Tumor Acidic Microenvironment Targeted Drug Delivery Based on

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Tumor Acidic Microenvironment Targeted Drug Delivery Based on pHLIP Modified Mesoporous Organosilica Nanoparticles Yunlei Zhang, Meng Dang, Ying Tian, Yefei Zhu, Wenfei Liu, Wei Tian, Yunyan Su, Qianqian Ni, Chaoli Xu, Nan Lu, Jun Tao, Yanjun Li, Shuang Zhao, Ying Zhao, Zhenlu Yang, Li Sun, Zhaogang Teng, and Guangming Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10840 • Publication Date (Web): 15 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Tumor Acidic Microenvironment Targeted Drug Delivery Based on pHLIP Modified Mesoporous Organosilica Nanoparticles

Yunlei Zhang,† Meng Dang,┬ Ying Tian,† Yefei Zhu,§ Wenfei Liu,† Wei Tian,† Yunyan Su,† Qianqian Ni,† Chaoli Xu,⊥ Nan Lu,† Jun Tao,┬ Yanjun Li,† Shuang Zhao,† Ying Zhao,† Zhenlu Yang,† Li Sun,§ Zhaogang Teng,*,† Guangming Lu*,†,‡

†

Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing, 210002 Jiangsu,

P.R. China ‡

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering, Nanjing University, Nanjing, 210093 Jiangsu, P.R. China ⊥

Department of Ultrasound Diagnostics, Jinling Hospital, Nanjing University School of Medicine,

Nanjing, 210002 Jiangsu, P.R. China §

Center of Laboratory Medicine, The Second Affiliated Hospital of Nanjing Medical University,

Nanjing, 210011 Jiangsu, P.R. China ┬

Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced

Materials (IAM), Jiangsu National Synergetic Innovation Centre for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, P.R. China

*Corresponding Author. Fax: +86 25 8480 4659. Tel: +86 25 8086 0185. E-mail: (Z.T.) [email protected]; (G.L.) [email protected]

ABSTRACT: Enhancing the tumor-targeting delivery of chemotherapeutic drugs is important yet challenging for improving therapeutic efficacy and reducing the side effects. Here, we firstly

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construct a drug delivery system for targeting tumor acidic microenvironment by modification of pH (low) insertion peptide (pHLIP) on mesoporous organosilica nanoparticles (MONs). The MONs has thioether-bridged framework, uniform diameter (60 nm), good biocompatibility and high doxorubicin (DOX) loading capacity (334 mg/g). The DOX loaded in the pHLIP modified MONs can be released responsive to glutathione and low pH circumstance, ensuring the chemotherapeutic drug exerts higher cytotoxic effects to cancer cells than normal cells because of high intracellular GSH of tumor cells and low pH of tumor microenvironment. Moreover, the engineered MONs exhibit higher cellular uptake in pH 6.5 medium by MDA-MB-231 and MCF-7 cells than the particles decorated with polyethylene glycol (PEG). Importantly, the pHLIP-mosaic MONs with DOX displays better cytotoxic effects against the breast cancer cells in pH 6.5 medium than pH 7.4 medium. The in vivo experiments demonstrate that the pHLIP modified MONs are accumulated in the orthotopic breast cancer via targeting to acidic tumor microenvironment while no serious pathogenic effects was observed. After loading DOX, the pHLIP modified MONs display better therapeutic effects than the control groups on the growth of MCF-7 breast cancers, showing promise for enhancing chemotherapy. KEYWORDS: Tumor acidic microenvironment, mesoporous organosilica nanoparticles, tumor targeting, drug delivery, breast cancer

1. INTRODUCTION Chemotherapy, as one of the typical methods for cancer treatment, remains the optimal choice for most cancer patients.1-2 However, chemotherapeutic drugs not only kill tumor cells but also damage normal organs.3 Therefore, chemotherapy is calling for the efficient tumor-targeting delivery systems to increase drug accumulation in tumors and lower their cytotoxicity to normal tissues. Nanotechnology actually provides an ideal pathway to deliver chemotherapeutic drugs to tumor and ACS Paragon Plus Environment


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enhance their anticancer activity.4-7 The tumor-targeting ability of nanoparticles is mainly based on the enhanced permeation and retention (EPR) effect or the specific ligands of tumor-associated antigens.8-12 Compared with free molecular tracers/therapeutic agents, the EPR effect can conspicuously increase the drug concentrations in solid cancers and enhance anticancer effects.13 Although the EPR effect efficiently increase the anticancer effects of chemotherapeutic drugs, achieving high specificity to differentiate tumors from healthy tissues remains a great challenge.14-15 Through using the ligands of cancer-associated antigens, such as Her2 affibody,16 EGFR nanobody/peptides,17 arginine–glycine–aspartic acid (RGD),18 folate19 and so on, the cancer specificity of nanoparticles can be efficiently elevated. However, the tumor-associated biomarkers are generally confined in only a small number of cancers and a subset patient (for example, 25-30% of patients with breast cancers over-express Her2 protein).20 Besides, ligand guided drug delivery system may induce side effects because biomarkers also exist in normal cells.21 Therefore, developing drug delivery system with the ability to target most cancers and low cytotoxicity becomes urgent. Different to the tumor-associated markers, acidosis is a typical feature of solid tumors and has been used as the hallmark for tumor-targeting imaging and therapy.22-23 A pH (low) insertion peptide (pHLIP), originally derived from the bacteriorhodopsin C helix, is characterized with the tumor acidic microenvironment targeting ability.24-25 Taking advantage of cancer acidic milieu, the accumulation of antisense oligomers in tumor is successfully increased by linking pHLIP, resulting in the tumor regression.26 Also, gold nanoparticles, superparamagnetic iron oxide and liposomes have been modified with pHLIP to increase the cellular uptake and tumor accumulation.27-29 However, to the best of our knowledge, pHLIP modified MONs have not been reported to deliver drugs for in vivo chemotherapy. MONs characterized with organic group incorporated frameworks, well-defined mesopores, ACS Paragon Plus Environment

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large surface areas, biodegradability and hemocompatible properties have attracted increasing attention for biomedical application, especially in cancer therapy.30-34 A great many of organic functional groups can be introduced into the frameworks of the MONs, which permits tuning of the surface hydrophilicity/hydrophobicity, providing active sites, loading anticancer drugs and linking imaging agents.35 For example, the disulfide bonds incorporated MONs could break up in the reducing microenvironment of tumor tissues, which allows for the quick tumor-responsive drug releasing from the particles.36 Therefore, the MONs is an ideal carrier to deliver chemotherapeutic drugs for cancer therapy. The particle size is a crucial parameter influencing biological processes. It is reported that relatively small size or size-changeable nanoparticles can achieve long blood-circulation and enhanced EPR effect.37-38 For instance, 80 nm nanoparticles exhibit longer blood-circulation lifetime than those with the size of 120, 200, and 360 nm.31 In addition, small size is beneficial for the penetration of particles into the deep center of tumors, of which the poorly vascularized microenvironments and high interstitial fluid pressure restrain the entry of large particles.37, 39 However, the previous reports mainly use the MONs with the size of over 100 nm for drug delivery. Herein, this study firstly synthesized tumor acidic microenvironment targeted drug delivery system by modification of pHLIP on the MONs with a diameter of about 60 nm. Both in vitro and in vivo assays demonstrated the good biocompatibility of the pHLIP modified MONs. Doxorubicin (DOX) was efficiently loaded into the mesochannels of the pHLIP decorated MONs and could be released via GSH and pH dual-responsive manner. Moreover, through the conjugated near-infrared fluorescence (NIFR) dye Cyanine (Cy5.5) on the MONs, confocal laser scanning microscopy and flow cytometry demonstrated that the pHLIP-mosaic MONs with DOX exhibited higher cell uptake by both basal like subtype cells (MDA-MB-231) and luminal A subtype cells (MCF-7) in pH 6.5 medium than the 7.4 medium. This acidic microenvironment targeting ability of the pHLIP modified ACS Paragon Plus Environment


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MONs was further confirmed using IVIS spectrum system. Due to the acidic microenvironment targeting ability, the pHLIP modified MONs with doxorubicin (DOX) showed better cytotoxicity in the low pH medium than in neutral medium against the breast cancer cells. What’s more, the in vivo experiments demonstrated that the MONs with the pHLIP exhibited highly tumor-targeting ability and therapeutic effects for the orthotopic MCF-7 breast cancers.

2. MATERIALS AND METHODS 2.1. Materials. Anhydrous ethanol, concentrated ammonia aqueous solution and 37% (w/w) HCl were provided by Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Cetyltrimethylammonium bromide (CTAB), dioxane, triphenylphosphine and N,N-dimethylformamide (DMF) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bis(triethoxysilylpropyl) tetrasulfide (TESPTS), 4',6-diamidino-2-phenylindole (DAPI), (3-aminopropyl) triethoxysilane (APTES), GSH and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma−Aldrich (St. Louis, MO, USA). Maleimide derivative cyanine dyes (Cy5.5-maleimide) and maleimide derivative polyethylene glycol 2000 (PEG 2000-maleimide) were bought from Seebio Biotechnology Co., Ltd. (Shanghai, China). Doxorubicin (DOX) in the form of hydrochloride salt was obtained from Sangon Biotech (Shanghai, China). Heat-inactivated fetal bovine serum (FBS) and pierce bicinchoninic acid (BCA) protein assay kit were bought from Gibco/ThermoFisher scientific (Waltham, MA, USA). The pHLIP-PEG 2000-maleimide was obtained from Nanjing Leon Biological Technology Co., Ltd. (Nanjing, China). Dulbecco’s modified Eagle’s medium (DMEM), 0.05% trypsin−EDTA and penicillin−streptomycin solution were obtained from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China). The ultrapure water used in the experiments was totally obtained using Millipore water purifier. 2.2. Synthesis of Thioether-Bridged MONs. Thioether-bridged MONs was prepared using a ACS Paragon Plus Environment

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CTAB-directed sol–gel process by using TEOS and TESPTS as precursors. In detail, 0.4 g CTAB, 150 mL of ethanol, 650 mL of water and concentrated ammonia aqueous solution (5 mL, 25 wt %) were mixed together and stirred at 35 °C at the speed of 500 rpm for 1 h. And then, 0.95 mL TEOS and 0.05 mL TESPTS were added and stirred for 24 h. The product was collected by centrifugation at 8000 rcf for 30 min. To remove the CTAB surfactants, the as-prepared materials were resuspended in 400 mL ethanol and 200 µL concentrated HCl and stirred at 500 rpm in 60 °C water for 3 h. The process was repeated three times to completely remove CTAB. In the end, the MONs was washed with ethanol three times and dispersed in water for the further modification. 2.3. Modification of pHLIP, Cy5.5 and PEG on MONs. In order to link the functional molecules, the disulfide bonds in the MONs were transformed into thiol groups. Typically, 0.02 g of the thioether-bridged MONs was suspended in a mixture of dioxane (1.1 mL) and water (0.3 mL). Next, 0.1 g of triphenylphosphine was added to the solution and heated to 40 °C. And then, two drops of concentrated HCl were added and exposed to nitrogen for 2 h. The MONs with thiol groups were gathered by centrifugation and washed with ethanol for three times. The final products were dispersed in 200 µL of ethanol for the further modification. To link near-infrared dyes, the obtained MONs suspension (200 µL), 500 µL of aqueous solution of Cy5.5 (0.1 mg/mL), 700 µL of water and 120 µL of DMF were mixed at pH 7.4 and shaken at room temperature for 12 h. Through the thiol-maleimide coupling reaction, Cy5.5-maleimide was grafted on the MONs. Finally, the reaction solutions were centrifuged and the obtained MONs-Cy5.5 was thoroughly washed with water to remove the free Cy5.5. The MONs-Cy5.5 was further modified with the pHLIP-PEG 2000-maleimide (pHLIP sequence: ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG) and PEG 2000-maleimide through the above method. Briefly, 0.01 g of the MONs-Cy5.5 was incubated with 0.002 g of pHLIP-PEG 2000-maleimide or PEG 2000-maleimide in phosphate buffer solution (PBS) to conduct the thiol-maleimide coupling reaction. The pHLIP-PEG 2000-maleimide ACS Paragon Plus Environment


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and PEG 2000-maleimide decorated MONs-Cy5.5 were designated as MONs-Cy5.5-pHLIP and MONs-Cy5.5-PEG, respectively. The pHLIP peptide sequence was Pierce BCA protein assay kit was used to quantify the pHLIP in the supernatant and washed solutions. 2.4. Loading Chemotherapeutic Drug. MONs-Cy5.5-PEG (0.01 g) or MONs-Cy5.5-pHLIP (0.01 g) was mixed with 5 mL of 1 mg/mL DOX-dissolved PBS solutions (pH 7.4) and shaken shielding from light at room temperature for 24 h. The DOX loaded MONs-Cy5.5-PEG (designated as [email protected]) or MONs-Cy5.5-pHLIP (named as [email protected]) were collected by centrifugation at 8000 rcf for 30 min. The products were further washed by PBS until the supernatant became colorless and no absorbance was found using UV-vis spectrometer compared with PBS. The obtained products were dried using vacuum and stored at –20 °C for further use. The DOX-loading capacity of MONs-Cy5.5-PEG and MONs-Cy5.5-pHLIP was calculated through quantifying the DOX in the supernatant using a UV–vis spectrometer at a wavelength of 490 nm. 2.5. Hemolysis Assay. A 0.5 mL sample of whole blood was added to 1 mL of 0.9 % NaCl, and the red blood cells (RBCs) were isolated from serum by centrifugation at 400 rcf for 5 min. The RBCs were further washed thrice using 0.9 % NaCl and diluted in 5 mL of 0.9 % NaCl. Then 0.2 mL of the above diluted suspensions was added to 0.8 mL of the MONs in 0.9 % NaCl at a concentration of 0.03, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 and 2.4 mg/mL. The suspension was gently vortexed and incubated for 2 h in the chamber of 37 °C static environment. And then, the mixture was centrifuged at 400 rcf for 5 min. A 100 µL of the supernatant was carefully transferred to the wells of 96-well-plate. The hemoglobin released from RBCs was determined using a microplate reader at 490 nm (BioTek Instruments, Winooski, VT, USA). 2.6. In vitro and in vivo Toxicity Assay. To evaluate the toxicity of the MONs, the nanoparticles were incubated with the normal cell lines (MCF-10A and 293T) for 48 h at the concentrations of 0.0625, 0.125, 0.25, 0.5 and 1 mg/mL, and the inhibitory effects of them on the growth of cells were ACS Paragon Plus Environment

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determined by MTT assay. The in vivo toxicity of the MONs-Cy5.5-pHLIP was conducted using ICR mice. In brief, 400 µL of PBS-dissolved MONs-Cy5.5-pHLIP (3 mg/mL) were weekly injected into 4-6 weeks female ICR mice (n = 10) via the tail vein. The activity of the mice was monitored daily, and the weight of mice was recorded every two days. After 30 days, the mice were sacrificed through cervical dislocation, and the organs (liver, spleen, heart, kidney and lung) were fixed in 4% paraformaldehyde while the livers and spleens were additionally weighed. The fixed tissues were performed using standard hematoxylin and eosin (H&E) staining. The sections were photographed under the Olympus microscope (Olympus IX71, Tokyo, Japan) 2.7. Drug Release Assay. 2 mg of the [email protected] or [email protected] was suspended in 2 mL PBS (pH 5.0 or pH 7.4). The solutions were shaken at a speed of 500 rpm at 37 °C in a thermomixer (Hangzhou Aosheng, China). At certain time intervals, the solution was centrifugated and the absorbance value of DOX in the supernatant was recorded at 490 nm in a microplate reader (BioTek Instruments, Winooski, VT, USA). Besides, the drug release was also performed in 2 mL of PBS containing different values of GSH (0, 5 and 10 mM). The release percentage of DOX in the nanoparticles was calculated according to the absorbance values of the supernatant. 2.8. Targeted Uptake Evaluation by Confocal Laser Scanning Microscopy. MDA-MB-231 and MCF-7 cells were seeded on glass coverslips overnight at 37 °C under 5% CO2. Then, the solutions were replaced with pre-warmed pH 6.5 or pH 7.4 medium containing 50 µg/mL of [email protected] or [email protected], and incubated for 2 h. Next, the coverslips were washed with PBS for three times, fixed in 4% paraformaldehyde for 15 min, and washed again thrice using PBS. The coverslip was carefully moved to the slides and covered with one drop of mounting medium with DAPI Hoechst (0.5 µg/mL) in a dark place to counter stain nuclei. Images of cellular uptake and distribution were captured using an inverted confocal laser scanning microscope (LSM 710，Carl Zeiss, Germany). ACS Paragon Plus Environment


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2.9. Targeted Cell Uptake Evaluation Through Flow cytometry and IVIS Spectrum System. MDA-MB-231 and or MCF-7 Cells were seeded on a 100 mm petri dish and cultured overnight. And then, the medium was replaced with the fresh medium of different pH values (pH 6.5 and pH 7.4) containing 50 µg/mL of [email protected] or [email protected], and incubated for 1 h, 3 h, 6 h and 24 h. Next, the cells were collected and washed thrice with PBS. In the end, the cells were immediately analyzed using flow cytometry analysis (CytoFLEX Flow cytometer, Beckman Coulter Inc., Miami, FL, USA). For Cy5.5 fluorescence analysis, each sample contains at least 30,000 single cells, and the results were processed through the mathematical Watson Pragmatic model with the FlowJo Analysis Software (Tree Star, Inc., Ashland, OR, USA). Simultaneously, the cells were seeded in a 96-well-plate and treated as the above. Alternatively, the NIFR of the cells were recorded using IVIS spectrum system. 2.10. Cytotoxicity of [email protected] Against the Breast Cancer Cells. Cells of MDA-MB-231 and MCF-7 at exponential phase were gathered and seeded in a 96-well-plate. After overnight incubation, the medium was replaced with fresh medium (pH 6.5/pH 7.4) containing different concentrations of DOX, [email protected] and [email protected]. The concentration range of DOX or equal amount DOX contained in [email protected] /[email protected] was 1, 1.5, 2, 2.5 and 3 µg/mL. The concentrations of the nanoparticles were calculated based on the free DOX and its DOX loading capacity. After 6 h incubation with the above medium, the cells were washed using PBS for three times and further cultured in fresh DMEM medium (pH 7.4). In the end, the cell viability was evaluated using MTT reagent kit according to manufacturer guideline. The absorbance values were recorded at 490 nm through a spectrophotometer. The cytotoxicity of the addressed drugs against the breast cancer cells was determined. 2.11. In vivo Distribution and Therapy. MCF-7 tumors in the fourth mammary pads of female ACS Paragon Plus Environment

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BABL/c nude mice, 6 to 8 weeks old, were established with subcutaneous injection of 5 × 106 tumor cells. At a defined time, the mice with orthotopic tumor models (n = 3) were i.v. administrated with 200 µL PBS containing [email protected] or [email protected] (1 µg/µL), respectively. After 24 h, the mice were sacrificed by cervical dislocation. The tumors, livers, spleens, lungs, hearts, kidneys and muscles were imaged and quantified using IVIS spectrum system. The average values of fluorescence intensity in the organs were calculated through subtracting the values of muscles. Next, the MCF-7 bearing mice (n = 5) were i.v injected with PBS, DOX (150 µg per mouse), [email protected] (214 µg per mouse) and [email protected] (224 µg per mouse), respectively. The DOX amount contained in 214 µg of the [email protected] and 224 µg of the [email protected] was equal to 75 µg. The treatments were performed every three days for three times after the tumor volume getting to ⁓200 mm3. Tumor volumes were recorded every 3 days using Vernier calipers. When the tumor volume in the control group reached 1400 cm3, the mice were sacrificed. 2.12. Characterization. Transmission electron microscopy (TEM) images were captured using an HT7700 microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV. Zeta potential and hydrodynamic size were recorded by a Brookhaven analyzer (Brookhaven Instruments Co., Holtsville, NY, USA).

UV–vis spectra were recorded through a Lambda 35 UV–vis

spectrophotometer (PerkinElmer, Inc., Waltham, MA, USA). Fourier transform infrared (FT–IR) spectra were obtained on a Nicolet Nexus 870 spectrometer (Nicolet Instruments Inc. Madison, WI, USA). NIRF imaging was performed using IVIS Lumina XR system (Xenogen Corporation–Caliper, Alameda, CA, USA) under the Cy5.5 filter (λex = 673 nm, λem = 707 nm). Raman spectrum was acquired on a Thermo Scientific DXR with the excitation wavelength of 633 nm. Nitrogen sorption isotherms were measured by a Micromeritics Tristar 3000 analyzer (Micromeritics Instruments Corporation, Atlanta, GA, USA) at –196 °C. The Brunauer-Emmett-Teller (BET) and nonlocal density functional theory (NLDFT) methods were used to calculate the specific surface areas and the



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pore sizes, respectively. The adsorbed amount at p/p0 = 0.995 was used to estimate the total pore volume. Solid state

29

Si magic-angle spinning (MAS) NMR and

13

C cross-polarization/MAS

(CP/MAS) NMR spectra were recorded at 9.47 T on a Bruker AVANCE III400 spectrometer.

3. RESULTS AND DISCCUSION

Scheme 1. Illustration of the construction of the thioether-bridged MONs-based acidic microenvironment targeted drug delivery system for cancer therapy. Under neutral conditions, the aspartic acids and glutamine acids in the pHLIP are negatively charged. Once the pH drops, the residues of the two kinds of amino acids in the pHLIP are protonated, which sharply increase the hydrophobicity of the peptide and form a transmembrane helix, mediating the internalization of the pHLIP-modified MONs by tumor cells. The loaded DOX in the pHLIP-decorated MONs will be released intracellularly to kill tumor cells.

The Scheme 1 depicted the construction of the [email protected] and the mechanisms underlying the selectively cytotoxic effects of the acidic microenvironment targeted drug delivery system against cancer cells. Firstly, thioether-bridged MONs was synthesized via a CTAB-directed sol–gel process using TEOS and TESPTS as precursors. Then, the disulfide bonds in the MONs were reduced to thiol groups in order to the conjugation of Cy5.5-maleimide and pHLIP-PEG ACS Paragon Plus Environment

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2000-maleimide. Finally, chemotherapeutic drugs (DOX) were loaded into mesochannels of the pHLIP modified MONs to form an acidic microenvironment targeted drug delivery vehicle. The pHLIP decorated MONs could selectively enter acidic microenvironments because of the low–pH responsive pHLIP, and further specifically flight tumor cells through its functionalized Cy5.5 fluorescence and kill them by releasing chemotherapeutic drugs (DOX).

Figure 1. (a) TEM image of the thioether-bridged MONs via a CTAB-directed sol–gel process using TEOS and TESPTS as precursors. (b) Hydrodynamic diameter of the MONs, MONs-Cy5.5, MONs-Cy5.5-PEG,

[email protected],

MONs-Cy5.5-pHLIP

and

[email protected]. (c) FT-IR spectrum of the thioether-bridged MONs. (d) Solid-state 29Si MAS NMR and (e)

13

C CP-MAS NMR spectra of the MONs. (f) Nitrogen sorption isotherms and ACS Paragon Plus Environment


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pore size distribution curves of MONs. (g) Zeta potentials of MONs, MONs-Cy5.5, MONs-Cy5.5-PEG, [email protected].

MONs-Cy5.5-pHLIP, (h)

UV-vis

[email protected]

spectra

of

Cy5.5,

MONs,

and

MONs-Cy5.5,

MONs-Cy5.5-pHLIP and [email protected].

The TEM image and DLS measurements demonstrated that the thioether-bridged MONs is well-dispersed with a uniform diameter of 60 nm (Figure 1a and b). This is the expected size for drug delivery because the cellular internalization of nanoparticles is size-dependent in the order 50 nm > 30 nm > 110nm > 280 nm.27 Nanoparticles with the size can not only be efficiently internalized by cells but also have longer blood-circulation lifetime.27,31 In addition, the FT-IR spectrum showed that the MONs have C-H bands at 2930 cm–1, 1450 cm–1 and 1410 cm–1 and C-S band at 694 cm–1, indicating their thioether-bridged frameworks (Figure 1c).40 The

29

Si MAS NMR spectrum of the

MONs shows six signals at -93, -102, and -110 ppm, corresponding to Q2 (Si(OSi)2(OX)2, X=H or Et), Q3 (Si(OSi)2(OX)), and Q4 (Si(OSi)4) species, and at -47, -58, and -66 ppm, corresponding to T1 (C-Si(OSi)1(OX)2, X=H or Et), T2 (C-Si(OSi)2(OX)), and T3 (C-Si(OSi)3 species (Figure 1d).36 Simultaneously, the 13C CP-MAS NMR spectra of the MONs exhibit the characteristic peaks at 10, 21 and 40 ppm, which can be assigned to the

1

C,

2

C and

3

C carbon species in the

-Si-1CH22CH23CH2-S-S-S-S-3CH22CH21CH2-Si- moiety (Figure 1e).36 The 29Si and 13C NMR results clearly demonstrated the organic-inorganic hybrid frameworks of the MONs. Through the formation of thiomaleimide, Cy5.5-maleimide was conjugated onto the MONs (named as MONs-Cy5.5). The zeta potential of the MONs-Cy5.5 turned to –13.96 mV from –8.46 mV of the MONs because of the negatively charged Cy5.5. The conjugation of the Cy5.5 on the MONs was very stable and no detached Cy5.5 was found in the supernatant, even after 72 h in PBS at room temperature (Figure S1a). The well-modified Cy5.5 on the MONs was further confirmed by the UV-vis absorbance (Figure 1h). The PEG 2000-maleimide and pHLIP-PEG 2000-maleimide were further linked on the MONs-Cy5.5 through the same method and called MONs-Cy5.5-PEG and MONs-Cy5.5-pHLIP, respectively. The zeta potential turned to –12.11 mV (MONs-Cy5.5-PEG) and –11.19 mV ACS Paragon Plus Environment

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(MONs-Cy5.5-pHLIP) from –13.96 mV (MONs-Cy5.5). The protein assay kit demonstrated that there were 0.32 µM pHLIP on per gram of the MONs. Moreover, FT-IR spectrum of the [email protected] displayed a band of amide I at around 1655 cm-1 and two characteristic peaks of PEG2000 at 1459 cm-1 and 1353 cm-1, further confirming the appearance of pHLIP peptides in the nanoparticles (Figure S1).41 The introduction of PEG-2000 between MONs and pHLIP in the MONs-Cy5.5-pHLIP will increase the biocompatibility of the nanoparticles and the flexibility of the pHLIP to attach the targeted object.42 Additionally, nitrogen sorption isotherms of the MONs depicted the uniform pore size of the MONs (3.2 nm) with a specific surface area of up to 870 m2/g (Figure 1f). The introduction of Cy5.5 and pHLIP-PEG 2000 decreases the surface area of the MONs to 513 m2/g (Figure S2a). Because of the high surface area and uniform pores of the thioether-bridged MONs, positively charged DOX was efficiently loaded into the mesochannels of the negatively charged nanoparticles (MONs-Cy5.5-PEG and MONs-Cy5.5-pHLIP) through electrostatic

interactions.

The

DOX-loading

capacity

of

the

MONs-Cy5.5-PEG

and

MONs-Cy5.5-pHLIP nanoparticles was measured to be 349 mg and 334 mg per gram, respectively. The nitrogen sorption isotherms of the [email protected] and [email protected] also revealed that the pores of the nanoparticles were occupied (Figure S2). Simultaneously, the zeta potential of [email protected] turned to 22 mV while [email protected] changed to 12.21 mV, suggesting the presence of DOX on the nanoparticles. Besides, the colorful particles and characteristic UV–vis peak at 490 nm further demonstrated that the DOX were efficiently and successfully loaded into the MONs-Cy5.5-pHLIP (Figure 1h). Furthermore, the absorption band at around 1700 cm–1 indicative of C=O carbonyl stretching provided another evidence for the above result (Figure S1c and d).43 MONs-Cy5.5-PEG,

DLS measurements found that the size of the MONs, MONs-Cy5.5, [email protected],

MONs-Cy5.5-pHLIP

and

[email protected] nanoparticles reach 58.5 nm, 61.5 nm, 63.8 nm, 65.1 nm, 75.5 nm and 71.5 nm, respectively (Figure 1b).



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Figure 2. (a) Hemolysis assay of the MONs. (b) Cytotoxicity of the MONs at different concentrations against MCF-10A and 293T cells. (c) Changes of body weights after weekly i.v administration of PBS and MONs-Cy5.5-pHLIP nanoparticles. (d) Comparison of liver and spleen weights between PBS group and MONs-Cy5.5-pHLIP treated group. (e) H&E staining of tissues from ICR mice after weekly i.v administration of MONs-Cy5.5-pHLIP

Hemolysis assay was firstly carried out to find whether the MONs could damage RBCs, the ACS Paragon Plus Environment

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membranes of which are typically representative of mammalian cells.44-45 The results demonstrated that hemoglobin slightly increased with the rise of MONs concentrations but did not exceed 3.07%, even at the maximum concentrations of 2.4 mg/mL (Figure 2a), indicating the thioether-bridged nanoparticles exhibited good biocompatibility to RBCs at the tested concentrations. Simultaneously, the MCF-10A normal breast cancer cell line and human embryonic kidney cells (293T) were employed to further assess the cytotoxicity of the MONs. After 48 h incubation, cell viability of the MCF-10A and 293T cells always stayed above 85%, although the concentrations of the MONs reached 1 mg/mL (Figure 2b). The relatively low cytotoxicity provided the basic foundations for the following functional assays. Further, the Figure 2c and 2d revealed that the weekly treated ICR mice by the MONs-Cy5.5-pHLIP had no effects on body, liver and spleen weights. Also, the typically anaphylaxis symptoms, such as scratching nose and coughing,46 was totally absent from the MONs-Cy5.5-pHLIP treated mice as judged by comparison with the PBS group. No death occurred during the experiments. Additionally, the liver, spleen, lung, heart and kidney had no distinct difference between the MONs-Cy5.5-pHLIP group and PBS group via H&E staining assay (Figure 2e). Therefore, the results demonstrated that weekly systemic administration of MONs-Cy5.5-pHLIP did not have any noticeable pathogenic effect on the ICR mice. Except the good biocompatibility, drug release profile is another key character for drug delivery system. Indeed, the DOX release rate and percentage of the [email protected] nanoparticles exhibited a GSH-dependent pathway in vitro assay (Figure 3a). In the absence of GSH, the DOX release in [email protected] nanoparticles proceeded in a very slow rate and only 29.12% percentage of DOX detached from the nanoparticles after 24 h incubation (Figure 3a). Contrarily, when the equal amount of the MONs-Cy5.5-pHLIP was added into 5 µM and 10 µM of GSH solutions, the DOX release rates sharply increased at 0 ~ 2 h and became slow in the following process (Figure 3a). This is due to that there were still residual disulfide bonds on the ACS Paragon Plus Environment


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[email protected] after the triphenylphosphine mediated reduction reaction. The Raman spectrum showed the characteristic stretching vibrations of disulfide bonds at the Raman shifts of 438 and 488 cm-1 (Figure S2d). The disulfide bonds on the MONs are physiologically responsive and can be efficiently reduced by GSH.47 Finally, the release percentage of DOX from the [email protected] in 5 µM and 10 µM of GSH solutions reached 52.34% and 63.67%, respectively, but the GSH-free group only got to 29.12% (Figure 3a). The intracellular GSH concentration is generally in the millimolar range, which is much higher than extracellular levels (20 – 40 µM) in common fluids 48 while cancer cells show much higher GSH concentration than normal cells.49 This indicated that the DOX release of the [email protected] in a GSH-dependent manner will maximum their cytotoxicity to cancer cells and lower their toxic effects to normal cells. Moreover, the in vitro test certified that the release percentage of DOX from the [email protected] in pH 5.0 medium (58.8 %) is much higher than in pH 7.4 medium (33.3 %), indicating their pH-responsive characterization of the engineered MONs (Figure 3a). This phenomenon could be explained by the quick increase of hydrogen ions in the low-pH solutions. The hydrogen ions can absorb on the MONs, which results in the mutual repulsion between the particles and DOX and the acceleration of drug release. Taking into account of the acidic milieu of tumors and lysosome, the DOX of the [email protected] could be quickly released in tumor microenvironment and exert their anticancer effects due to the pH responsive character. Considering tumor

cells

have

higher

intracellular

GSH

concentrations

and

low-pH

values,

the

[email protected] are expected to selectively release DOX in tumor acidic microenvironment and tumor cells, avoiding the considerable leakage of the drugs to normal organs and lowering their side effects. To evaluate whether pHLIP can exhibit its acidic microenvironment targeting ability and increase the internalization of the [email protected] by tumor cells at low-pH medium, the ACS Paragon Plus Environment

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[email protected] were respectively incubated with MDA-MB-231 and MCF-7 cells in medium with different pH values (pH 6.5 and pH 7.4). The cellular uptake and intracellular distribution of the [email protected] were traced and recorded by confocal laser scanning microscopy. Figure 3b clearly showed that both MDA-MB-231 and MCF-7 cells in the medium with different pH values display green (DOX) and red (Cy 5.5) fluorescence after 2 h incubation. The NIFR (Red) mainly locates in the cytoplasm of the cells while DOX almost fully enter nucleus. This result was completely consistent with the former assay that GSH and low-pH induce the quick releasing of DOX from [email protected] within 2 h. Although both cell lines in the medium of different pH values could take up the nanoparticles, there were obviously significant differences of fluorescence intensity between pH 7.4 and pH 6.5 groups (Figure 3b). Considering the fluorescence intensity is proportional to the absorption of the nanoparticles, there were much more [email protected] taken up by the cells at pH 6.5 than pH 7.4 (Figure 3b). However, this phenomenon was not found in the [email protected] treated group (Figure S2). The result revealed that it was pHLIP that contributed to the acidic microenvironment targeting ability of the MONs. It also demonstrated that pHLIP has potential to be an acidic microenvironment “guider”, enhancing chemotherapeutic effect of nanoparticle-based delivery system.



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Figure 3. (a) Dox release profiles from the [email protected] at different concentrations of GSH and different pH values. (b) Confocal laser scanning microscopy of MDA-MB-231 and MCF-7 cells incubation with 50 µg/mL [email protected] or [email protected] in the medium with different pH values for 2 h. 7.4 and 6.5 stands for pH 7.4 and pH 6.5, respectively. The black bar represents 25 µm. ACS Paragon Plus Environment

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The acidic microenvironment targeting ability of the pHLIP-conjugated MONs for MDA-MB-231 and MCF-7 cells were further evaluated via flow cytometry and IVIS spectrum system. After 2 h and 6 h incubation, the NIFR fluorescence intensity of the cells at 6 h was conspicuous higher than 2 h regardless of pH values through the analysis of flow cytometry (Figure 4a). However, the [email protected] treated group showed stronger fluorescence intensity at pH 6.5 than pH 7.4 in both MDA-MB-231 and MCF-7 cells at defined time points (2 h and 6 h) (Figure 4a). The acidic microenvironment targeting ability of the [email protected] increased with the extension of time. In comparison to the acidic microenvironment targeting ability of the [email protected], this effect was not observed in the [email protected] treated groups (Figure 4a). However, the low-pH driven tumor-targeting ability of the nanoparticles displayed no significant differences with the control groups after 24 h incubation (Figure S3). This phenomenon could be due to the saturation of the cells to the nanoparticles after long time incubation and the proper diameters of the MONs (~60 nm) that are the perfect sizes for cellular uptake. To quantify the amount of nanoparticles internalization by the cells, IVIS spectrum system was used to record fluorescence intensity of the [email protected] in MDA-MB-231 and MCF-7 cells. After fresh medium of 50 µg/mL nanoparticles were added into cells, the fluorescence signals of the cells was analyzed at 1 h, 3 h and 6 h, and the fluorescence intensity were quantified using living imaging software. The fluorescence intensity of all the cells rose with time extension. Cells that cultured in pH 6.5 medium showed stronger fluorescence than the groups incubated in the pH 7.4 medium (Figure 4b). The significant differences among them were found at 3 h and 6 h after incubation. The low-pH driven targeting effects of the [email protected] reached the highest in MDA-MB-231and MCF-7 after 6 h incubation (Figure 4b). The fluorescence values were 0.76 × 107 photos s–1 cm–2 sr–1 in MDA-MB231 cells and 2.04 × 107 photos s–1 cm–2 sr–1 in MCF-7 cells at pH 7.4 after 6 h incubation, which turned to 1.26 × 107 photos s–1 cm–2 sr–1 and 3.49 × 107 ACS Paragon Plus Environment


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photos s–1 cm–2 sr–1 at pH 6.5, respectively (Figure 4b). Moreover, MDA-MB-231 cells absorbed less nanoparticles than MCF-7 cells (Figure 4b), which was consistent with the former results of flow cytometry (Figure 4a). This signified that the low-pH (pH 6.5) medium could efficiently drive the [email protected] to quickly enter the tumor cells while other nanoparticles just performed a general internalization process by the cells in pH 7.4 medium. Therefore, the results demonstrated that pHLIP could efficiently guide the MONs to preferably enter tumor cells under a low–pH condition. Namely, acidic microenvironment targeting ability of other nanoparticles could be achieved through the pHLIP modification to produce low–pH driven delivery systems that have potential application in cancer therapy.

Figure 4. (a) Flow cytometry analysis of MDA-MB-231 and MCF-7 cells after incubation with 50 µg/mL [email protected] or [email protected] at 2 h and 6 h in pH 7.4 and pH 6.5 medium, respectively. Solid lines represent [email protected] while dotted lines stand for [email protected]. (b) Quantification of [email protected] internalization at 1 h, 3 h and 6 h after co-incubation of MDA-MB-231 and MCF-7 cells in pH 7.4 and pH 6.5 medium with 50 µg/mL [email protected].


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The [email protected] was expected to deliver chemotherapeutic drugs to selectively kill cancer cells under low-pH conditions. Therefore, triple-negative breast cancer cells (MDA-MB-231) and estrogen/progesterone receptor positive cells (MCF-7), typically standing for basal like subtype and luminal A subtype, respectively,50 were used to evaluate the cytotoxic effects of the [email protected] against the tumor cells. As shown in Figure 5a, the inhibitory effects of the [email protected] on the growth of tumor cells in low–pH medium were obviously higher than other groups (Figure 5a). Importantly, the [email protected] exhibited better cytotoxic effects against the tumor cells in low-pH medium than the neutral medium (Figure 5b). However, the inhibitory effects of the [email protected] on both the tumor cells showed no significant difference in pH 6.5 medium and pH 7.4 medium. The cell viability of the [email protected] treated groups was only 44.07% for MDA-MB-231 and 55.98% for MCF-7 cells while other groups were significantly higher than 66% at maximum concentrations (Figure 5a). Additionally, Compared to free DOX, all nanoparticle-treated groups exhibited higher inhibitory effects on the growth of the tumors cells than free DOX (Figure 5a). This mainly resulted from that the nanoparticles dominate the releasing of the DOX via a relatively slow rate in case that the drugs were expelled by cells after reaching a certain concentration. This demonstrated the low-pH condition could drive the [email protected] to target and kill tumor cells whatever the cells are basal like subtype (MDA-MB-231) or luminal A subtype (MCF-7).



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Figure 5. (a) Cytotoxicity of [email protected] and [email protected] against MDA-MB-231 and MCF-7 cells in pH 7.4 (solid shape) and pH 6.5 medium (hollow shape) after 6 h treatment and 48 h further culture with fresh medium. (b) Selective inhibitory effects of [email protected] at the maximum concentration in pH 7.4 and pH 6.5 medium on the growth of the tumor cells.

Furthermore, an orthotopic tumor model was used to evaluate the in vivo tumor-targeting ability and therapeutic effects of the [email protected]. The results demonstrated that the MONs with or without pHLIP mainly retarded in the lung and liver while the fluorescence intensity in the spleen and kidney slightly increased after 24 h (Figure 6a). The average value of fluorescence intensity in the lungs for [email protected] (2.41 × 108 photos s–1 cm–2 sr–1) was significantly higher than that of the [email protected] (1.79 × 108 photos s–1 cm–2 sr–1) after 24 h (P < 0.05). This would favor the prevention of pulmonary metastasis because the lungs are the most common site of tumor dissemination. Importantly, the fluorescence intensity in the tumors for ACS Paragon Plus Environment

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the mice injected with [email protected] (0.58019 × 108 photos s–1 cm–2 sr–1) is two times higher than that with [email protected] (0.2906 × 108 photos s–1 cm–2 sr–1) (P < 0.05). This result demonstrated the pHLIP can greatly enhance the accumulation of the MONs in the solid tumors. The excellent tumor-targeting ability of the [email protected] provides a favorable

foundation

for

cancer

therapy.

Figure

6b

clearly

showed

that

the

[email protected] and [email protected] display better therapeutic effects than the DOX and PBS groups for MCF-7 breast cancers (P < 0.05). It is worth meaning that the DOX contained in the [email protected] and [email protected] was only half of the amount of free DOX. Notably, the [email protected] displays the best anticancer effects compared to the [email protected], owing to acidic microenvironment could efficiently increase the accumulation of the [email protected] in the tumor.

Figure 6. (a) NIFR imaging and quantification of the fluorescence intensity in the kidney, heart, lung, spleen, liver, muscle and tumor after i.v. administration of the engineered MONs into the MCF-7 bearing mice (n = 3) for 24 h (i: [email protected]; ii: [email protected]). (b) ACS Paragon Plus Environment


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Relative tumor volumes of the orthotopic MCF-7 tumors (n = 5) after i.v injected with PBS, free DOX, [email protected] and [email protected], respectively (i: PBS; ii: free DOX; iii: [email protected]; iv: [email protected]). * P < 0.05.

4. CONCLUSIONS This was the first attempt to construct the pHLIP modified MONs with acidic microenvironment targeting ability and GSH and pH dual-responsive drug release manner as chemotherapeutic drug delivery system to selectively kill two subtypes of breast cancer cells. The pHLIP modified MONs not only showed good biocompatibility to normal cells but also did not have any noticeable pathogenic effect in ICR mice after weekly i.v administration of the nanoparticles. The stably conjugated Cy5.5 on the [email protected] greatly benefited the detection of the particle internalization by cells and successfully found the acidic microenvironment targeting ability of the engineered nanoparticles. Additionally, the [email protected] displayed low-pH condition chemotaxis to target triple-negative breast cancer cells (MDA-MB-231) and estrogen/progesterone receptor positive cells (MCF-7) and kill the tumor cells via releasing DOX with a GSH and pH dependent

manner.

Furthermore,

the

in

vivo

experiments

showed

that

the

[email protected] has highly tumor-targeting ability and therapeutic efficacy for the orthotopic MCF-7 breast cancers, further demonstrating the potential value of the engineered MONs in cancer therapy. Overall, this study provides a promising strategy for constructing drug delivery system with tumor acidic microenvironment targeting ability by the pHLIP modification, which will greatly benefit most drug delivery systems to achieve tumor-targeting therapy. ASSOCIATED CONTENT Supporting Information

FT-IR spectra, fluorescence intensity, nitrogen sorption isotherms, Raman spectrum, confocal imaging and flow cytometry. ACS Paragon Plus Environment

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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Fax: +86 25 8480 4659. Tel: +86 25 8086 0185 *E-mail: [email protected] Present Addresses †

Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing, 210002 Jiangsu, P.R. China

Author Contributions The manuscript was prepared through contributions of all authors. 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 Key Basic Research Program of the PRC (2014CB744504 and 2014CB744501), the National Natural Science Foundation of China (81530054, 81501538, 81601556, 81601556), the China Postdoctoral Science Foundation (2016M593035), Jiangsu Planned Projects for Postdoctoral Research Funds (1501122c), and the Natural Science Foundation of Jiangsu Province (BK20160017).

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Graphic for manuscript


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Tumor Acidic Microenvironment Targeted Drug Delivery Based on

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