoparticles TEM were used to evaluate the morphology of the different HMSNs samples. As the images indicated that the bare HMSNs displayed a spherical shape and uniform porous structure. The average diameter of HMSNs was 110 ± 10 nm and the thickness of shell was about 20 ± 2 nm (Fig. 1a). Comparing with the bare HMSNs, the particle size of HMSNs-PDA increased by about 30 nm, the surface became rough and the mesoporous channels were partially masked by a thin PDA film. Besides, the particle size became about 170 ± 5 nm when conjugated with the mPEG-NH2, and the particles had better dispersion than HMSNs-PDA nanoparticles (Fig. 1b and c). The zeta potential values of the different HMSNs samples were measured to investigate charge changes and the surface modification. The mean surface charge of bare HMSNs was ‒10.0 ± 1.3 mV, for the large number of silane alcohols on the silica surface. When HMSNs were coated with PDA, the potential of the HMSNs-PDA decreased to ‒18.6 ± 2.0 mV, due to the presence of hydroxyl groups of the polydopamine. The zeta potential became more negative when conjugated with mPEG-NH2, which decreased to ‒27.7 ± 0.7 mV and this zeta potential made HMSNs-PDA-PEG more stable due to the electrostatic repulsion between nanoparticles. Moreover, DLS measurements were revealed that the average hydrodynamic diameter of HMSNs-PDA-PEG increased from 180.6 ± 2.4 nm to 238.1 ± 4.3 nm when compared with HMSNs which were consistent with the TEM results basically. Additionally, different HMSNs samples exhibited narrow size distributions with appropriate polydispersity index (PDI) values of 0.268 and 0.201 (Fig. 2a, Fig. S1, see the supporting information, SI). As shown in Fig. 2b, the thermal gravimetric analysis (TGA) was performed for quantitative analysis. The weight loss of bare HMSNs was only 19.34%, but that of HMSNs-PDA increased to 32.25%, which was due to the existence of PDA films. After functionalization with mPEGNH2, the weight loss of HMSNs-PDA-PEG increased to 38.62%. This

2.5. Cell culture HCT-8/TAX cells were cultured in RPMI 1640 cell culture medium, which containing 10% fetal bovine serum (FBS) and 1 μM taxol under a humidified atmosphere with 5% CO2 at 37 °C. HCT-8 cells were cultured under the same conditions except with no taxol. For cell passage, 0.25% trypsin solution was used for digesting cells. 2.6. In vitro cell uptake Confocal laser scanning microscopy (CLSM) (Olympus, Japan) was used to observe cell uptake behavior by treating HCT-8/TAX cells with free drugs and different HMSNs samples. Briefly, HCT-8/TAX cells were separately seeded and cultured in a 35 mm confocal dishes (Glass Bottom Dish) at a density of 1 × 104 per dish at 37 °C for 24 h. Then, the culture medium was discarded and replaced by a fresh medium containing free DOX, DOX + QUR, HMSNs-PDA-PEG@QD or HMSNsPDA-PEG@DOX (10.0 μg/mL DOX equiv.). After 12 h incubation, the cells were stained with 17.8 μM Hoechst 33342 for 15 min, rinsed three times with PBS (pH 7.4) to perform fluorescence imaging with a CLSM. 2.7. In vitro cytotoxicity assay The cytotoxicity of different HMSNs carriers against HCT-8/TAX cells and HCT-8 cells were quantitatively evaluated by the MTT assay. HCT-8/TAX cells and HCT-8 cells were seeded into 96-well plates at 5.0 × 104 cells per well and incubated for 24 h at 37 °C. Afterwards, the 3

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Fig. 1. TEM images of HMSNs (a), HMSNs-PDA (b) and HMSNs-PDA-PEG (c). Scale bares are 100 nm for (a), and 200 nm for (b) and (c) respectively.

decreased further, which were 76 m2 g−1, 0.34 cm3 g−1 respectively, and the pore diameter were hardly be measured. These results confirmed that the DOX and QUR occupied the mesoporous channels and the successful surface modification by the PDA film. X-ray photoelectron spectroscopy (XPS) analysis with high sensitivity was studied to further determine the surface chemical composition. Both HMSNs-PDA and HMSNs-PDA-PEG exhibited a N1s signal at the binding energy of 396–402 eV in the XPS spectrum, but the bare HMSNs did not exhibit any N1s peaks, which could verify the presence of PDA films [44]. Moreover, the narrow XPS scan for N1s peaks also proved the success of the PDA coating and mPEG-NH2 conjunction (Fig. 3), while the N1s peaks of HMSNs-PDA-PEG was more intense than those of HMSNs-PDA due to the conjugation of the mPEG-NH2 onto the surface of HMSNs-PDA through the Michael addition reaction [45]. The silicon peaks (Si2p), which the signals only from silica structures in HMSNs, had decreased intensity when comparing HMSNsPDA and HMSNs-PDA-PEG to bare HMSNs at 101–105 eV (Fig. S3, SI), and this results also could prove the existence of PDA films. The surface characterization of prepared nanoparticles was also

results further demonstrated that HMSNs-PDA and HMSNs-PDA-PEG were successfully constructed. Small X-ray diffraction patterns (XRD) of HMSNs and HMSNs-PDA were depicted in Fig. S2 (SI). A definite diffraction peak showed the well-ordered mesostructure of HMSNs at 2.1° (2θ). After modification with PDA, the diffraction peak almost disappeared at the same place attributing to the coating effect of PDA membranes. To confirm the mesoporous features of the different HMSNs samples, the N2 adsorption–desorption isotherms with the corresponding BJH pore size distribution were examined and shown in Fig. 2c and d. The BET surface area, the average pore volume and pore diameter of the related HMSNs samples were listed in Table S1 (SI). N2 adsorptiondesorption isotherms of HMSNs displayed a typical IV isotherm, which exhibited a great mesoporous feature. The BET surface area was 1194 m2 g−1, the pore volume was 1.65 cm3 g−1 and the pore diameter estimated by BJH method was about 4.68 nm. After loading drugs, the BET surface area of HMSNs@QD decreased to 1044 m2 g−1, which the pore volume and the pore diameter were decreased to 1.07 cm3 g−1 and 3.94 nm. When coating with PDA, the values of HMSNs-PDA@QD

Fig. 2. Physical characterization: (a) zeta potential values of various HMSNs; (b) TGA curves of various HMSNs; (c) BET N2 adsorption/desorption isotherms and (d) BJH pore size distributions of various HMSNs. 4

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Fig. 3. XPS spectra of various HMSNs: (a) - (c) surveys of all tested peaks. (d) - (f) narrow scan for N1s peaks.

within144 h in pH 6.0 and 7.4 PBS, respectively. Moreover, the drug release rate of QUR was 61.1% within 144 h, while the drug release rate of QUR was only separately reached 32.6% and 17.2% in pH 6.0 and pH 7.4 PBS. Different release curves showed that PDA film could coat nanomaterials well under physiological conditions to prevent the early leakage of DOX and QUR. When the acidic environment is close to the tumor cell environment, the drug release rate increased, which was attributed to the dropping of PDA coatings from HMSNs surface. As a result, the HMSNs-PDA-PEG@QD system had higher drug release ability at pH 5.0, and the acid-base-dependent release behavior can greatly enhance the therapeutic effect of tumors and reduce the potential damage to normal cells [47].

evaluated by FT-IR spectra. As shown in Fig. S4 (SI), the characteristic absorption peaks for HMSNs were found at 1085, 803, 462 cm−1, corresponding to bending vibration and stretching vibration of Si-O-Si bond. Moreover, the peak appearing at 3432, 1637 cm−1 were attributed to the stretching and bending vibration of Si−OH respectively. In the case of HMSNs-PDA nanoparticles, the typical absorption peak at 1510 cm−1 was the stretching vibration of aromatic ring C]C on PDA. For HMSNs-PDA-PEG nanoparticles, the peaks at 2921 cm−1 and 1320 cm−1 were assigned to the C–H stretching vibration of mPEGNH2, which indicated that mPEG-NH2 was successfully conjugated on the surface of HMSNs-PDA nanoparticles. 3.2. Drug loading and in vitro release

3.3. In vitro cell uptake The DL values of HMSNs-PDA-PEG@QD calculated using the equations were 12.17% (DOX) and 12.01% (QUR) respectively, which were higher than conventional MSNs system due to the large hollow interior drug storage volume of HMSNs [46]. The accumulative drug release experiments of HMSNs-PDA-PEG@QD were carried out under pH 7.4, 6.0 and 5.0 PBS solutions at 37 °C. The results showed that this system had a higher pH sensitivity and the release of QUR was slightly higher than that of DOX. As shown in Fig. 4. The DOX release amount under pH 5.0 PBS reached 26.5% within 24 h and then increased to 30.3% in 144 h, while only 16.5% and 5.3% of DOX was released

The cellular uptake and intracellular distribution of free drugs and drug-loaded HMSNs samples in HCT-8/TAX cells were evaluated by measuring the fluorescence intensity of DOX, which further reflected the effect of QUR on DOX resistance. The results are shown in Fig. 5. The free DOX + QUR showed stronger red fluorescence than the free DOX, which was attributed to the decrease of drug efflux caused by the QUR-meditated inhibition of P-gp expression and leaded to the increase of intracellular DOX uptake. Similarly, the fluorescence of the HMSNsPDA-PEG@QD was stronger than that of HMSNs-PDA-PEG@DOX,

Fig. 4. In vitro DOX (a) and QUR (b) release profiles of HMSNs-PDA-PEG@QD in PBS with different pH values. 5

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increasing the time of DOX residence in cells and enhancing the therapeutic effect. 3.4. In vitro cytotoxicity In vitro cytotoxicity of different samples to HCT-8 cells and HCT-8/ TAX cells was determined by MTT assay. As shown in Fig. 6a and b, the prepared drug-free HMSNs and HMSNs-PDA-PEG did not show obvious toxicity to both HCT-8 cells and HCT-8/TAX cells after treatment for 48 h, even at a relatively high concentration, which demonstrated the good safety and biocompatibility of HMSNs and HMSNs-PDA-PEG. The cytotoxicity results of different samples at a tested concentration against HCT-8 cells and HCT-8/TAX cells for 48 h and 24 h were also shown in Fig. 6c, d and Fig. S5 (SI), from which it was easy concluded that all of the samples showed a dose-dependent cytotoxicity to both types of cells, and the 48 h results were significantly better than 24 h. The half maximal inhibitory concentration (IC50) of free DOX on HCT-8 and HCT-8/TAX cells was 0.20 μg/mL and 10.90 μg/mL, respectively. The resistance index (RI) was 54.5, indicating that the HCT-8/TAX cells met the requirement of doxorubicin resistant cell line (Table S2, SI). For HCT-8 cells, the IC50 of free DOX, free DOX + QUR, HMSNsPDA-PEG@DOX and HMSNs-PDA-PEG@QD was 0.20, 0.21, 3.40 and 2.02 μg/mL, respectively (Table S2, SI). There was no obviously difference between two samples, i.e., free DOX and free DOX + QUR, which was likely due to no multidrug resistance in HCT-8 cells. In terms of HCT-8/TAX cells, when some chemotherapy drug such as DOX entering cells will be recognized and pumped out by the overexpressed P-gp on cell membrane. As a result, the free DOX was less cytotoxic than free DOX + QUR, because QUR could promote the

Fig. 5. CLSM images of HCT-8/Tax cells after incubation with free DOX, DOX + QUR, HMSNs-PDA-PEG@DOX and HMSNs-PDA-PEG@QD for 12 h. The nuclei stained with Hoechst 33342 were observed as blue fluorescence. DOX was observed as red fluorescence. Merged images were obtained by overlapping the red and blue fluorescence. The scale bar is 50 μm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

which further confirmed the effect of QUR in reversing MDR. It is noteworthy that the HMSNs-PDA-PEG@QD exhibited the strongest fluorescence intensity, because the system released drugs slowly and continuously relative to free DOX + QUR, which is conducive to

Fig. 6. In vitro cell viability of HCT-8 cells (a) and HCT-8/Tax cells (b) incubated with different concentrations of free HMSNs and HMSNs-PDA-PEG for 48 h. In vitro cell viability of HCT-8 cells (c) and HCT-8/Tax cells (d) incubated with different concentrations of free DOX, DOX + QUR, HMSNs-PDA-PEG@DOX and HMSNs-PDAPEG@QD for 48 h. Error bars represent means ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. 6

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Fig. 7. Assessment of P-gp knockdown in HCT-8/TAX cells: a) Western blot analysis. Lane 1: HMSNs-PDA-PEG@QD (10 μg/mL); Lane 2: HMSNs-PDA-PEG@QD (5 μg/mL); Lane 3: free DOX + QUR; Lane 4: HMSNs-PDA-PEG@DOX; Lane 5: free DOX; Lane 6: cells without any treatment. b) Quantitative analysis of relative P-gp expression (*p < 0.05).

Acknowledgements

down-regulation of P-gp level and avoid DOX efflux effect. The trend of anti-tumor effect of drug-loaded nanoparticles was consistent with free drug. The increased IC50 of HMSNs-PDA-PEG@QD relative to free DOX + QUR at high concentrations was probably attributed to the slower internalization and the slowly release of free drug from the nanoparticles.

This work was financially supported by the National Natural Science Foundation of China (51773162, 21204071) and the Open Fund Project of Hubei TCM Standardization Engineering and Technology Center (ZDSYS201802). Appendix A. Supplementary data

3.5. Western blot assay Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110427.

To assess the potential of prepared HMSNs-PDA-PEG@QD for MDR reversal in HCT-8/TAX treatment, western blot assay was used to detect the level of p-gp expression. As shown in Fig. 7a, the free DOX and HMSNs-PDA-PEG@DOX showed no significant inhibition on p-gp expression in HCT-8/TAX cells, indicating that the cell beads were resistant to DOX. However, the expression of p-gp in both the free DOX + QUR and HMSNs-PDA-PEG@QD was down-regulated to some extent (Fig. 7b). In particular, HMSNs-PDA-PEG@QD (10 μg/mL) showed the highest P-gp down-regulation efficiency compared to the control groups, which was about 52% decrease of the P-gp expression level. These results confirmed that HMSNs-PDA-PEG@QD could effectively knockdown P-gp protein expression and exhibit a certain dosedependent, which was consistent with the conclusion of the in vitro cytotoxicity and cell uptake.

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4. Conclusion In summary, we have developed an effective HMSNs-PDA-PEG@QD drug delivery system, which is used to combat MDR of drugs by coating pH-sensitive PDA on the surface of HMSNs and grafting mPEG-NH2 to enhance dispersion. This therapeutic diagnostic nanocarrier exhibited a high ability to simultaneously transport DOX and QUR into HCT-8/TAX cells and stimulate drug release in its specific acidic environment. Western blotting experiments showed that QUR can effectively inhibit the P-gp protein on the surface of HCT-8/TAX cells. In addition, HMSNs-PDA-PEG@QD showed high cytotoxicity on HCT-8/TAX cells and blank HMSNS-PDA-PEG is biocompatible and basically non-toxic. Therefore, we believe that the HMSNs-PDA-PEG@QD presented here can provide new possibilities and guidance for reversing the treatment of MDR cancer.

Declaration of Competing Interest The authors declare no conflict of interest. 7

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