Super-pH-sensitive mesoporous silica nanoparticles-based drug

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Controlled Release and Delivery Systems

Super-pH-sensitive mesoporous silica nanoparticles-based drug delivery system for effective combination cancer therapy Yin-Jia Cheng, Si-Yong Qin, Yihan Ma, Xiao-Sui Chen, Ai-Qing Zhang, and Xian-Zheng Zhang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00099 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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ACS Biomaterials Science & Engineering

Super-pH-sensitive mesoporous silica nanoparticles-based drug delivery system for effective combination cancer therapy

Yin-Jia Cheng,*,† Si-Yong Qin,† Yi-Han Ma,† Xiao-Sui Chen,† Ai-Qing Zhang,† Xian-Zheng Zhang‡

†School

of Chemistry and Materials Science, South-Central University for Nationalities, 182 Minyuan

Road, Hongshan District, Wuhan, Hubei 430074, P. R. China ‡A

Key Laboratory of Biomedical Polymers of Ministry of Education and Department of Chemistry,

Wuhan University, 299 Bayi Road, Wuchang District, Wuhan, Hubei 430072, P. R. China

* Corresponding author. E-mail address: [email protected] (Y.-J. Cheng).

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ABSTRACT: A multifunctional nanoplatform based on mesoporous silica nanoparticles (MSNs) was developed for combinational tumor therapy. Doxorubicin (DOX) was chosen as an anti-tumor drug and loaded into mesopores of MSNs via physical absorption. Then, a tumor-targeted fusion peptide conjugated with 2,3-dimethylmaleic anhydride (DTCPP) and a therapeutic peptide conjugated with 2,3dimethylmaleic anhydride (DTPP), were introduced to the surface of MSNs as super-pH-sensitive nanovalves through disulfide linkages. The BSA adsorption assay confirmed the charge-reversal property of MSN-ss-DTPP&DTCPP nanoparticles at slightly acidic condition (pH 6.8) and superior stability in physiological environment (pH 7.4). According to the drug release research, both glutathione (GSH) and acidic condition are required for the accelerated drug release from DOX@MSN-ssDTPP&DTCPP nanoparticles. Moreover, in vitro studies demonstrated the significantly reinforced tumor cellular uptake efficiency and mitochondrial disruption ability of DOX@MSN-ss-DTPP&DTCPP nanoparticles in tumor environment, in which DOX@MSN-ss-DTPP&DTCPP nanoparticles exhibited the preferred cytotoxicity toward αvβ3-positive Human cervical carcinoma (HeLa) cells. We believe that the multifunctional dual-stimuli-sensitive MSN could provide an effective strategy for combinational tumor therapy.

KEYWORDS: Mesoporous silica nanoparticles, Multifunctional peptide, Charge-reversal, Redoxsensitive drug release, Combinational tumor therapy

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INTRODUCTION For cancer treatment, advancement in nanotechnology provides considerable opportunities to improve the anticancer efficacy of therapeutic agents.1 Currently, researchers have been searching and designing multifunctional nanoparticles (NPs) for efficient drug loading and thus desired cancer treatment, including gold nanoparticles (AuNPs), polymeric micelles, magnetic nanoparticles and mesoporous silica nanoparticles (MSNs).2-7 The superiority of multifunctional nanoparticulate drug delivery systems (DDSs) includes their capacity to improve drug solubility, enhance drug accumulation in targeting sites and reduce the unwanted side toxicity to a large extent.8,9 MCM-41 type based MSNs are selected as a kind of leading candidates for controlled drug delivery and attracted much interest in biomedical applications.5,10-13 Due to their advantageous properties, such as excellent biocompatibility, ease of surface modification and large pore volume, MSNs can encapsulate drug in pores with high drug loading capacity, precisely control drug release behavior, and co-deliver different therapeutic agents to different sites after surface modification. However, there still exists various physiological barriers for MSNs before arriving at the specific target site, which could result in a heterogeneous distribution of NPs and subsequently severe side toxicity.11 Therefore, it is a good choice to develop various kinds of therapeutic strategies to surmount all those physiological obstacles. Recently, combination therapy involving different therapeutic agents with synergism, has been proposed as an innovative strategy for enhancing therapeutic efficiency in cancer treatment.12-21 For example, Zhang’s group established a one-pot strategy constructing a pH-responsive MSN (CPT@MSN-hyd-DOX), where doxorubicin (DOX) was chemically conjugated with MSNs through tumor-acidity-cleavable hydrazone bond and camptothecin (CPT) was physically entrapped into the pores of MSNs. This work demonstrated that the multi-drugdelivery system could keep a relatively “stealth” behavior during blood circulation, but achieve intracellular release of synergistic drugs (CPT/DOX) in tumor acidic environment (~ pH 6.8), resulting in an enhanced chemotherapy of glioblastoma.13 Nel’s group reported a lipid capped MSN nanoplatform (PTX/GEM LB-MSNP) for co-delivery of gemcitabine (GEM) and paclitaxel (PTX) to human pancreatic tumor in mice.14 The in vivo research highlighted the therapeutic efficacy of PTX/GEM LBMSNP compared with free drugs, and indicated an effectively primary inhibition of tumor growth as well as elimination of metastatic tumor foci. Moreover, a great many of conventional chemotherapeutic drugs have poor pharmacokinetics and spread all over the body, which would produce serious side effects. The tumor-targeted drug delivery systems (TDDSs) that can target to the tumor site have been developed to solve the problems mentioned above. In recent years, active targeting strategy refers to incorporate affinity ligands to the surface of NPs to provide the cell-type specificity and thus reduce 3



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systemic toxicity, which has been proposed to promote the accumulation of NPs to tumor cells.22 Several different kinds of targeting ligands have been developed and employed for this strategy, such as folate acid (FA), transferrin (Tf), vascular endothelial growth factor (VEGF) and RGD peptide et al.23-29 It is well-known that RGD peptide and RGDS peptide are widely used as the targeting ligand for quick recognition of αvβ3 integrin receptor over-expressed on the membrane of HeLa cells, and thereby increasing the therapeutic effect of anti-tumor drugs.25-27 Representative mitochondria penetrating peptide (TPP), C-GKGG-D(KLAKLAK)2, was previously employed as the therapeutic agent to accelerate the mitochondria-anchoring accumulation of NPs as well as induce mitochondria-dependent apoptosis.5-30 However, these targeting ligands cannot help nanocarriers penetrate cellular membrane, so additional decorations are needed to promote the cellular uptake of therapeutic agents. It is worth noting that cell-penetrating peptides (CPPs), such as the transactivator of transcription (TAT) peptide, CGRKKRRQRRRPPQ, can facilitate the internalization of NPs into cells due to the electrostatic interactions between negatively charged cellular membranes and positively charged peptides, and therefore have been exploited in many targeting DDSs.31,32 For the sake of pH difference between the microenvironments of tumor tissues and normal tissues, and the pH difference between the intracellular environments and extracellular matrix (ECM), the strategy of dual-pH-response could effectively avoid the premature release of payloads from DDSs.33-35 Moreover, an ideal nanoplatform modified with 2,3dimethylmaleic anhydride (DMA) has been proven to be relatively stable under neutral and alkali conditions, and therefore possess original negative charge during the blood circulation to avoid nonspecific protein adsorption. However, it degrades rapidly at slightly acidic pH value and convert to positive charge once reaching the tumor extracellular environment (pHe ~6.8) due to the detachment of DMA.20,32,36-40

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Scheme 1. Schematic design of the delivery process: (a) charge-reversion of DOX@MSN-ss-DTPP&DTCPP NPs in acidic tumor microenvironment; (b) RGDS-targeted accumulation of DOX@MSN-ss-DTPP&DTCPP NPs in acidic tumor microenvironment; (c) adsorption-interaction between positively charged DOX@MSN-ss-TPP&TCPP NPs and negatively charged cellular membrane; (d) TCPP peptide mediated tumor cellular penetration of drug loaded NPs; (e) GSH-triggered removal of TPP and TCPP peptides from DOX-loaded NPs; (f) GSH-triggered intracellular DOX release into nucleus, resulting in DNA damage; (g) glutathione-triggered intracellular TPP peptide release into the mitochondria, causing a specific mitochondrial disruption.

Herein, the aim of this work is developing a type of multifunctional MSN (DOX@MSN-ssDTPP&DTCPP) for tumor-targeting delivery with dual stimuli responsive capacities (super-pH and redox stimuli) as demonstrated in Scheme 1. Doxorubicin hydrochloride (DOX), a very popular antitumor model drug, was selected to be encapsulated into pores of MSNs. Then, the lysine amino residue of tumor-targeted fusion peptide (C-GRKKRRQRRRPPQ-RGDS, TCPP) and therapeutic peptide (CGKGG-D(KLAKLAK)2, TPP)

were amidated by DMA into the super-pH-responsive β-carboxylic

amide, and the formed negatively charged multifunctional peptides (DTCPP and DTPP) were further conjugated with MSNs via redox-sensitive disulfide linkages. The DOX@MSN-ss-DTPP&DTCPP NPs were capable of maintaining “stealth” in physiological environment, which could sequentially overwhelm biological barriers. However, a charge conversion from negative to positive charge occurred in DOX@MSN-ss-DTPP&DTCPP NPs after RGDS-induced an enhanced accumulation in the specific tumor site, which could largely prompt the cellular uptake efficiency. After then, drug-loaded DOX@MSN-ss-DTPP&DTCPP NPs would release DOX and TPP peptide in response to the overexpressed GSH (10 mM) in cytoplasm, owing to the rupture of disulfide linkages and the removal of

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peptide shells. More importantly, the synergistic delivery of therapeutic peptide and anti-tumor drug from multifunctional DOX@MSN-ss-DTPP&DTCPP NPs could induce dysfuction of nucleus as well as mitochondria in tumor cells (Scheme S1), and thus enhance the treatment efficacy against tumor cells. For comparison, another kind of anhydride, succinic anhydride (SA), were conjugated with the side chain of multifunctional peptides, and acted as super-pH-insensitive “nanovalves” on DOX@MSN-ssSTPP&STCPP NPs via disulfide linkages. The concept of this work will provide the possibility of a multistage targeting strategy on the basis of multifunctional MSNs, for intracellular co-delivery of antitumor drug and therapeutic peptide for combination tumor therapy. EXPERIMENTAL SECTION Materials and reagents Rink-Amide resin (100-200 mesh, 0.59 mmol/g), 2-chlorotrityl chloride resin (100-200 mesh, 1.21 mmol/g), N-fluorenyl-9-methoxycarbonyl (Fmoc) protected D-amino acids (Fmoc-D-Ala-OH, Fmoc-dLeu-OH,

Fmoc-D-Lys(Boc)-OH,

Fmoc-Lys(Dde)-OH),

1,2-ethanedithiol

(EDT),

N-

hydroxybenzotriazole (HOBt), o-benzotriazol N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), triisopropylsilane (TIS) and diisopropylethylamine (DiEA) N-fluorenyl-9-methoxycarbonyl (FMOC) protected L-amino acids (Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH, FmocArg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Lys(mtt)-OH, and Fmoc Lys(Boc)-OH) were provided by GL Biochem Ltd and used directly. N-Cetyltrimethylammonium bromide (CTAB), 3-mercaptopropyltrimethoxysilane (MPTMS), tetraethylorthosilicate (TEOS), trifluoroacetic acid (TFA), phenol, piperidine and dichloromethane (DCM) were obtained from Shanghai Reagent Chemical Co. and used without any purification. JC-1 and Mitochondria probe (Mito Tracker® Red CMXRos) were brought from Qcbio Science & Technologies Co. Ltd. (China). (3mercaptopropyl) trimethoxysilane, succinic anhydride (SA), dimethylmaleic anhydride (DMA) and N, N-dimethylformamide (DMF) were purchased from Aladdin Reagent Co. Ltd. and DMF was used after distillation. Doxorubicin hydrochloride (DOX) and 2,2-dipyridyl disulfide were brought from Zhejiang Hisun Pharmaceutical Co. (China). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin-streptomycin and trypsin were brought from Invitrogen. Other reagents and solvents are of analytical grade and used without any purification.

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Synthesis of DMA-conjugated peptides (DTCPP and DTPP) The multifunctional fusion peptide TCPP (C-GRKKRRQRRRPPQ-RGDS), endowing tumortargeting and cellular membrane-penetrating abilities, was manually synthesized on 2-chlorotrityl chloride resin through standard solid phase peptide synthesis (SPPS) technique [25]. First, FmocSer(tBu)-OH was linked to the resin in the presence of 4.5 equiv. DiEA and 3 equiv. amino acid. After reaction for 2 h, the Fmoc protecting groups were removed with a 25% piperidine/DMF solution twice (10 min+10 min). Next, the following coupling of Fmoc protected L-amino acid was proceeded in the presence of 8 equiv. DiEA, 6 equiv. HBTU, 6 equiv. HOBt and 4 equiv. amino acid, and reacted for 2 h at room temperature. The successful coupling of amino acids and deprotection of the Fmoc groups was proven by ninhydrin assay. The resin was purified with DMF, CH3OH and DCM for each after completion of peptide synthesis, and reacted with a mixture of TFA, phenol, distilled water, TIS and EDT at the volume ratio of 82.5: 6: 4.5: 4.5: 2.5 to cleave the peptide from resin. After rotary evaporation, precipitation in cold ether and drying under vacuum, the peptide product was obtained. The synthesis procedure to DTCPP peptide was taken as follow: 100 mg TCPP (0.1 mmol NH2 groups) and 0.16g DMA (1.0 mmol) were dissolved in 2 mL phosphate-buffered saline (PBS) buffer (pH ~ 10). Then, the pH value of the reaction solution was adjusted to 10 by adding NaOH. After 12 h to ensure the complete reaction between DMA and TCPP peptide, the product DTCPP was obtained after dialyzing against PBS buffer (pH~ 10) and freeze drying. The therapeutic TPP peptide (C-GKGG-D(KLAKLAK)2), which consists of D-amino acids, was synthesized on the Rink-Amide resin according to previous reports.5,25 The synthesis procedure to DTPP peptide was taken as follow: 100 mg TPP (0.78 mmol NH2 groups) and 1.2 g DMA (7.8 mmol) were dissolved in 2 mL phosphate-buffered saline (PBS) buffer (pH ~ 10). Then, the pH value of the reaction solution was adjusted to 10 by adding NaOH. After 12 h to ensure the complete reaction between DMA and TPP peptide, the product DTPP was obtained after dialyzing against PBS buffer (pH~ 10) and freeze drying. Besides, The DMA-conjugated peptides (DTPP and DTCPP) were analyzed by using reversephase high performance liquid chromatography (HPLC) after incubation for different times at pH 6.8. Synthesis of SA-conjugated peptides (STPP and STCPP) For comparison purposes, SA-conjugated peptides (STPP and STCPP) were prepared due to its superpH-insensitive property. The synthesis of STPP peptide was carried as follow: 100 mg TPP (0.78 mmol NH2 groups) and 0.8 g SA (7.8 mmol) were dissolved in 5 mL PBS buffer (pH 8.0 ~ 9.0). Then, the pH value of the reaction solution was adjusted to 10 by NaOH addition. After 12 h reaction to ensure the 7



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complete modification of TPP peptide with SA, the product STPP was obtained after dialyzing against PBS buffer (pH~10) and freeze drying. Additionally, STCPP peptide was synthesized in a similar way as STPP peptide. The product, SA-conjugated peptides (STPP and STCPP), were analyzed using MALDI-TOF MS: STCPP (Figure S1) , 2659.4 [M+H]+ and STPP (Figure S2), 2567.6 [M+H]+. Synthesis of MSN-SH NPs Bare MSNs were prepared in a similar approach to our previous work [22,36]. Then, solid MSNs (400 mg) were dispersed in methanol (40 mL) containing 3 mL of MPTMS, and the mixture was vigorously stirred for 12 h. Finally, the solid MSN-SH was purified by centrifugation (11000 rpm/min, 15 min), washed with methanol and DI water, and dried under vacuum. The extraction of surfactant template (CTAB) was finished by refluxing solid MSN-SH NPs in a solution of methanol (80.0 mL) containing 1.0 mL of HCl (37%) at 60 oC for 48 h. The CTAB-removed MSN-SH NPs were purified by centrifugation (11000 rpm/min, 15 min), washed with methanol, and collected by vacuum drying. Synthesis of MSN-ss-Pyridyl NPs MSN-SH NPs (300 mg) were refluxed in 20 mL of methanol containing 60 mg of 2, 2′-dipyridyl disulfide, and the reaction was conducted in the dark at 25 oC for 24 h. After centrifugation (11000 rpm/min, 15 min), the precipitate was washed with methanol, and dried under vacuum. Synthesis of drug-unloaded MSN-ss-DTPP&DTCPP NPs 50 mg of MSN-ss-pyridyl was dispersed in 20mL of DI water, and negatively charged peptides (DTPP and DTCPP) were added to the above solution to finish pore-sealing of MSN-ss-pyridyl NPs via disulfide bonds. The MSN-ss-DTPP&DTCPP NPs were obtained after centrifugation (11000 rpm/min, 15 min), wash with PBS buffer (pH 7.4, 10 mM) and ethanol, and finally vacuum drying. Synthesis of drug-loaded DOX@MSN-ss-DTPP&DTCPP NPs MSN-ss-pyridyl NPs (100 mg) were dispersed in 10 mL PBS buffer (pH ~ 11.0, 10 mM). DOX (10 mg) was added under stirring, and the mixture was reacted for 24 h in the dark at 25 oC. Subsequently, DTPP and DTCPP peptides were added into the suspension above to finish the pore-sealing of drug8


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loaded DOX@MSN-ss-DTPP&DTCPP NPs via disulfide linkages. Finally, the DOX@MSN-ssDTPP&DTCPP NPs were obtained after centrifugation (11000 rpm/min, 15 min), wash with PBS buffer (pH ~ 11.0, 10 mM), and drying under vacuum. Drug loading content (DLC) and drug loading efficiency (DLE) determination A 0.1 M HF solution was used to destroy the mesoporous silica structure of DOX-loaded NPs (0.1 mg), and the pH value of the above mixture was changed to ∼7.4 with 0.1 M NaOH solution for further fluorescence detection.5 The DOX intensity was evaluated via fluorescence spectroscopy (excitation wavelength: 480 nm; emission wavelength: 555 nm). The DLC and DLE were defined and calculated as previous literature reported.38 pH-triggered zeta potential changes of MSN-ss-DTPP&DTCPP NPs The zeta potential changes of DOX-unloaded NPs (MSN-ss-DTPP&DTCPP and MSN-ssSTPP&STCPP) in response to physical or mildly acidic conditions were observed by dynamic light scattering (DLS) measurements. MSN-ss-DTPP&DTCPP NPs were dispersed in 10 mM PBS buffer (pHs 7.4 and 6.8) at the final concentration of 0.5 mg mL−1. Each sample was taken at a predetermined time and measured for zeta potential. All experiments were launched in triplicate, and the average data were obtained. Stability assay of MSN-ss-DTPP&DTCPP NPs 1mg MSN-ss-DTPP&DTCPP NPs was dispersed in DMEM culture medium (pH 7.4, 10 mM) in the presence of 10% FBS, and the final concentration of NPs was 0.1 mg/mL.41 At predetermined time points, the particle size change was assessed by Nano-ZS ZEN3600 (MALVERN Instrument) at 37 °C. All experiments were launched in triplicate, and the average data were obtained. Protein adsorption of DOX-unloaded NPs at different pHs MSN-ss-DTPP&DTCPP NPs (0.15 mg/mL) were dispersed in 2 mL BSA/PBS solution (0.25 mg mL−1/ 50 mM) at pHs 7.4 and 6.8.41 After then, each sample was centrifuged (12000 rpm/min, 10 min) to collect the supernatant at each predetermined time interval for UV-vis spectroscopy analysis. The protein concentration of supernatant was obtained by detecting its feature UV absorbance peak (280 9



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nm), and the amount of adsorbed proteins in the samples was calculated by the standard calibration obtained according to previous study.38 The protein adsorption study of MSN-ss-STPP&STCPP NPs was conducted in similar procedure as described above. In vitro drug release In vitro drug release was studied in different media including PBS (pHs 5.0 and 7.4) in the absence of 10mM GSH, and PBS (pHs 5.0 and 7.4) in the presence of 10 mM GSH. The drug-loaded DOX@MSNss-DTPP&DTCPP NPs (2.5 mg/mL) were suspended in four different media, and dialysis against 10 mL of PBS. Then, 3 mL of the sample solution outside the dialysis bag was withdrawn for fluorescence spectrophotometry observation and equal volume of fresh medium was added at selected time intervals. Cumulative DOX release from drug-loaded MSNs inside the dialysis bag was obtained in accordance with the emission intensity of DOX at 555 nm and to the previous literature.5 Cellular uptake of DOX-loaded NPs at different pHs The pH-activated cellular uptake of DOX-loaded NPs (DOX@MSN-ss-DTPP&DTCPP and DOX@MSN-ss-STPP&STCPP) was investigated by confocal laser scanning microscopy (CLSM). HeLa cells were cultured in a glass-bottom dish and seeded in fresh DMEM (pH 7.4) with 10% FBS, 1% streptomycin and 1% penicillin for 24 h at 37 oC. To explore the cellular uptake efficacy in tumor microenvironment, DOX@MSN-ss-DTPP&DTCPP NPs (DOX concentration: 5 µg/mL) were incubated in DMEM with the pH values from 7.4 to 6.8 for 1, 4 and 8 h. Moreover, the super-pH-insensitive DOX@MSN-ss-STPP&STCPP NPs were used as control. After incubation for specific time intervals, the cells were washed with cold PBS for 5 times, and their nuclei were stained with blue-fluorescent Hoechst 33342 solution (10 μg/mL) for 15 min. Finally, the cells were washed with cold PBS for several times, cultured in fresh DMEM (pH 7.4 or 6.8), and observed under CLSM. In order to investigate the specific recognition of surface-modified RGDS towards αvβ3-positive HeLa cells, spread HeLa cells were exposed to the addition of 100 µM soluble RGDS peptide. After 30 min of co-incubation, HeLa cells were treated with DOX-loaded NPs (DOX concentration: 5 μg/mL) at pH 6.8 for 4 h. The following treatment was conducted as described above.36 In vitro flow cytometry

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The quantification of cellular uptake was analyzed by using flow cytometry. First, HeLa cells were seeded in a six-well plate and cultured in 1 mL DMEM in the presence of 1% penicillin-streptomycin and 10% FBS for 24 h. Then, DOX@MSN-ss-DTPP&DTCPP NPs (DOX concentration: 5 μg/mL) were dissolved in 1 mL DMEM (pH 7.4 or 6.8) and treated with cells. After 4 h incubation, the cells were washed with PBS, digested by trypsin, and centrifuged (1000 rpm/min, 3 min). After removing the supernatant, each sample was analyzed using flow cytometry (BD FACS Aria III, USA). HeLa cells without any treatment were regarded as the blank control. For quantitative analysis of the specific recognition of surface-modified RGDS peptide toward αvβ3positive HeLa cells, HeLa cells were pre-incubated with 100 μM free RGDS peptide. After 30 min coincubation, DOX@MSN-ss-DTPP&DTCPP NPs (DOX concentration: 5 μg/mL) were dissolved in 1mL DMEM (pH 6.8) and seeded with HeLa cells. After 4 h incubation, the subsequent procedure was taken out as described above, and HeLa cells without any treatment were used as the blank control. Study on mitochondrial membrane potential HeLa and COS 7 cells were cultured in discs and seeded in 1mL DMEM with 10% FBS and 1% antibiotics, respectively. After the treatment with DOX-unloaded NPs (10 mg/L) at different pH levels (pHs 7.4 and 6.8) for 24 h, JC-1 (2.5 μg/mL) was added to the cells and cultured for 30 min.25 Finally, the confocal laser scanning microscopy (CLSM, Nikon C1-si TE2000, BD Laser) was employed to analyze the mitochondrial membrane potential of HeLa cells. Cytotoxicity evaluated by MTT assay αvβ3-Positive HeLa cells were incubated in 96-well plates and maintained in 100 µL DMEM with 10% FBS and 1% antibiotics for 24 h. Then, drug-loaded MSNs were treated under different conditions (pHs 7.4 and 6.8) for 48 h. Next, cells were treated with 20 μL MTT (5 mg/mL) and incubated with for further 4 h. Finally, the supernatant was replaced with 150 μL DMSO and the microplate reader was employed to determine the optical density (OD). The mean OD value of eight replicates was evaluated, and cell viability was obtained in a similar calculation to that in the previous work.25 Statistical analysis

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The statistical analysis was performed by a three-sample Student’s t-test unless otherwise noted,, and the acquired quantitative data were reported as mean ± standard deviation (S.D.). A p value < 0.05 is regarded of significance.

RESULTS AND DISCUSSION Synthesis and characterization MSNs and multifunctional peptides (TPP and TCPP) were constructed according to previous reports with modifications.25 After loading DOX, DMA-conjugated peptides (DTPP and DTCPP) were decorated with DOX@MSN-ss-pyridyl NPs via disulfide linkages to fabricate drug-loaded DOX@MSN-ss-DTPP&DTCPP (Scheme S1). The morphology and size of blank MSNs were examined by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and DLS. From SEM image in Figure 1A, bare MSNs showed spherical shapes with an approximately 118 nm. DLS observation demonstrated excellent colloidal dispersity of MSNs with a polydispersity index (PDI) of 0.15, and the average hydrodynamic diameter was around 140 nm (Figure 1A). Besides, zeta potential of bare MSNs was about -19.7 mV. After surface modification and drug loading, no obvious size increase of DOX@MSN-ss-DTPP&DTCPP NPs was observed via TEM measurement (Figure 1B). The successful preparation of DTCPP and DTPP peptides was indirectly confirmed by monitoring their hydrolysis behaviour at pH 6.8 by HPLC according to the previous report.30,36 As revealed from Fig. S5, TCPP and TPP peptides were regenerated without DMA modification after the incubation of DTCPP and DTPP peptides for 15 min. We postulated that the rapid hydrolysis of β-carboxyl imide linker in acidic environment produces the primary amines, consequently changing the charge of super-pHsensitive MSN-ss-DTPP&DTCPP NPs. Then, we measured zeta potential of two NPs (MSN-ssDTPP&DTCPP and MSN-ss-STPP&STCPP) at pHs 7.4 and 6.8, respectively. As revealed in Figure 1C, the zeta potential of super-pH-sensitive MSN-ss-DTPP&DTCPP NPs (approximately -15.5 mV) was stable at pH 7.4 after 300 min, but quickly increased to a high positive value of 16.3 mV at pH 6.8. This result indicated the excellent charge stability in physiological pH environment and charge-convertible ability in mildly acidic tumor environment. However, super-pH-insensitive MSN-ss-STPP&STCPP NPs still retained the negative charge (approximately -16.5mV) at different pHs within 300 min (Figure 1D).

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Figure 1. (A) Size distribution and SEM image of blank MSNs in 10 mM PBS (pH 7.4); (B) TEM image of DOXloaded NPs; Zeta potential of MSN-ss-DTPP&DTCPP NPs (C) and MSN-ss-STPP&STCPP NPs (D) at pH 7.4 or 6.8 in 10 mM PBS as a function of incubation time. The concentration of NPs was 0.5 mg/mL for each sample. Data are shown as mean ± SD (n=3).

The sequential fabrication and functionalization of DOX@MSN-ss-DTPP&DTCPP NPs were also confirmed by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) measurements and thermogravimetric analysis (TGA). As presented in Table S1 and Figure S3, BET surface area and BJH pore size decreased gradually during modification and drug loading. Furthermore, successful functionalization of the MSN surface was confirmed by TGA. The mass loss values of blank MSNs, MSN-SH, MSN-ss-DTPP&DTCPP and drug-loaded DOX@MSN-ssDTPP&DTCPP NPs were 10.8%、21.1%、37.7% and 43.3%, respectively, when the temperature up to 800°C (Figure S4). Moreover, the weight percentages of peptide plug and DOX were calculated as 16.6% and 5.6% according to TGA curves, and the mass of drug loading agree with the fluorescence detection result (5.8%). Besides, the DLE of DOX@MSN-ss-DTPP&DTCPP NPs were calculated as 37.5%. Notably, the designed MSN-ss-DTPP&DTCPP NPs displayed a notable inhibition property against FBS adsorption under simulated physiological environment that neglectable particle size change of MSN-ss-DTPP&DTCPP NPs happened after 48 h (Figure 2A), owing to the repulsive interaction between the negatively charged FBS and the negatively charged MSN-ss-DTPP&DTCPP NPs at pH 7.4. This finding indicated the good stability of MSN-ss-DTPP&DTCPP NPs in physiological pH

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environment. For further evaluating the protein adsorption ability of super-pH-sensitive MSN-ssDTPP&DTCPP NPs at different pH levels (pH 7.4 and 6.8), BSA was utilized as the model protein for measurement. As shown in Figure 2B, MSN-ss-DTPP&DTCPP NPs absorbed merely less than 10% BSA at pH 7.4. However, the BSA absorption amount reached to around 74% when pH value dropped to 6.8. By comparison, MSN-ss-STPP&STCPP NPs absorbed less than 5% BSA over 2 h after incubation at either pH 7.4 or 6.8. These BSA adsorption results further demonstrated the charge reversal ability of MSN-ss-DTPP&DTCPP NPs under slightly acidic condition, and revealed an additional superiority in bloodstream stability.

Figure 2. (A) Particle size of charge-reversal MSN-ss-DTPP&DTCPP nanoparticles after incubation under physiological conditions at 37oC for 48 h; (B) BSA adsorption of negatively charged MSN-ss-DTPP&DTCPP and MSN-ss-STPP&STCPP NPs at pH 7.4 or 6.8 after incubation at 37oC for 0.5, 1 and 2 h. Data are shown as mean ± SD (n=3).

In vitro redox and pH induced drug release profiles The in vitro release behavior of DOX from DOX@MSN-ss-DTPP&DTCPP NPs in response to physiological, tumor endosomal/lysosomal and/or cytoplasmic conditions was studied to confirm the dual-stimuli-responsibility of NPs. As shown in Fig. 3, the pH declines slightly increased the DOX release from DOX@MSN-ss-DTPP&DTCPP NPs. After 12 h incubation, the cumulative drug releases in different PBS solutions (pHs 7.4 and 5.0) was around 10% and 40%, respectively. An increasing drug release was observed at pH 5.0, which was caused by the enhanced electrostatic repulsion between the positively charged nanovalves decorated on MSNs due to the ionization of tertiary amines in DTCPP 14


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and DTPP peptides, thus resulting in a drug diffusion from pores of MSNs. Furthermore, 10 mM GSH can cleave disulfide bonds and thus open the pores of MSNs for an accelerated drug release. The DOX release amount from DOX@MSN-ss-DTPP&DTCPP NPs at pH 7.4 with 10 mM GSH was 40% in 1 h. Furthermore, DOX was released rapidly (over 70%) at pH 5.0 with 10 mM GSH within 1 h, indicating that the DOX@MSN-ss-DTPP&DTCPP NPs can accelerate the DOX release in simulated tumor intracellular environment as compared with that in physical environment. These results proved the onoff gatekeeping property of DOX@MSN-ss-DTPP&DTCPP NPs, which can display the simultaneous delivery of DOX and therapeutic TPP peptide in response to the specific stimuli.

Figure 3. In vitro quantitative DOX release profiles of DOX@MSN-ss-DTPP&DTCPP NPs under different conditions. Data are shown as mean ± SD (n=3)

Cellular uptake research of DOX-loaded NPs To assess the impact of pH value on the cellular uptake and drug release of DOX-loaded NPs, the fluorescence of DOX in super-pH-sensitive DOX@MSN-ss-DTPP&DTCPP NPs was detected, and the images were shown in Figure 4. From the bright field images, the normal morphology of HeLa cells could be observed, which was regarded as an important proof in determining the cellular state.42 The fluorescence field images (blue and red) revealed the normal morphology of cellular nuclear and the intracellular uptake of DOX, respectively. Moreover, the biocompatibility of drug-loaded NPs towards cells and intracellular uptake of DOX were further demonstrated by the overlapped images of bright 15



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field image and fluorescence field images. As depicted in Figures 4A-C, a faint red fluorescence of DOX was observed after treating DOX@MSN-ss-DTPP&DTCPP NPs with αvβ3-positive HeLa cells at pH 7.4, demonstrating the inefficient uptake of negatively charged DOX@MSN-ss-DTPP&DTCPP NPs under neutral condition. However, a substantially enhanced red fluorescence appeared in a timedependent manner in HeLa cells after DOX@MSN-ss-DTPP&DTCPP NP treatment at pH 6.8 (Figures 4D, 4E and 4G). This result indicated that the hydrolysis of the negative DMA group under tumor extracellular environment (pH 6.8) facilitates internalization of DOX@MSN-ss-DTPP&DTCPP NPs into tumor cells, and the over-expressed GSH in cytoplasm can accelerate drug release because of the breakage of disulfide linkages. However, DOX@MSN-ss-DTPP&DTCPP NPs showed a weakened red fluorescence in HeLa cells (pH 6.8) pre-treated with additional free RGDS peptide (Figure 4F), which was mainly caused by the competition between free RGDS peptide and the surface-immobilized RGDS peptide of DOX@MSN-ss-DTPP&DTCPP NPs towards αvβ3-positive HeLa cells, and thereby leading to considerable cellular uptake inhibition. For comparison, the HeLa cell uptake of super-pH-insensitive DOX@MSN-ss-STPP&STCPP NPs under different conditions (pHs 7.4 and 6.8) was evaluated at predetermined incubation times (1, 4 and 8 h). It was amazing that exceedingly low cellular uptake of DOX@MSN-ss-STPP&STCPP NPs was observed under different conditions (pHs 7.4 and 6.8) after different incubation times (Figures S6A-F). This result was mainly attributed to the weakend membrane affinity by negatively charged STPP and STCPP peptide in DOX@MSN-ss-STPP&STCPP NPs. All of these results demonstrated the enhanced tumor cell penetrating ability of super-pH-sensitive DOX@MSN-ss-DTPP&DTCPP NPs.

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Figure 4. CLSM images of HeLa cells incubated with DOX@MSN-ss-DTPP&DTCPP NPs at pH 7.4 (A-C) and 6.8 (D-G) in the absence of free RGDS peptide for 1 h (A and D), 4 h (B and E) and 8 h (C and G), and in the presence of free RGDS peptide at pH 6.8 for 4 h (F). (A1, B1, C1, D1, E1, F1, G1) Blue fluorescence images of Hoechst 33342. (A2, B2, C2, D2, E2, F2, G2) red fluorescence images of DOX; (A3, B3, C3, D3, E3, F3, G3) overlap of confocal fluorescence images. (A4, B4, C4, D4, E4, F4, G4) overlap of confocal fluorescence image and bright field images. Scale bar = 20 μm.

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Encouraged by the effective accumulation of super-pH-sensitive DOX@MSN-ss-DTPP&DTCPP NPs in tumor cells, we employed flow cytometer for qualitative analysis of the cellular uptake of DOXloaded NPs towards HeLa cells under different conditions (Figure S7). At pH 6.8, the mean fluorescence intensity (MFI) of DOX in DOX@MSN-ss-DTPP&DTCPP NPs was 4138 (Figures S7A and 7B). However, the intracellular MFI of DOX in DOX@MSN-ss-STPP&STCPP NPs under the same condition (pH 6.8) was only 1139 (Figures S7C and 7D). Furthermore, the intracellular MFI of DOX in DOX@MSN-ss-DTPP&DTCPP (1587) and DOX@MSN-ss-STPP&STCPP NPs (1173) at pH 7.4 was virtually the same. The flow cytometry results acquired above was in accordance with CLSM observation toward HeLa cells (Figure S6).

Investigation of targeted mitochondria damage by JC-1 assay In general, the therapeutic TPP peptide can target mitochondria and disrupt the mitochondrial membrane, resulting in a drop in mitochondrial trans-membrane potential (Ym) and subsequent cellular apoptosis.25 We employed JC-1 assay to evaluate the therapeutic efficacy of TPP peptide towards mitochondria. As depicted in Figures 5B and 5C, an increasingly strong green fluorescence intensity of MSN-ssDTPP&DTCPP NPs in cytoplasm was detected at pH 6.8 as compared with that at pH 7.4, indicating the severe mitochondrial injury. However, the green fluorescence intensity was obviously weakened after incubating HeLa cells with MSN-ss-DTPP&DTCPP NPs at pH 6.8 in the presence of free RGDS peptide (Figure 5D). This discovery demonstrated that the addition of free RGDS peptide can inhibit the binding affinity by RGDS peptide of MSN-ss-DTPP&DTCPP NPs with αvβ3-positive HeLa cells and therefore markedly decrease the mitochondrial damage. As control, MSN-ss-STPP&STCPP NPs did not result in the significant damage of mitochondrial function of HeLa cells at pHs 7.4 and 6.8 (Figures S8B and 8C), further verifying that MSN-ss-DTPP&DTCPP NPs should undergo complete reduction upon Ym in intracellular environment (pH 6.8) and subsequent mitochondria damage.

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Figure 5. CLSM images with JC-1 assay of HeLa cells incubated with free culture medium (A) and MSN-ssDTPP&DTCPP NPs at pH 7.4 (B); CLSM images with JC-1 assay of HeLa cells incubated with MSN-ssDTPP&DTCPP NPs in the absence (C)/presence (D) of free RGDS peptide at pH 6.8 for 4 h. Scale bar = 20 μm.

In vitro cytotoxicity research of DOX-loaded NPs In vitro antitumor activities of DOX@MSN-ss-DTPP&DTCPP NPs under different conditions (pHs 7.4 and 6.8) were evaluated by MTT assays on αvβ3-positive HeLa cells (Figure 6). Compared with the cytotoxicity against HeLa cells at pH 7.4, DOX@MSN-ss-DTPP&DTCPP NPs possessed an enhanced dose-dependent cytotoxicity at pH 6.8. Table S2 exhibited the half-inhibitory concentration (IC50) of DOX-loaded NPs at different pH levels. The IC50 value of DOX@MSN-ss-DTPP&DTCPP NPs at pH 6.8 (1.4 µg/mL) was around three-fold lower than that at pH 7.4 (~ 3.7 µg/mL). Meanwhile, IC50 value of DOX@MSN-ss-STPP&STCPP NPs at pH 6.8 was ~ 4.3 µg/mL, which was comparable with that at

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pH 7.4 (∼ 4.5 μg/mL). These results confirming that the released DOX and TPP peptide from chargereversal DOX@MSN-ss-DTPP&DTCPP NPs could simultaneously target to the nucleus and mitochondria of αvβ3-positive HeLa cells, displaying the dual-stimuli-sensitive tumor cell apoptosis.

Figure 6. In vitro cytotoxicity of DOX@MSN-ss-DTPP&DTCPP NPs (A) and DOX@MSN-ss-STPP&STCPP NPs (B) incubated with HeLa cells at different conditions.

CONCLUSION In summary, a plain strategy has been developed for efficient multistage combinational drug delivery. In normal physiological environment (pH ~7.4), the DOX@MSN-TPP&TCPP NPs showed a negatively charged surface, ensuring low adsorption of protein in blood. However, the super-pH-sensitive DMA layer was broken off under tumor extracellular microenvironment (pHe ~6.8), and the formed positively charged DOX@MSN-TPP&TCPP NPs displayed the enhanced tumor cellular internalization and subcellular mitochondria orientation. After internalization into the cytoplasm, the over-expressed GSH could trigger the cleavage of disulfide linkages, and subsequent release of the anti-tumor drug (DOX) and therapeutic peptide (TPP), generating a considerable apoptotic cell death of HeLa tumor cells. This strategy would be promising for synergetic treatment in cancer research.

■ ASSOCIATED CONTENT

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

AUTHOR INFORMATION *E-mail: [email protected] ORCID Yin-Jia Cheng: 0000-0001-7012-9174 Si-Yong Qin: 0000-0001-5034-3309 Xian-Zheng Zhang: 0000-0001-6242-6005 Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (Nos. 51703251, 51503227, 51502352 and 51703249) and the Fundamental Research Funds for the Central Universities (No. CZY17003). REFERENCES [1] Zhao, C. Y.; Cheng,R.; Yang, Z.; Tian, Z. M. Nanotechnology for cancer therapy based on chemotherapy. Molecules 2018, 23 (4), 826. DOI: https://doi.org/10.3390/molecules23040826. [2] Brigger, I.; Dubernet, C.; Couvreur, P. Nanoparticles in cancer therapy and diagnosis. Adv. Drug Delivery Rev. 2012, 64, 24-36. DOI: https://doi.org/10.1016/j.addr.2012.09.006. [3] Kalimuthu, K.; Lubin, B. C.; Bazylevich, A.; Gellerman, G.; Shpilberg, O.; Luboshits, G.; Firer, M. A. J. Gold nanoparticles stabilize peptide-drug-conjugates for sustained targeted drug delivery to cancer cells. Nanobiotechnol. 2018, 16 (1), 34. DOI: https://doi.org/10.1186/s12951-018-0362-1. 21



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For Table of Contents Use Only Super-pH-sensitive mesoporous silica nanoparticles-based drug delivery system for effective combination cancer therapy

Yin-Jia Cheng,*,† Si-Yong Qin,† Yi-Han Ma,† Xiao-Sui Chen,† Ai-Qing Zhang,† Xian-Zheng Zhang‡

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Super-pH-sensitive mesoporous silica nanoparticles-based drug

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