Supramolecular Modular Approach toward Conveniently Constructing

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Functional Nanostructured Materials (including low-D carbon)

Supramolecular Modular Approach towards Conveniently Constructing and Multifunctioning a pH/ Redox Dual Responsive Drug Delivery Nanoplatform for Improved Cancer Chemotherapy Jia Liu, Xingxin Liu, Ye Yuan, Qilin Li, Bingcheng Chang, Luming Xu, Bo Cai, Chao Qi, Cao Li, Xulin Jiang, Guobin Wang, Zheng Wang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b05232 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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ACS Applied Materials & Interfaces

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Supramolecular Modular Approach towards Conveniently Constructing and Multifunctioning a pH/ Redox Dual Responsive Drug Delivery Nanoplatform for Improved Cancer Chemotherapy Jia Liu#a, Xingxin Liu#a, Ye Yuana, Qilin Lia, Bingcheng Changa, Luming Xua, Bo Caia, Chao Qia, Cao Lid, Xulin Jiange, Guobin Wang*b, Zheng Wang*a,b, Lin Wang*a,c

a. Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China b. Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China c. Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China d. Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Key Laboratory for the Green Preparation and Application of Functional, Materials of Ministry of Education, Hubei University, Wuhan, 430062, China e. Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry, Wuhan University, Wuhan, 430072, China

#

These authors contributed equally to this work.

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ABSTRACT Since heterogeneity affects many functional aspects of a tumor, a way to overcome it is to arm nanosized drug delivery systems (nanoDDS) with diverse functions required to shatter heterogeneity. However, it remains technically challenging to fabricate a nanocarrier possessing all required functions. Here we propose a modular strategy for generating a supramolecular, multifunctional, stimuli-responsive nanoDDS through docking a parental core nanoDDS with various daughter function-prebuilt modules. Doxorubicin loaded mesoporous silica nanoparticles (MSNs) as the parental nano-core are wrapped by poly(β-cyclodextrin) (PCD) as a gatekeeper through host-guest interactions between cyclodextrin and pyridine groups of pyridine-disulfide bonds that confers pH/redox dual responsiveness, thus constructing stimuli-responsive nanoDDS (DOX@PRMSNs).

Meanwhile,

PCD’s

free

cyclodextrin

is

tightly

caged

by

adamantyl-terminated daughter modules via host-guest interactions, achieving convenient multi-functionalization of this nanoDDS. DOX@PRMSNs rapidly released DOX in lysosomal pH/redox microenvironment, potently killing drug-resistant cancer cells. Further, three different types of adamantyl (Ad-) terminated daughter modules: two targeting ligands (Ad-PEG-FA and Ad-PEG-LA), a cationic polymer (Ad-PEI), and a fluorescence agent (Ad-FITC), are utilized to functionalize PRMSNs via cyclodextrin-adamantyl self-assembly, endowing the nanoDDS with cell-targeting capability, gene co-delivery property, and imaging function. Thus, this work develops

a

supramolecular

modular

self-assembly

approach

for

constructing

and

multifunctionalizing stimuli-responsive “smart” nanoDDSs.

KEYWORDS: mesoporous silica nanoparticles, pH/redox dual responsiveness, host-guest interaction, drug delivery platform, cyclodextrin


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INTRODUCTION An increasing number of studies have demonstrated that cancer reoccurrence, drug resistance, immune evasion and distant metastasis are rooted in tumor heterogeneity that dwells in nearly every facet of cancer.1-3 Even within a single tumor, different neoplastic cells can vary drastically in phenotypic and morphological characteristics, such as motility, metabolic rate, growth rate, gene expression patterns, mutation profiles, and metastasis capability. 4 These heterogeneous features of a tumor pose a formidable hurdle to therapeutic regimens hoping to eradicate cancer. Despite of being a major treatment option for cancer, conventional chemotherapy is known to be ineffective in destructing cancer heterogeneity. This is largely because conventional chemotherapy is vulnerable to complicated intratumoral biochemical heterogeneity (such as low pH, high redox, and hypoxia); it lacks targeting drug delivery capability required to surmount the complex composition of cell types within a tumor (genetic heterogeneity); it easily succumbs to drug resistance (genetic heterogeneity); its drug distribution within a tumor is difficult to monitor and control due to varying degree of intratumoral vasculature and stiffness (physical heterogeneity).1,5,6 Aiming to solve these problems that tumor heterogeneity presents to conventional chemotherapy, ever-growing efforts have been channeled to nanotechnology where diverse nano-sized drug delivery systems (nanoDDSs) have been developed.7,8 For instance, “smart” stimuli-responsive nanoDDSs are developed to utilize low pH and/ or redox potential as biochemical cues for triggering precise drug release, thus circumventing biochemical heterogeneity;9,10 ligand-grafted nanoDDSs are fabricated to actively target specific groups of cells, improving chemotherapy’s selectivity;11,12 assisted by co-delivering siRNAs (gene therapy agents) against key genes underpinning drug resistance, nanoDDSs restore killing effectiveness when facing resistant cells, thus dismantling genetic heterogeneity; 13,14 the conjugation of


3


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nanoDDSs with fluorescence dyes allows kinetics tracking, feeding back valuable information for optimizing design towards shattering physical heterogeneity.15,16 Certainly, it would be ideal that a smart nanoDDS simultaneously possesses all these functions required for tumor eradication. However, most of nanoDDSs are functionalized to individually cope with single aspect of aforementioned tumor heterogeneity, because it is methodologically and technically challenging to fabricate an “all-in-one” multifunctional nanosystem. Here we propose a modular self-assembly strategy to construct such a nanosystem in a “function-on-demand” fashion. This nanosystem is designed to have a stimuli-responsive smart nanoDDS as a parental core that can be freely functionalized with various types of daughter functional modules, each of which self-assembles onto this core nanoDDS endowing it with a capability of handling a given tumor heterogenous feature. A key to achieve this strategy is to chemically transform the parental core nanoDDS to be a “host” and an array of functionally distinct daughter modules to be the “guests”. In light of this design rationale, we utilized the supramolecular “host-guest” interactions between β-cyclodextrin and adamantyl groups to mediate such modular self-assembly (Scheme 1). Owing to the good biocompatibility, large pore volume for drug loading, and easily modifiable surface,17,18 mesoporous silica nanoparticles (MSNs) were chosen to be the core nanoDDS that would be wrapped with polymerized β-cyclodextrin (PCD) via pH/redox dual sensitive linkage (Scheme 1), which would make this nanoDDS stimuli-responsive. This linkage is designed to consist of a pyridine group (for host-guest interacting with β-cyclodextrin units) immediately followed by a disulfide bond stemming from the MSNs’ surface (Scheme 1a). Of note, PCD as a corona wrapping the MSNs’ surface would be expected to play two functional roles: capping the MSNs’ pores for preventing drug leakage and turning the core MSNs to be the supramolecular “host”. PCD on the surface of the “host” MSNs supplied cyclodextrin units that were caged by an adamantyl group that were


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universally added to “guest” functional modules beforehand regardless their bearing functions (Scheme 1b). Guided by this design rationale, we have successfully synthesized pH/ redox dual responsive MSNs (MSNs-SS-Py). With PCD as a gatekeeper blocking premature drug leakage, this nanosystem (PRMSNs) had a high doxorubicin (DOX) loading content (29.0%). The acidic and redox conditions expectedly weakened the host-guest interactions between PCD and pyridine groups and cleaved disulfide linkages, leading to PCD removal, jointly accelerating DOX release within lysosomes, resulting in enhanced DOX nuclear accumulation even in drug resistant cancer cells, thereby effectively overcoming drug resistance. Importantly, using three different types of adamantyl(Ad)-modified functional modules, we provided three working examples clearly demonstrating that this smart PRMSNs nanoDDS could be conveniently functionalized via modular self-assembly to acquire active cell-targeting ability, gene co-delivery capability, and optically trackable property, which were all valuable for effectively combating tumor heterogeneity. Thus, our modular building strategy offers convenience and simplicity for customizing a multifunctional drug delivery nanosystem capable of overcoming tumor heterogeneity towards tumor eradication, which would help pave the way for personalized and precise nanomedicine against cancer.

EXPERIMENTAL SECTION Materials. Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), benzoyl chloride, amantadine hydrochloride, (3-mercaptopropyl)trimethoxysilane (MPTMS), nicotinic acid, cysteamine hydrochloride, 2,2’-dithiodipyridine, DL-dithiothreitol (DTT), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide 3-aminopropyltriethoxysilane

(APTES)

and

hydrochloride fluorescein5(6)-isothiocyanate

(EDC), (FITC)

were

purchased from Aladdin Industrial Corporation (Shanghai, China). Sodium hydroxide, ACS Paragon Plus Environment

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hydrofluoric acid, dimethyl sulfoxide (DMSO), β-cyclodextrin, N, N-dimethylformamide (DMF), ethanol, acetone, methanol, N-hydroxysuccinimide (NHS) were obtained from Sinopharm chemical regent Co., Ltd (Shanghai, China). Doxorubicin hydrochloride (DOX) was purchased from Meilun Biology Technology Co., Ltd (Dalian, China). Poly(β-cyclodextrin) (PCD), adamantyl terminated poly(ethylene glycol) (Ad-PEG), adamantyl and folate terminated poly(ethylene glycol) (Ad-PEG-FA), adamantyl and lactobionic acid terminated poly(ethylene glycol) (Ad-PEG-LA), and adamantyl terminated polyethylenimine (Ad-PEI) were synthesized in previous works.

19 , 20

Adamantyl terminated fluorescein isothiocyanate (Ad-FITC) was

synthesized as the literature.21


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Scheme 1. (a) Synthetic procedure, doxorubicin (DOX) loading and release mechanisms of pH and

redox

dual

responsive

MSNs

(DOX@PRMSNs).

(b)

Post-functionalization

of

DOX@PRMSNs using adamantyl-terminated guests (targeting ligands, cationic polymer and fluorescence agent) by self-assembly. Synthesis of MSNs-SH. MCM-41 mesoporous silica nanoparticles with template (CTAB@MSNs) were synthesized as previous procedure.22 The obtained CTAB@MSNs (1.0 g) were dispersed in methanol (200 mL) containing MPTMS (5 mL) and stirred for 24 h at room temperature. Subsequently, the particles were collected and washed with water and methanol. Then the product was refluxed in the mixture of hydrochloric acid (36%-38%, 10 mL) and ACS Paragon Plus Environment

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methanol (160 mL) at 80°C for 48 h to remove the CTAB template. The product was washed with water and methanol, and collected by centrifugation. Finally, the thiol-functionalization MSNs (MSNs-SH) were dried under vacuum.

Synthesis of MSNs-SS-Py. MSNs-SH (160 mg) were dispersed in methanol (40 mL) containing 2,2'-dithiodipyridine (480 mg), and the suspension was stirred at room temperature for 24 h. The product (MSNs-SS-Py) was washed (methanol and deionized water), and dried by lyophilization.

Drug loading and PCD capping. MSNs (200 mg, MSNs-SS-Py, MSNs-Py and MSNs-SS-Bz) were dispersed respectively in PBS (180 mL) and mixed with DOX solution (100 mg, in 20 mL water). These dispersions were stirred at room temperature for 24 h. Then, PCD (400 mg) was added and the dispersions were stirred for 24 h. The DOX-loaded MSNs (DOX@PRMSNs, DOX@PMSNs, DOX@RMSNs) were collected by centrifugation, washing, and dried by lyophilization. MSNs@PCD (PRMSNs, PMSNs, RMSNs) were prepared using the same method without the procedure of DOX loading. The MSNs-SS-Py@PCD-FITC were prepared using PCD-FITC as the gatekeeper with the same method. To evaluate the DOX loading efficiency, DOX-loaded MSNs were dissolved in hydrofluoric acid (4%) and the absorbance of solution at 480 nm was determined using a UV-Vis spectrophotometer (Lambda Bio40 UV/Vis spectrometer, Perkin–Elmer).

In vitro gatekeeper dissociation dynamics. MSNs-SS-Py@PCD-FITC (0.5 mg) were dispersed in the buffers (1.5 mL) with different pH values (acetate buffer, pH 5.0; PBS, pH 6.8 or 7.4) or in the buffers (pH 5.0 or 7.4) containing 10 mM DTT, respectively. Then these dispersions were shaken (200 rpm) at 37 °C. At certain time intervals, the samples were centrifuged, the ACS Paragon Plus Environment

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supernatants were collected and replaced with fresh buffers. The concentrations of PCD-FITC in these supernatants were determined using a RF-5301 PC fluoro spectrophotometer (Shimadzu) (Ex: 495 nm, Em: 520 nm).

In vitro drug release behaviors. The drug loaded nanoparticles (0.5 mg, DOX@PRMSNs, DOX@PMSNs, or DOX@RMSNs) were dispersed in the buffers (1.5 mL) with different pH values (5.0, 6.8 or 7.4) or in the buffers (pH 5.0 or 7.4) containing 10 mM DTT. Subsequently, the dispersions were shaken (200 rpm) in the dark at 37 °C. At given time intervals, the release media were withdrawn and replaced with fresh buffers. The DOX concentrations of these media were determined using a UV-Vis spectrophotometer at 480 nm.

Characterizations. 1H nuclear magnetic resonance (NMR) spectra were recorded on a Mercury 300 MHz spectrometer (Varian Associates Inc. NMR instruments, Palo Alto, CA) with D2O or DMSO as solvents. 2D 1H nuclear Overhouser effect spectroscopy (NOESY) spectra were obtained on a Bruker Avance III-600 MHz spectrometer (Bruker Biospin, Germany). Transmission electron microscope (TEM, HITACHI H-7000FA, Japan) was used to characterize the morphologies of nanoparticles. The zeta potential of the MSNs was determined using Nano-ZS ZEN3600 (Malvern Instruments). The thermal gravimetric analysis (TGA) was carried out on METTLER TOLEDO TGA/DSC 1 actions. The N2 adsorption/desorption isotherm was performed using an adsorption analyzer (JW-BK112, Beijing JWGB Sci.&Tech. Co.,Ltd.), and the surface area and pore size were calculated using the Brunauer–Emmett–Teller (BET) model.

Hemolysis assay. The hemolysis experiments were performed as previous study.22 In detail, 900 µL rabbit red blood cells suspension (2% in PBS, v/v) was mixed with nanoparticles (100 µL


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in PBS), and then shaken (100 rpm) at 37 oC for 4 h. Finally, these samples were centrifuged (10000 rpm, 10 minutes), and the absorbance of hemoglobin in supernatants was measured using a microplate reader (Infinite F50, Tecan, Switzerland) at 545 nm. Triton X-100 and PBS were used as the positive and negative controls, respectively.

Cell Culture. Human breast cancer cells (MCF-7 cells) and the DOX-resistant counterpart cells (MCF-7/ADR cells) were cultured in Roswell Park Memorial Institute (RPMI) 1640 media containing 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C. Human liver hepatocellular cancer cells (HepG2 cells), human cervical cancer cells (HeLa cells) and human embryonic kidney cells (293T cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin at 37 °C.

In vitro cytotoxicity assay. The cytotoxicity of nanoparticles was evaluated using the MTT assay. HeLa, HepG2, MCF-7, MCF-7/ADR and 293T cells were seeded respectively at a density of 8000 cells/well in 96-well plates and cultured for 24 h. Then the media were replaced with fresh media containing free DOX or nanoparticles. After incubation for 48 h or 96 h, the media were replaced with fresh media (200 µL). Next, the MTT reagent (5 mg/mL, 20 µL in PBS) was added and incubated for 4 h at 37 °C. Then, the formed formazan crystals were dissolved in DMSO (150 µL) and the absorbance was measured using a microplate reader (Infinite F50, Tecan, Switzerland) at 570 nm.

Intracellular DOX release and cellular uptake. HepG2 cells were seeded on 10 mm2 glass coverslips in 12-well plates at a density of 6×104 per well and cultured for 24 h. Then the media


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were replaced with fresh media containing nanoparticles (4 µg/mL of DOX). After incubation for 1 h, all the media were removed and washed with PBS for three times, and fresh media were added and incubated for another 1 or 3 h. After that, the cells were fixed (4% paraformaldehyde) and stained with Hoechst 33342. Finally, the coverslips were taken out and observed on Nikon Ti-U microscope equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa) and an EM-CCD camera (iXon+; Andor). The fluorescence intensity of DOX in nuclei were analyzed by ImageJ software from 100 randomly selected cells. The cellular uptake of these nanoparticles was studied by flow cytometry. The HepG2 cells were seeded in 6-well plates (106 cells/well) and cultured for 24 h. Then the cells were treated with the DOX-loaded nanoparticles. After incubation for 1 h, the cells were washed, collected and analyzed by flow cytometry (Canto II, BD Company, USA). To study the intracellular release mechanisms, HepG2 cells were treated with DOX@PRMSNs in the media containing NH4Cl (10 µM), BSO (1 µM), or NH4Cl (10 µM) plus BSO (1 µM) at 37 °C for 4 h. After incubation, the cells were fixed, stained with Hoechst 33342, and observed as described above. For flow cytometry analysis, the HepG2 cells were washed, collected and analyzed by flow cytometry. To study the targeting functions of PRMSNs/Ad-PEG-LA and PRMSNs/Ad-PEG-FA, HepG2

cells

(or

HeLa

cells)

were

treated

with

DOX@PRMSNs/Ad-PEG,

DOX@PRMSNs/Ad-PEG-LA (or DOX@PRMSNs/Ad-PEG-FA) in the media without or with 10 mM lactobionic acid (or 1 mM folic acid) at 37 °C for 4 h (4 µg/mL of DOX). After incubation, the cells were imaged by confocal or analyzed by flow cytometry as described above. To study the imaging function of PRMSNs/Ad-FITC, HepG2 cells were incubated in the media containing PRMSNs/Ad-FITC (or PRMSNs/FITC, FITC, Ad-FITC; 0.4 µg/mL of FITC) for 4 h. Then, the cells were imaged by confocal or analyzed by flow cytometry as described


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above.

Agarose gel electrophoresis. PRMSNs/Ad-PEI and pDNA (pcDNA3-Luc) were mixed at weight ratios from 0 to 50, and incubated for 30 minutes to obtain polyplexes. The agarose gel electrophoresis was carried out in a 1% (w/v) agarose gel containing ethidium bromide (EB, 0.25 mg/mL) in Tris–acetate (TAE) buffer at 100 V for 40 minutes. The pDNA bands were photographed using a Gel Doc™ XR+ imaging system (Bio-Rad, USA).

In vitro transfection activity assay. HeLa and 293T cells were seeded respectively at a density of 8000 cells per well in 96-well plates and cultured for 24 h. Then, the media were replaced with fresh media containing nanoparticles/pDNA polyplexes (0.25 µg pcDNA3-Luc in 25 µL PBS). After incubation for 4 h, the media were replaced by fresh DMEM and the cells were incubated for another 44 h. After that, the cells were washed with PBS and lysed with 1× Reporter Lysis buffer (50 µL). The luciferase expression was measured using a luminometer (GloMax®20/20, Promega, USA), and the total protein was detected using a BCA protein assay kit (Beyotime, China). The luciferase activity was expressed as RLU/mg protein.

RESULTS AND DISCUSSION Preparation and characterizations of DOX@PRMSNs. The synthetic procedure of the pH/ redox dual responsive MSNs drug delivery platform was summarized in Scheme 1a, including four steps: (1) the MCM-41 MSNs were synthesized via hydrolysis of tetraethyl orthosilicate using the cetyltrimethylammonium bromide (CTAB) template according to previous work;22 (2) the external surface of CTAB@MSNs was modified with sulfhydryl (-SH) followed by template extraction to obtain sulfhydryl-modified MSNs (MSNs-SH); (3) the MSNs-SH were


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reacted with 2, 2’-dithiodipyridine through disulfide exchange to prepare pyridine functionalized MSNs with disulfide linkage (MSNs-SS-Py); (4) the anticancer drug (doxorubicin, DOX) was loaded into the pores of the nanoparticles that were capped with PCD through host-guest interactions between CD units and pyridine groups. The DOX-loaded nanoparticles were denoted as DOX@PRMSNs (Scheme 1), while the empty nanoparticles (without DOX) were referred to as PRMSNs. The nanoparticles of MSNs-SS-Py exhibited a uniform spherical shape with an average diameter of 102.2 nm and a well-defined mesostructure (Figure 1a and b). PCD formed a corona wrapping the MSNs (Figure 1c), drastically reduced the BET (Brunauer–Emmett–Teller) surface area from 719.2 m2/g to 128.3 m2/g (Figure 1d), and switched the BJH (Barrett-Joyner-Halenda) pore size from 2.4 nm in MSNs-SS-Py to be undetectable in PRMSNs (Figure 1e), indicating an effective PCD coating on MSNs-SS-Py. These chemical modifications on the MSNs’ surface were confirmed by a decrease in zeta-potential (MSNs-SH, -18.6 mV; MSNs-SS-Py, -23.5 mV; PRMSNs, -27.4 mV) (Figure 1f), and an increasing weight loss across MSNs-SH (20.5%), MSNs-SS-Py (27.8%) and PRMSNs (39.8%) determined by thermal gravimetric analysis (TGA) (Figure 1g). These results indicate that PRMSNs are successfully synthesized. In addition, the mono-responsive MSNs coated with PCD (pH-responsive MSNs-Py@PCD, termed as PMSNs; redox-responsive MSNs-SS-Bz@PCD, termed as RMSNs) were synthesized (see details in the Supporting Information, Scheme S1) and confirmed also by zeta potential measurements and TGA analysis (Figure S1). These mono-responsive nanoparticles were used as the controls in this study.


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Figure 1. Characterizations of MSNs-SS-Py and PRMSNs. (a-c) TEM images of MSNs-SS-Py (a, b) and PRMSNs (c). Scale bars, 50 nm. (d-e) The BET surface area (d) and the BJH pore size (e) of MSNs-SS-Py and PRMSNs. (f-g) Zeta potential (f) and TGA (g) of corresponding silica nanoparticles (MSNs-SH, MSNs-SS-Py, PRMSNs and PRMSNs treated with DTT or acidic buffer).


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Figure 2. In vitro pH/redox dual responsive properties of PRMSNs. (a) 2D 1H NOESY spectra of PCD/pyridine solution at pH 7.4 and 5.0 in D2O. (b) In vitro release profiles of PCD-FITC from PRMSNs in the buffers (pH 5.0, 6.8 or 7.4) and the buffers (pH 5.0 or 7.4) containing 10 mM DTT at 37 °C. (c) In vitro DOX release profiles from DOX@PRMSNs in the buffers (pH 5.0, 6.8 or 7.4) and the buffers (pH 5.0 or 7.4) containing 10 mM DTT at 37 °C.

In vitro PCD dissociation behaviors in response to acidic and redox conditions. Given that pKa value of pyridine (5.23)23 is close to lysosomal microenvironment (pH 4.5~5.0),24 the host-guest interactions between PCD and the pyridine groups from MSNs would be stable in ACS Paragon Plus Environment

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neutral conditions, but weakened by lysosomal acidity due to the protonation of PRMSNs’ pyridine groups, which would lead to dissociation of PCD corona from the MSNs, opening up the pores for drug unloading. Additionally to acidity, redox conditions within lysosomes (2-10 mM glutathione) would cleave disulfide bonds between pyridine groups and the surface of the MSNs, which would similarly free PCD.24 To test whether this design rationale could be realized in vitro, we analyzed the host-guest interactions between PCD and pyridine at different pH conditions (pH 7.4 and 5.0) using 2D 1H nuclear Overhauser effect spectroscopy (NOESY) in D2O (Figure 2a). The NOE cross peaks between the inner protons of β-CD units (3.5 to 4.1 ppm) and the protons of pyridine (7.2 to 8.6 ppm) were clearly detected at pH 7.4, while the cross peaks nearly disappeared at pH 5.0, indicating that pyridine is within the cavity of CD units forming a non-covalent bond at the neutral condition, but physically away from the CD units at the acidic condition (lower than pKa). Moreover, after incubation at acidic buffer (pH 5.0, 12 h), the weight loss of PRMSNs measured by TGA decreased from 39.8% to 30.7%, close to that of MSNs-SS-Py (27.8%), suggesting nearly complete removal of PCD (Figure 1g). Next, we tested the efficacy of PCD removal in redox conditions. The weight loss of PRMSNs in redox conditions (10 mM DTT, 12 h) decreased from 39.8% to 23.3%, approaching that of MSNs-SH (20.5%), indicating PCD’s full dissociation resulting from effective cleavage of disulfide bonds (Figure 1g). Then, we used FITC to label the PCD gatekeeper (PCD-FITC) in order to dynamically monitor PCD dissociation behavior in response to acidic and redox conditions (Figure 2b). While under pH 7.4 and 6.8 conditions less than 10% of PCD-FITC was dissociated over 12 h, the higher amount of PCD-FITC was detached from the nanoparticles either at the acidic environment (50% for pH 5.0) or in the presence of a reducing agent (60% for 10 mM DTT). Notably, the PCD dissociation was drastically enhanced under the acidic and redox combined


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condition (pH 5.0 with 10 mM DTT) as 90% of PCD-FITC fell off within 8 h (Figure 2b). Taken together, these results indicate that PCD tightly attaches to pyridine groups on the surface of MSNs via host-guest interactions at neutral conditions, while swiftly detaches from the surface of the MSNs in response to the acidic and/ or redox conditions.

In vitro drug release kinetics in response to pH and/ or redox conditions. Doxorubicin (DOX) was chosen as a model drug to be loaded into the MSNs. The loading content (LC) of DOX in DOX@PRMSNs was determined to be 29.0% (408 mg DOX per 1000 mg PRMSNs), a high drug-loading efficacy. The mono (pH)-responsive DOX@PMSNs (LC: 27.2%) and the mono (redox)-responsive DOX@RMSNs (LC: 28.3%) were synthesized as the controls. The pH responsive drug release behavior of these nanoparticles was studied in the buffers with different pH values, mimicking blood and normal tissues (pH 7.4), tumor microenvironment (pH 6.8), and lysosomes (pH 5.0). Under pH 7.4 and 6.8 conditions where PCD securely capped the MSNs’ pores, less than 20% of DOX was released from PRMSNs, PMSNs and RMSNs over 72 h (Figure 2c, S2a and S2b), suggesting a good stability in circulation. In contrast, the DOX release from PRMSNs (57.3% within 72 h) and PMSNs (60.3% within 72 h) was significantly accelerated at pH 5.0 (Figure 2c and S2a), consistent with the rapid PCD dissociation in response to the acidity. We then tested the drug release triggered by redox conditions. The addition of DTT (10 mM) significantly increased the DOX release rate from the disulfide-linkage containing MSNs (PRMSNs and RMSNs) at both pH 7.4 and 5.0, but did not affect the DOX release from PMSNs (no disulfide linkage), indicating that the cleavage of disulfide linkages in redox environment accelerates drug release. More importantly, in pH 5.0 with DTT (mimicking lysosomal microenvironment), the DOX release from the pH/redox dual responsive MSNs (PRMSNs, 77.1% within 24 h) was significantly faster than the two mono-responsive MSNs (PMSNs, 54.2%; ACS Paragon Plus Environment

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RMSNs, 62.8%) (Figure 2c and S2C), indicating that the pH/ redox dual responsiveness enhances drug release. Together, these results indicate that acidic or redox conditions can effectively trigger DOX release, while their combination leads to a significant enhancement on drug release rate.


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Figure 3. Intracellular DOX release mechanisms and behaviors in HepG2 cells. (a) Confocal images of HepG2 cells after incubation with DOX@PRMSNs (4 µg/mL of DOX) for 4 h in the


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media without (control) or with BSO (B), NH4Cl (N), or BSO plus NH4Cl (B+N). The cell nuclei were stained with Hoechst 33342 (blue). Scale bar, 10 µm. (b) The mean DOX fluorescence intensity in the nuclei of HepG2 cells after incubation with DOX@PRMSNs (4 µg/mL of DOX) for 4 h in the media without (control) or with BSO, NH4Cl, or BSO plus NH4Cl. Data are shown as Mean ± SD, n=4, *p

Supramolecular Modular Approach toward Conveniently Constructing

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