Hybrid MesoporousâMicroporous Nanocarriers for Overcoming

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Hybrid Mesoporous-Microporous Nanocarriers for Overcoming Multidrug Resistance by Sequential Drug Delivery Liucan Wang, Haidi Guan, Zhenqiang Wang, Yuxin Xing, Jixi Zhang, and Kaiyong Cai Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01096 • Publication Date (Web): 16 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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Molecular Pharmaceutics

via the encapsulation during ZIF-8 growth. The sustained release of DOX was observed to follow earlier and faster release of CUR by acid-sensitive dissolution of the ZIF-8 shell. Furthermore, the nanoparticles showed good biocompatibility and effective cellular uptake in invitro evaluations using drug-resistant MCF-7/ADR cancer cells. More importantly, the preferentially released CUR inhibited the drug efflux function of the membrane P-glycoprotein (P-gp), which subsequently facilitated the nuclear transportation of DOX released from the PDAMSN core, and in turn, the synergistic effects on killing MDR cancer cells. The hybrid mesoporous-microporous nanocarrier holds great promise for combination chemotherapy applications on the basis of sequential drug release.

1. INTRODUCTION Multidrug resistance (MDR) is a leading cause for treatment failure in cancer chemotherapy.1, 2 The overexpression of P-glycoprotein (P-gp) encoded by MDR1 gene, has been identified to be one of the most relevant reasons for MDR.3, 4 This glycoprotein belongs to the ATP-binding cassette (ABC) family, 1 and can act as a drug efflux pump to reduce the accumulation of the intracellular drugs.5 In light of this, the combination therapy by the co-delivery of anticancer drugs and MDR modulators was developed to maximize the therapeutic efficacy and reduce side effects. Nanoparticles represented by mesoporous silica nanoparticles (MSNs) with high surface areas are typical drug delivery systems (DDSs) for this application.6, 7 Recently, the sequential delivery of therapeutic cargo has been emerging as a promising route in combination therapy. In the enlightening studies, a faster and earlier release should be achieved for the modulator/chemosensitizer, so that the functional inhibition of drug efflux pump

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can be realized in advance before the effective accumulation of chemotherapeutic drugs within cells.8 The design rationale for sequential release systems is to retain drugs with different strength inside the carriers. For instance, composite nanomaterials with a distinct PLGA-casein core/shell structure was reported to encapsulate the hydrophobic and hydrophilic drugs within different storage compartments, thereby leading to a sequential release behavior regulated by diffusion and dissolution.9 Li et al. has developed a polymeric micelle system prepared by conjugating therapeutic drugs to polymers via an acid-cleavable bond and encapsulating a drug sensitizer (disulfiram) via the self-assembly of the polymer-drug conjugate.10 However, challenges in achieving high drug loading capacities and controlled release still remain as urgent research subjects in these systems, due to the difficulties in efficient utilization and partition of the intra-carrier space. Drug carriers with multiple storage areas/compartments that can be loaded with different cargo drugs are expected to be a highly potential strategy for addressing the challenge above.11 In the past decade, an intriguing type of supramolecular soft matter, i.e. metal-organic frameworks (MOFs) built from assembling metal ions or clusters with organic ligands, has been reported to be a promising DDS with porous structure.12 Composite nanoparticles based on MOFs have been reported to possess ultrahigh drug payload and pH-responsive drug release, due to the outstanding advantages from uniform cavities and large specific surface area.13-15 Most importantly, MOFs are ideal candidates for regulating the travelling of the in-coming cargo by the molecular sieving effects of their defined pore apertures, especially when they are loaded with guest molecules.16, 17 In a pioneering study, MOF particles armored with a mesoporous silica coating (MOF@MSN) was observed to be suitable for the separation of small molecules by MSN enhanced mechanical properties.18 Regarding the specific requirements for drug


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delivery, an alternative design, i.e. mesoporous particles armored with a microporous shell, may offer superior capabilities and even synergistic properties for building sequential release systems to tackle the above-mentioned challenges. To achieve the goal of integrating MOF with other functional nanomaterials, concerns on the elegant regulation of interfacial interactions were widely raised and hence warrant further investigations. In the past several years, selective capping ligands (e.g., polyvinylprolidine (PVP), poly(sodium 4-styrene-sulfonate) (PSS)) were thus employed to facilitate the surface growth of MOF on nanoparticles.19-21 However, the most exciting breakthrough in this field is the exploitation of mussel-inspired polydopamine (PDA) for tailored structural/function integration, owing to the interactions of PDA toward both organic and inorganic (e.g., metal ions) compounds.22, 23 The combination of physicochemical characteristics of PDA makes it the most versatile surface modifier to enhance drug loading,23 material hybridization,24 as well as heterogeneous nucleation and growth of MOFs.25 So far, however, most of the research focused on PDA-mediated MOF coatings on nonporous nanocatalysts, while very few on porous nanomaterials for constructing multi-compartmental DDSs. Herein, a core-shell structured nanohybrids with sequential drug release properties was constructed by PDA mediated integration of the mesoporous MSN core and the microporous zeolite imidazolate frameworks-8 (ZIF-8) shell. PDA was hybridized in the framework of MSN by the organic-inorganic interactions during particle formation (PDA-MSN),24 which facilitates the high-capacity loading of DOX by π-π stacking interactions. A P-gp inhibitor, i.e. curcumin (CUR) was loaded insitu during the growth of ZIF-8 shell. The microporous shell loaded with CUR helps to prevent anticancer drugs (DOX) in the mesoporous core from premature release. Under acidic conditions inside MDR cancer cells, CUR in the shell was preferentially released to


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cytoplasm to inhibit P-gp related drug efflux, followed by DOX releasing from core PDA-MSN to exert the efficient therapeutic function (Figure 1). In general, the core-shell hybrid is expected to pave a new avenue for combination therapy in MDR.

Figure 1. Schematic diagram showing the preparation of dual drug (DOX and CUR) loaded core-shell structured PDA-MSN@ZIF-8 nanocarriers composed of a microporous ZIF-8 shell and a mesoporous PDA-MSN core, the mechanism of drug release, as well as the synergistic MDR reversal therapy. 2. EXPERIMENTAL SECTION 2.1. Materials. Unless otherwise noted, distilled water was used in the preparation of all aqueous solutions. Cetylmethylammonium bromide (CTAB, AR), methanol (AR), and ethanol (AR) were purchased from Fluka. NH4OH (30 wt%, AR), 3-aminopropyltriethoxysilane (APTES, AR), and tetraethyl orthosilicate (TEOS, AR) were purchased from Sigma. 2Methylimidazole (2-MIM, RG, 98%) was purchased from Adamas (Shanghai). Doxorubicin hydrochloride (DOX, 98%), zinc nitrate hexahydrate (Zn(NO3)2• 6H2O, AR), Rhodamine 123,


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and dopamine hydrochloride (98%) were purchased from Aladdin Industrial Inc (Shanghai). Curcumin (CUR) was purchased from TCI (Shanghai) Development Co., Ltd. The DOX resistant MCF-7 (MCF-7/ADR) cell line and DOX sensitive MCF-7 cell line were obtained from Bogoo Biological Technology Co., Ltd (Shanghai). The cell culturing medium (RPMI-1640) was purchased from Canspec Scientific Instrument Co., Ltd (Shanghai). Cell counting Kit-8 (CCK-8, for cell viability evaluation) was obtained from Dojindo Chemical (Shanghai). The nuclear-staining dye (Hoechst 33258) was obtained from Yeasen Biological Technology Co., Ltd (Shanghai). 2.2. Synthesis of PDA-MSN composite nanoparticles. PDA-MSN nanoparticles were synthesized according to our previous work by a facial one-pot synthesis at 70 °C,

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in which

the molar ratio of the reaction solution was 1 TEOS: 0.18 CTAB: 0.06 APTES: 77.36 ethanol: 1249 H2O: 5.9 NH3: 0.043 dopamine. After the synthesis, the template of the particles was removed by using an efficient ion-exchange method. 27 Finally, the products were suspended in ethanol (5 mg mL-1) for further use. 2.3. Synthesis of DOX/CUR co-loaded nanoparticles (PDA-MSN@ZIF-8/DOX+CUR). In a typical procedure for generating the first ZIF-8 coating without CUR loading, PDA-MSN (0.5 mg) was dispersed into 72.8 µL of a drug (DOX) solution in deionized water by sonication. The mixture was stirred at 37 °C for 10 h, then 13.5 µL of a Zn(NO3)2• 6H2O solution (74.3 mg mL-1 in methanol) and 48.5 µL methanol were added and stirred. After 1 h of chelation reaction between PDA and Zn2+, 107.9 µL of a 2-MIM solution (41.1 mg mL-1 in methanol) was added to initiate the coordination reaction and ZIF-8 coating (12 h). Finally, the drug loaded PDA-


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MSN@ZIF-8/DOX nanoparticles were retrieved by centrifugation and the drug loading amount was quantified by the UV-vis absorbance at 340 nm. For a two-step ZIF-8 coating with CUR encapsulation, PDA-MSN@ZIF-8/DOX was dispersed into 50 µL of a drug (CUR) solution in methanol (8 mg mL-1), followed by the addition of 13.5 µL of Zn(NO3)2• 6H2O (74.3 mg mL-1 in methanol), 52.4 µL methanol, and 72.8 µL deionized water. The chelation reaction was sustained for the same period of 1 h, followed by the addition of 54 µL 2-MIM (164.2 mg mL-1 in methanol). Finally, after reacted at 37 °C for 12 h, the dual drug-loaded hybrid core-shell DDSs (PDA-MSN@ZIF-8/DOX+CUR) was collected by centrifugation at 11000 rpm for 15 min. 2.4. Drug release experiment. Briefly, dual drug-loaded PDA-MSN@ZIF-8/DOX+CUR (0.5 mg) was ultrasonically dispersed in 2 mL of release media with different pH values at pH 7.4 (phosphate buffered saline, PBS) and pH 5.0 (sodium acetate buffer, 20 mM). After different time intervals, 0.2 mL of drug containing supernatant was taken out for analysis. Subsequently, 0.2 mL of fresh medium was added to keep a constant release volume. The evaluation of CUR and DOX released from PDA-MSN@ZIF-8/DOX+CUR was carried out by UV-vis spectrophotometer (NanoDrop One Microvolume UV-Vis Spectrophotometer, Thermo) and fluorescence spectrophotometer (RF5301PC, Shimadzu, Japan), respectively. The final results were expressed as an average from three repeated experiments. An equation for concentration calculation of DOX and CUR is listed as follows: ୲ିଵ

ν Cୡ = C୲ + ෍ C୲ V ଴


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where Cc is the corrected concentration at the release time of t; Ct is the apparent concentration at t; ν is the volume of sample taken (0.2 mL); and V is the total volume of the release fluid (2 mL). 2.5. Cell culture. Drug-resistant MCF-7/ADR cancer cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Biological Industries), 100 U mL-1 penicillin (Invitrogen), 100 U mL-1 streptomycin (Invitrogen), as well as DOX (1 µg mL-1), and maintained at 37 °C in a humidified incubator containing 5% CO2. The culture medium has to be changed every other day to keep the nutrition supply before cell experiments. A PBS solution of trypsin (0.25%) was used to harvest cancer cells, and then cells were resuspended in fresh medium before plating for further use. 2.6. Cytotoxicity assay. The cytotoxicity of MCF-7/ADR and MCF-7 cancer cells was evaluated by CCK-8 assay in 96-well plates (8×103 cells per well). After incubated overnight at 37 °C, the cancer cells were treated with various concentrations of free DOX, PDA-MSN@ZIF8/DOX, or PDA-MSN@ZIF-8/DOX+CUR. At the predetermined time intervals (24 h and 48 h), each well was washed with PBS after the removal of the original culture medium. Subsequently, 200 µL of fresh medium containing 20 µL of CCK-8 was added into each well and incubated for another 2 h at 37 °C, followed by the detection of the absorption values of each well at 450 nm by using a microplate reader (Bio-Rad 680, USA). 2.7. DOX delivery analyzed by confocal laser scanning microscopy (CLSM) and flow cytometry assay. Briefly, a proper density (1 × 105 cells) of MCF-7/ADR cancer cells were seeded and incubated overnight in 1 mL of RPMI-1640 medium. Then, the cells were incubated with 1 mL of fresh culture medium containing DOX, PDA-MSN@ZIF-8/DOX, or PDA-


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MSN@ZIF-8/DOX+CUR at an equivalent DOX concentration (10 µg mL-1). At predetermined time periods, PBS was used to wash excess drugs or nanoparticles. For CLSM (Leica, SD AF, Germany) experiments, cells were fixed with 0.5 mL of 4% paraformaldehyde (in PBS, 30 min), stained by Hoechst 33258, and visualized by fluorescence imaging. For flow cytometry (Beckman Coulter) experiments, cells were washed, harvested, and suspended in PBS for analysis. 2.8. P-gp activity assessment. P-gp efflux pump activities were measured by determining the intracellular accumulation of a P-gp probe, i.e. a fluorescent dye Rhodamine 123 (Rh 123). A proper density (1 × 105 cells) of MCF-7/ADR cancer cells were seeded and incubated overnight in confocal dishes containing 1 mL of RPMI-1640 medium. Then, 1 mL of fresh medium containing 5 µg mL-1 Rh 123 or drug loaded PDA-MSN@ZIF-8/CUR particles at a CUR concentration of 5 µg mL-1 was used to replace the original medium. After particle treatment for another 6 h, PBS was used to wash extracellular particles. Then, 1 mL of RPMI-1640 medium containing 5 µg mL-1 Rh 123 was added and incubated with cells for additional 1 h. Another PDA-MSN@ZIF-8/CUR group without Rh 123 was set as a control. The intracellular Rh 123 accumulation was visualized and analyzed by CLSM and flow cytometry, respectively. 3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of PDA-MSN and PDA-MSN@ZIF-8. The mesoporous PDA-MSN nanoparticles were synthesized by a one-pot procedure reported by our previous work.24 The typical scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images of the as-prepared PDA-MSN are shown in Figure 2a and b. The uniform spherical nanoparticles with radially aligned mesopores are about 100 nm in diameter.


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As shown in Figure 2c and d, PDA-MSN@ZIF-8 nanoparticles synthesized by one step ZIF-8 coating in a mixture solution of H2O and methanol, are core-shell structured nanoparticles with a ∼5 nm thick shell of lower contrast surrounding the PDA-MSN particles. In comparison, PDAMSN@ZIF-8 particles synthesized by two steps of ZIF-8 coating (Figure 2e and f) showed a thickness increase of ∼5 nm in the ZIF-8 shell, thereby indicating a stepwise growth of ZIF-8. Moreover, polycrystalline and fractured structures, stemming from the rhombic dodecahedron morphology of ZIF-8 nanocrystals, were generated in the shell. The hydrodynamic diameters of PDA-MSN@ZIF-8 in HEPES, as obtained by dynamic light scattering (DLS) measurements, showed an increase of ∼10 nm after ZIF-8 coating (Figure S1a). The average diameters of both particles were shifted to higher values (~300 nm) in PBS (pH 7.4) due to some slight agglomerations (Figure S1a). Moreover, the obtained nanoparticle resulted in a surface charge changing from negative (-33.4±1.3 mV) to positive (+28.7±1.1 mV, +27.2±1.2 mV) that can be seen in Figure S1b. The charge reversion further suggested that the ZIF-8 growth on the PDAMSN surface was successful.28


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Figure 2. TEM and SEM images of PDA-MSN (a, b), the core-shell structured PDAMSN@ZIF-8 with a ZIF-8 synthesized by one step ZIF-8 coating (c, d), and PDA-MSN@ZIF-8 synthesized by two steps of ZIF-8 coating (e, f). The strong metal-chelating ability of catechol groups in PDA, as well as the hydrophobic interaction between aromatic groups of PDA and the organic ligands, are expected to be the two pivotal factors facilitating the heterogeneous nucleation and growth of ZIF-8 on PDA surfaces.23, 29

To initiate the deposition of a new material phase on a previous one (PDA-MSN) in our study,

as proposed in this stepwise integration of nano ZIF-8, fundamental aspects on the influences of synthesis conditions need to be addressed. The X-ray photoelectron spectroscopy (XPS) spectra of Zn 2p (Figure S2a) for Zn(NO3)2•6H2O, PDA-Zn2+ and PDA-MSN@ZIF-8 revealed peak shifts in the binding energies. This can be attributed to the chelation between catechol group and Zn2+,25 as well as the metal-ligand coordination.15 While an extremely thin coating (Figure S2b)


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was obtained in the synthesis with pure water as the solvent, partially coated Janus structures (Figure S2c) were obtained in methanol synthesis, indicating the importance of the methanol/water ratio in obtaining the ZIF-8 coating. The mechanism behind these phenomenon should be ascribed to the fast and slow transfer/complexation speed of Zn2+ in water and methanol,21 respectively. Hence, the proper solvent ratio is a prerequisite for the successful ZIF-8 coating in our PDA-MSN@ZIF-8 system. Typical nitrogen adsorption isotherm of PDA-MSN exhibited in Figure 3a shows a type-IV isotherm with a H1 hysteresis loop, supporting the mesoporous structure of PDA-MSN nanomaterials. The corresponding pore size distribution in Figure 3 b exhibits a peak mesopore size at 4.1 nm. In comparison, a mixed isotherm was observed for PDA-MSN@ZIF-8, suggestive of the mesoporous PDA-MSN and microporous ZIF-8.18 Specifically, obvious microporosity (peak pore size: 1.7 nm) can be seen from the steep increase of N2 uptake at low relative pressures (0-0.1), while a continuous increase of the adsorbed N2 at higher relative pressure (0.2-0.4) confirms the mesoporous property from the PDA-MSN core. There are reductions in the BET surface area (from 1043 to 62 m2 g-1), as well as the pore volume (from 0.93 to 0.46 cm3 g-1), possibly because the microporous ZIF-8 shell prevent the access of N2 into the inner pore space of the PDA-MSN core.17, 30 The result further implies that the ZIF-8 phase was continuously grown and coated on the external surface of PDA-MSN core. Moreover, the Xray diffraction (XRD) pattern of PDA-MSN@ ZIF-8 nanoparticles is shown in Figure 3c. For comparison, the XRD patterns for PDA-MSN and simulated ZIF-8 were also presented. The diffraction peaks of PDA-MSN@ZIF-8 at 2θ = 7.24º, 10.29º, 12.62º, 14.95º, 16.65º and 17.93º should be attributed to the typical lattice planes of (011), (002), (112), (022), (013) and (222) in


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ZIF-8. These results confirm that the PDA-MSN@ ZIF-8 particles observed in TEM possess core-shell mesoporous-microporous structure.

Figure 3. Nitrogen-sorption isotherms (a), the corresponding pore size distributions derived from the desorption branches of the isotherms (b), and XRD patterns (c) for PDA-MSN and PDAMSN@ZIF-8. The XRD pattern of the simulated ZIF-8 was used for comparison of peaks in c. Component analysis was then applied to verify the success in ZIF-8 coating. Signals of Si, O, C and Zn atoms were observed in elemental analysis on the basis of energy-dispersive X-ray


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spectroscopy (EDS, Figure 4a) associated with SEM. In FTIR spectra (Figure 4b), the typical absorption bands at 464 cm−1, 800 cm−1, 953 cm−1, 1098 cm−1, and 3450 cm−1 are attributed to silica, while the characteristic adsorption peak of PDA can be seen at 1630 cm−1 (stretching vibration of aromatic ring and bending vibration of N-H).23 In comparison, the C-H stretching vibrations resulted from the aromatic and aliphatic C-H of the imidazde in PDA-MSN@ZIF-8, were observed at 3140 and 2934 cm-1.31 Additionally, the peak at 1585 cm-1 can be assigned to the C=N stretching, and the band at 421 cm-1 belongs to Zn-N stretching mode.31, 32 These typical characteristic peaks of PDA-MSN@ZIF-8 indicated the existence of ZIF-8 shell on the coreshell nanoparticles. Thermogravimetric analysis (TGA, Figure 4c) was used to identify the increment of organic matters in PDA-MSN@ZIF-8 after ZIF-8 coating. As compared with PDAMSN, PDA-MSN@ZIF-8 synthesized by one step ZIF-8 coating possesses a substantial weight loss increment of 31.6% in the temperature range of 200 °C-500 °C, which was a consequence of ZIF-8 decomposition. Resulting from the two steps of ZIF-8 coating, a further weight loss increment (3%) from TGA was indicative of the ZIF-8 thickness adjustment.


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Figure 4. EDS elemental analysis of PDA-MSN@ZIF-8 (a); comparison of FTIR spectra for PDA-MSN and PDA-MSN@ZIF-8 (b); TGA curves (c) of the as-synthesized PDA-MSN, PDAMSN@ZIF-8 synthesized by one step and two steps. 3.2. Drug loading and pH-responsive sequential release from PDA-MSN@ZIF-8. Acknowledging that the core-shell structured mesoporous-microporous nanoparticles can be constructed based on the PDA-MSN@ZIF-8 system, we then investigated the potential of the nanoparticles in loading and release dual cargo drugs. Furthermore, DDSs based on drug combination therapy with DOX and CUR have been developed to reversion MDR.33-35 DOX and


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CUR were selected as the model drugs, and they were sequentially loaded in the mesoporous core via π-π stacking interactions mediated by PDA,24 as well as the microporous shell via the encapsulation in the ZIF-8 framework in one-step and two-steps coating of ZIF-8, respectively (Figure 1). Ultraviolet-visible (UV-vis) absorption was measured to verify the drug loading in the co-loaded nanoparticles (PDA-MSN@ZIF-8/DOX+CUR). As shown in Figure 5a, free DOX and CUR have characteristic absorbance peaks at 482 nm and 430 nm, respectively, which could be clearly observed on the spectrum of PDA-MSN@ZIF-8/DOX+CUR nanoparticles. Furthermore, the loading capacity in PDA-MSN@ZIF-8 nanoparticles were determined to be 607 µg mg-1 (DOX) (37 mg less than the loading amount evaluated after one step coating of ZIF8, due to some inevitable release in the coating solution) and 778 µg mg-1 (CUR), respectively.


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Figure 5. The UV-vis absorption spectra of DOX, CUR, PDA-MSN@ZIF-8, and PDAMSN@ZIF-8/DOX+CUR (a); cumulative release curves (b) for DOX and CUR from drug loaded PDA-MSN@ZIF-8 nanoparticles in different release media at pH 5.0 and pH 7.4 (with the presence of 5‰ tween 20); release of Zn2+ ions from PDA-MSN@ZIF-8 nanoparticles at different times (c) in buffer solutions. Data was expressed as mean ± SDs (n=3). Inspired by the pH-stimulated drug release properties of ZIF-8 materials,36-39 cumulative drug release profiles (Figure 5b) of PDA-MSN@ZIF-8 were measured in different buffer solutions at pH 5.0 (endosomal environment) and pH 7.4 (physiological environment). Herein, the UV-vis


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absorbance at 340 nm and fluorescence at 594 nm were used to evaluate the released amounts of DOX and CUR, respectively (Figure S3). Approximately 61% of the loaded CUR was released from the PDA-MSN@ZIF-8/DOX+CUR nanoparticles in only 0.5 h at pH 5.0, then the release amount gradually accumulated to 86% in 32 h. In contrast, there was only 30% of CUR released in the same time period at pH 7.4, while the release of DOX was found to be significantly slower. Only a slight release of 6% in the first 0.5 h at pH 5.0, a sustained release up to 40% within 32 h ensued, was observed. Besides, the release profile showed only a small amount of DOX (14%) released in 32 h at pH 7.4. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) was used to test and verify the cleavage of coordination bonds between Zn2+ and 2-MIM by evaluating the amount of Zn2+ ions released from PDA-MSN@ZIF-8 at different times in buffer solutions. As shown in Figure 5c, a burst release of Zn2+ was observed at pH 5.0, where the released amount reached up to 67% in an hour, followed by a sustained increase in 12 h (84%) and 24 h (88%). However, it showed a very less release (only 10%) at pH 7.4 after the same incubation times. Furthermore, a TEM micrograph for PDA-MSN@ZIF-8 nanoparticles incubated at pH 5.0 for 2 h shows the dissosation of ZIF-8 shell in acidic conditions (Figure S4). The result demonstrated an acid-sensitive dissolution property of the ZIF-8 shell in agreement with the previous reports,37 which should be responsible for the fast release of CUR and sequential release of DOX. Additionally, a faster release behavior of DOX (Figure S5) from PDA-MSN@ZIF-8/DOX nanoparticles synthesized by one-step growth strategy was observed. Obviously, a thicker ZIF-8 shell and CUR encapsulation of PDAMSN@ZIF-8/DOX+CUR synthesized by two-steps coating of ZIF-8 facilitated an efficient sequential drug release. The sequential release behavior of this new DDS was highly attractive for reversion of MDR. Specifically, the nanocarriers could not only significantly protect the co-


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loaded drugs from premature drug release before cellular uptake of the particles, but also sequentially provide sufficient amount of drugs to effectively kill the cancer cells after particle internalization. 3.3. Drug delivery property of PDA-MSN@ZIF-8. The success in achieving high payloads of both hydrophilic (DOX) and hydrophobic (CUR) drugs by PDA-MSN@ZIF-8, as well as the sequential release behavior in acidic conditions, encouraged us to examine whether the DDS would work in living cells. DOX-resistant cancer cells (MCF-7/ADR) were selected to assess the therapeutic efficiency of PDA-MSN@ZIF-8 for reversing MDR of cancer cells. Firstly, the nonspecific toxicity of the nanoparticles was examined by co-incubating drug resistant cancer cells with PDA-MSN@ZIF-8. As shown in Figure S6, the results by the CCK-8 assay revealed a high viabilities (above 95% after 24 h, and 80% after 48 h) of MCF-7/ADR cancer cells at high particle concentrations up to 80 µg mL-1, indicating the high biocompatibility of the nanohybrids. Generally, the uptake of nanoparticles by cells is driven by receptor-mediated specific endocytosis or non-specific adsorption pathways.40 Confocal laser scanning microscope (CLSM, Figure 6) was used to analyze the intracellular release behavior by using the intrinsic red ﬂuorescence of DOX (10 µg mL-1). There were only few scattered red spots inside the freeDOX-treated cells after 24 h. Moreover, there was still a little amount of DOX within MCF7/ADR cancer cells after 48 h co-incubation, owing to the overexpressed P-gp in cell membrane which can easily pump the free intracellular DOX out of the cells and cause MDR.41 Comparatively, the PDA-MSNs@ZIF-8/DOX group exhibits slightly enhanced intracellular DOX concentrations, especially in perinuclear region after 48 h. However, without functional inhibition of P-gp, the intracellular DOX would still be actively transported to the extracellular environment. DOX exerts its pharmacological functions through interaction with DNA and


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inhibiting the biosynthesis of macromolecules in cell nucleus.42 The distribution of DOX in perinuclear region is expected to hamper its therapeutic efficiency.

Figure 6. Typical CLSM images (left panel) of fluorescence intensity for drug resistant cancer cells after treated with DOX, PDA-MSN@ZIF-8/DOX for 24 h and 48 h at 37 °C (DOX concentration: 10 µg mL-1), and the intensity profiles along the white lines in the overlay images (right panel). All the scale bars are 25 µm. Cell nucleus were stained with Hoechst 33258, and the red fluorescence was from DOX. In contrast, Figure 7 shows significant differences for drug resistant cancer cells co-incubated with PDA-MSN@ZIF-8/DOX+CUR. Time-dependent DOX accumulation was observed and the fluorescence image obtained after 24 hours of incubation showed strong DOX signals in the cells. Notably, after 48 h, the cells showed a significantly stronger red fluorescence in the cell nucleus than that in cytoplasm. Meanwhile, qualitative results of the line scanning profiles (in the right


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panel in Figure 6 and Figure 7), as derived from CLSM images, also confirmed the above trend. For a further verification of this conclusion, the DOX uptake efficiency within cells was also quantitatively analyzed by using flow cytometry (Figure S7). As shown, the PDA-MSN@ZIF8/DOX+CUR group had a much greater cellular uptake than either the free DOX group or PDAMSN@ZIF-8/DOX group, suggesting that both CUR and the PDA-MSN@ZIF-8 nanocarrier played essential roles in cellular internalization. The results above indicate that the combination therapy of DOX and CUR by PDA-MSN@ZIF-8/DOX+CUR is beneficial for inhibiting multiple MDR pumps in cancer cells.

Figure 7. Typical CLSM images (left panel) of fluorescence intensity for drug resistant cancer cells after incubated with PDA-MSN@ZIF-8/DOX+CUR for 6 h, 12 h, 24 h and 48 h (DOX concentration: 10 µg mL-1), and the intensity profile along with the white line drawn in the overlay image (right panel). All the scale bars are 25 µm.


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The higher intracellular accumulation of DOX induced significant death of DOX resistant cancer cells in the PDA-MSN@ZIF-8/DOX+CUR group (Figure 8), whereas no significant decreases in cell viabilities can be observed for the treatment of free drugs with concentrations up to 100 µg mL-1 for 24 h (Figure 8a). With the identical DOX dosage, PDA-MSN@ZIF8/DOX group showed intermediate cell viabilities. Meanwhile, a greater cytotoxicity can be seen with additional function of CUR (Figure S8) in PDA-MSN@ZIF-8/DOX+CUR. The fast release of CUR exerts a dual function of P-gp inhibition and intrinsic chemotherapy. Furthermore, PDAMSN@ZIF-8/DOX+CUR also showed a small dosage effect especially at 40 µg mL-1 (30% viable cells) after 24 h and 20 µg mL-1 (21% viable cells) after 48 h, respectively. In contrast, there is very less difference among groups when the same treatment and progress was applied to MCF-7 cancer cells without DOX resistance (Figure S9). The above results illustrated that PDAMSN@ZIF-8 nanoparticles will be a promising DDS candidate with enhanced therapeutic efficacy for MDR cancer cells.


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Figure 8. Cell viability assay of DOX, PDA-MSN@ZIF-8/DOX and PDA-MSN@ZIF8/DOX+CUR on drug resistant cancer cells at elevated DOX concentrations for 24 h (a) and 48 h (b). All the data were expressed as mean ± SDs (n=3). Having demonstrated the enhanced cytotoxicity of the loaded drugs, we then investigated the effects of the sequential release on the synergistic cancer cell inhibition. Specifically, in some in vitro and in vivo models, curcumin has been reported as a P-gp function inhibitor.43 Rhodamine 123 (Rh 123) is a well-known tracer probe for examining the membrane transport of P-gp,44 where the inhibition of P-gp function leads to its increased intracellular accumulation. The inhibition by PDA-MSN@ZIF-8/CUR was thus investigated at an early uptake stage (6 h) when the preferential release of curcumin had happened. As shown in Figure 9b, fluorescence was seldomly detected in drug resistant cancer cells treated with Rh 123 for 6 h, which may be


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attributed to the strong pumping-out function of P-gp in MCF-7/ADR cells. In comparison, PDA-MSN@ZIF-8/CUR group (Figure 9c) exhibited a slight increase in the green signal stemming from the intrinsic fluorescence of curcumin. Significantly, the strongest fluorescence was observed for the cells treated with PDA-MSN@ZIF-8/CUR (Figure 9c) and Rh 123. The flow cytometry results shown in Figure 9e are in good agreement with the trend in the fluorescence images. All these results indicate that the accumulation of Rh 123 increases, confirming the inhibitory effect of curcumin on P-gp.


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Figure 9. Typical fluorescence microscopy images of MCF-7/ADR cancer cells incubated with medium (control) (a), free Rh 123 (b), PDA-MSN@ZIF-8/CUR (6 h, c), and PDA-MSN@ZIF8/CUR (6 h) followed by Rh 123 (1 h, d); the corresponding flow cytometry analysis (e). All the scale bars are 50 µm. 4. CONCLUSION In conclusion, an efficient drug delivery system consisting of mesoporous-microporous PDAMSN@ZIF-8 nanohybrids was developed with sequential drug release properties for combination therapy. Especially, after the construction of a ZIF-8 coating mediated by PDA in the core, the nanoparticles possess a core-shell structure with a high surface area, which facilitates the high-payload and encapsulations of DOX and CUR molecules independently. More attractively, the two drugs exhibited significantly different release kinetics resulted from the discrepancy in the loading compartments (core vs. shell) inside the nanohybrid, as well as the molecular sieving effects from the CUR loaded ZIF-8 coating. The acid-sensitive burst release of CUR from the ZIF-8 shell has a strong effect on the inhibition of P-gp, while a sustained release of DOX from the core (PDA-MSN) subsequently exerts the efficient therapeutic function. The unique core-shell nanostructure, as well as sequential drug release characteristic would make the particle design potentially promising for drug nanocarriers toward the treatment of cancer, especially for reversing MDR of cancer cells. ASSOCIATED CONTENT Supporting Information. Particle characterization methods, hydrodynamic diameter and zeta potential measurements, high resolution XPS spectrum of Zn 2p, TEM images, UV-vis absorption spectra and fluorescence emission spectra, DOX release profiles, ﬂuorescence


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intensity analysis from the flow cytometry experiments, and cell viability tests. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *J. Zhang. Tel.: +86 2365102507. E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported in part by National Natural Science Foundation of China (NSFC, Grant No. 51773022, 51502027, 21734002), National Key R&D Program of China (grant No. 2016YFC1100300), the Basic Advanced Research Project of Chongqing (Grant No. cstc2015jcyjA10051), 100 Talents Program of Chongqing University (J. Z.), and Innovation Team in University of Chongqing Municipal Government (CXTDX201601002). National Engineering Research Center for Nanotechnology (Shanghai) is greatly acknowledged for the help for TEM characterization. REFERENCES (1) Gottesman, M. M.; Fojo, T.; Bates, S. E. Multidrug resistance in cancer: role of ATPdependent transporters. Nat. Rev. Cancer 2002, 2 (1), 48-58. (2) Riganti, C.; Mini, E.; Nobili, S. Editorial: Multidrug Resistance in Cancer: Pharmacological Strategies from Basic Research to Clinical Issues. Front. Oncol. 2015, 5, 105.


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Table of Contents Graphic and Synopsis


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Hybrid MesoporousâMicroporous Nanocarriers for Overcoming

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