DNA-Functionalized Hollow Mesoporous Silica Nanoparticles with

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DNA-Functionalized Hollow Mesoporous Silica Nanoparticles with Dual Cargo Loading for Near-Infrared Responsive Synergistic Chemo-Photothermal Treatment of Cancer Cells Luo Hai, Xin Jia, Dinggeng He, Anman Zhang, Tianzheng Wang, Hong Cheng, Xiaoxiao He, and Kemin Wang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00657 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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ACS Applied Nano Materials

DNA-Functionalized Hollow Mesoporous Silica Nanoparticles with Dual Cargo Loading for NearInfrared Responsive Synergistic ChemoPhotothermal Treatment of Cancer Cells Luo Hai†, Xin Jia†, Dinggeng He, Anman Zhang, Tianzheng Wang, Hong Cheng, Xiaoxiao He* and Kemin Wang*

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China. Corresponding Author * Kemin Wang, E-mail: [email protected]; * Xiaoxiao He, E-mail: [email protected]. Tel/Fax: +86 731 88821566

† These authors contributed equally.

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ABSTRACT: Low drug loading, premature drug leakage and intratumoral genetic heterogeneity are three main challenges that hinder the successful nanotherapeutics-based oncotherapy, as they provide insufficient therapeutic efficacy, systematic adverse effects and multidrug resistance, respectively. To address these challenges, we herein develop a near-infrared (NIR) lasersensitive DNA-modified hollow mesoporous silica nanoparticles (HMSNs) with dual cargo loading for chemo-photothermal combined treatment of tumors. Starting from the zeolitic imidazolate framework-8 (ZIF-8) template, a layer of mesoporous silica is coated on ZIF-8 (ZIF8@MSNs) and subsequently the template is self-degraded under acidic conditions to obtain HMSNs. It is demonstrated that the as-made HMSNs possesses well-defined morphology, large hollow cavities and abundant mesoporous structures. After loading of indocyanine green into HMSNs core (ICG@HMSNs), the DNA strands, which are composed by sequential cytosineguanine (CG) base pairs, are then grafted onto the ICG@HMSNs (DNA-ICG@HMSNs) to provide loading sites for anticancer drug doxorubicin (DOX). The as-prepared DOX-inserted DNA-ICG@HMSNs (DOX@DNA-ICG@HMSNs) shows highly efficient transformation of light energy into thermal energy. Additionally, the loading amount of ICG is determined to be 930 mg g-1 SiO2, which is more than 30 times compared to that in MCM-41-type MSNs. In vitro experiments using HeLa cells demonstrate that this NIR-responsive drug delivery system (DDS) enable triggerable cargo release, presumably by heat-induced disruption of the modified DNA double-strands. Most importantly, in vitro evaluation and preliminary in vivo investigations independently verified that the combination of triggered chemotherapy and NIR laser based hyperthermal therapy result in better therapeutic effect than individual monotherapies. With these superior properties, we expect that these multifunctional DDS would promote the application of HMSNs in nanomedical applications.


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KEYWORDS: Hollow mesoporous silica nanoparticles (HMSNs); Doxorubicin (DOX); Indocyanine green (ICG); Dual cargo delivery; Synergistic therapy of cancer

INTRODUCTION Chemotherapy is the predominant therapeutic modalities for effective cancer therapy.1 However, cancer patients still suffer systematic adverse effects associated with overdose because safe dosages may not completely ablate cancer cells.2 Tremendous attempts have been devoted to the design of drug delivery system (DDS),3,4 aiming at increasing the local effective therapeutic concentration in tumor but not in normal tissue. Several promising strategies have been proposed to achieve this objective.5-7 One very encouraging solution is to develop a stimuli-responsive DDS responding to particular stimuli, including endogenous stimuli like pH,8 ROS,9,10 and enzymes,11-13 as well as external stimuli like ultrasound,14,15 magnetic field,16-18 temperature19 and light.20 Among all stimulation methods, near-infrared (NIR) laser responsive DDS have attracted intensive research preference because of their greater tissue penetration capacity, high precision to control exposure doses and location, as well as negligible influence on health tissues within the safe power density.21-23 Moreover, the NIR-triggered DDS can realize a “zero premature release” and release their loaded cargo into desired sites.24 Another hopeful approach is the combination of conventional chemotherapy regimens with additional

therapeutics such as

radiotherapy,25,26 magnetic hyperthermia,27 photodynamic therapy28,29 and photothermal therapy (PTT),30,31 generated by exposure to a physical stimulus. As one promising candidate, PTT employs a light photosensitizing agent to generate hyperthermia from light, resulting in raise the tumor local temperature and eradicate tumor. The combination of chemotherapy and PTT not


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only can be applied directly to burn the cancerous tissue, but also is useful for sensitizing neoplastic cells to the effect of cytotoxic drug and trigger other therapeutic approaches, aiming at the synergistic anti-tumor effect.32 For example, Zhu et al.33 demonstrated the application of graphene quantum dots (GQD) as caps to attach the surfaces of mesoporous silica nanoparticles (MSNs) by electrostatic forces and acid-cleavable hydrogen bonds. The GQD act as doublepurpose entity that behave as local photothermal agents and capping agents, and the multifunctional doxorubicin (DOX)-loaded GQD-MSNs exhibited much more effective inhibition of neoplastic cells proliferation owing to a synergistic effect of chemo-photothermal therapy. As one of the promising DDS, MSNs have unique properties like large pore volume (0.6-1 cm3 g-1) and high specific surface area (700-1000 m2 g-1), tailorable mesoporous channels, and remarkable diversity in surface modification.34 In addition, the preparation of MSNs is facile, controllable, cost-effective and scalable.35 All of these merits contribute to the widespread studies and applications of MSNs in stimuli-responsive anti-cancer drug delivery.36,37 Nonetheless, despite remarkable progress in the past years, MSNs cannot satisfy the extension of application in the field of nanomedicine because of the limited loading capacity.38 For the purpose of integrating the advantages of MSNs and microcapsules into a single drug carrier, the fabrication of hollow mesoporous silica nanoparticles (HMSNs) has been proposed.39 Compared with conventional MSNs, HMSNs has vast empty interior cavities to accommodate a large amount of therapeutic agents, which provides opportunities for an ultrahigh cargo loading capacity.40,41 Meanwhile, the mesoporous shell behave as a protective covering so as to prevent degradation of loaded-cargo.42 Furthermore, both inner core and the shell can be easily functionalized with designed functional groups.43 All of which are ideal as next generation DDS.


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Hence, many attempts are ongoing to prepare HMSNs with controlled size and homogeneous morphology to exploit their drug delivery application.44 Template-eliminated methods have been considered as the most representative and straightforward approach toward hollow structures. Specifically, soft- and hard-templating approach are widely applied synthesis methods for preparing HMSNs, which mostly involve a two-step process: fabrication of the core-shell architectures then removal of the interior templates.45 Herein, we propose a strategy that using HMSNs as the scaffold to rationally develop a cooperative therapy as utility of NIR laser-triggered controlled drug release, and simultaneous utility of the synergistic effect of PTT and chemotherapy. Details of this process are demonstrated in Scheme 1A. Firstly, ZIF-8 nanocrystal is synthesized as a sacrificial template for the hollow structure. Thereafter, as-prepared ZIF-8 is wrapped in a mesoporous silica layer (ZIF-8@MSNs) and subsequently self-degrade under acidic conditions to obtain HMSNs. The obtained HMSNs possesses huge inner cavities and narrow-distributed pore structures that oriented perpendicular to the surface of HMSNs. Thereupon, indocyanine green (ICG), a cargo loaded into HMSNs core (ICG@HMSNs), act as the model NIR laser-to-heat energy converter for cancer PTT and for promoting drugs release upon NIR laser illumination. Additionally, the nanoassembly of DNA functionalized ICG@HMSNs (DNA-ICG@HMSNs) is assembled through the hybridization of one strand of the DNA (namely DNA1) attached on the HMSNs and the partly-complementary DNA (namely DNA2). Meanwhile, the as-prepared DNA doublestranded architectures provide scaffolds for DOX intercalation. Thus, their final product of DOX-inserted DNA-ICG@HMSNs (DOX@DNA-ICG@HMSNs) has obtained. As shown in Scheme 1B, once the nanoassembly enter into cells by endocytosis, DOX@DNA-ICG@HMSNs is encapsulated in an endosome. Under NIR light exposure, the photothermal effect of


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DOX@DNA-ICG@HMSNs not only results in endosomal disruption, but also induces DNA melting and the rapid drug release in cancer cells. Thereafter, the cytosolic DOX released from DOX@DNA-ICG@HMSNs can enter the cell nucleus to cause apoptosis. Simultaneously, photoinduced hyperthermia by DOX@DNA-ICG@HMSNs can also induce apoptosis. In consequence, synergistic chemo-photothermal therapy is realized. Combining these excellent synergistic effects, we believe that it should become a favorable on-demand DDS for therapeutic applications in cancer.

Scheme 1. Schematic demonstration of the synthesis of multifunctional DOX@DNAICG@HMSNs (A) and its utilization in NIR laser-controlled chemo-photothermal combined


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therapy of neoplastic cells (B). Once the nanoassembly entered into cancer cells by endocytosis (1), DOX@DNA-ICG@HMSNs was encapsulated in an endosome. Under NIR light illumination, the photothermal effect of DOX@DNA-ICG@HMSNs not only induced endosomal escape (2), but also resulted in DNA denaturation and the rapid released of DOX in cancer cells (3). Thereafter, the cytosolic DOX released from DOX@DNA-ICG@HMSNs could cause chemotherapy (4). Simultaneously, photoinduced hyperthermia by DOX@DNAICG@HMSNs could also induce PTT (5).

EXPERIMENTAL SECTION Materials. Heochst-33342, 1,3-Diphenylisobenzofuran (DPBF), Doxorubicin hydrochloride Calcein

(DOX),

acetoxymethyl

esteror

(Calcein-AM),

2-Methylimidazole

and

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) were provided by Sigma Aldrich (Missouri, US). Copper(I) bromide (CuBr, ≥99.9%), Zinc nitrate hexahydrate (Zn (NO3)2 · 6H2O,

≥ 98%),

Hexadecyltrimethylammonium

chloride

(CTAC,

≥ 98%),

3-

chloropropyltrimethoxysilane (Cl-TMS), Sodiumazide (NaN3, ≥99%), Indocyanine green (ICG) and Propidium iodide (PI) were obtained from Alfa Aesar (Tianjing, China). Sodium hydroxide (NaOH), tert-butyl alcohol (tBuOH) and Tetraethylorthosilicate (TEOS, 28%) were obtained from Dingguo Biotech. Co., Ltd (Beijing, China). Other solvents and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All the chemicals were of analytical-reagent grade and used as received. All aqueous solutions were generated using deionized distilled water (ddH2O), which was purified by passage through a Millipore MilliQ Biocel ultrapure water system (electric resistance >18.2 MΩ cm-1) (Iowa, US). The oligonucleotides were obtained from Takara (Dalian, China). The oligonucleotide sequences are as follows: DNA1: 5’ -Alkyne-TCT GCC TGC TCA TCT AAC TG-3’


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DNA2: 5’-TAG CAG GCA GT-3’. Human cervical cancer (HeLa) cell line was supplied by the cell center of our group. HeLa cells were cultivated in Dulbecco's Modified Eagles Medium with high glucose (DMEM) containing 12% fetal bovine serum (FBS), 1% L-glutamine and 1% streptomycin-penicillin. Cells were maintained in a humidified incubator (37 °C, 5% CO2). Characterization. The elemental components, size and shape of different samples were measured at Tecnai G2 F20 S-TWIN transmission electron microscopy (FEI Inc., US) equipped with X-ray energy dispersive spectroscopy (EDS) at an accelerating voltage of 200 kV. The zeta potentials and hydrodynamic sizes were obtained on a ZetaSizer Nano-ZS apparatus (Malvern, UK), equipped with a He/Ne laser light source (633 nm wavelength, 4 mW) and a fixed detector angle of 90°. Powder X-ray diffraction (XRD) patterns were obtained using Cu Kα (λ = 0.154 nm) radiation on an XDS 2000 powder diffractometer (Scintag Inc., US). The nitrogen adsorption/desorption isotherms were measured at -196 °C on a sorptometer ASAP 2010 (Micromeritics Inc., US). Prior to analysis, sample was degassed at 100 ℃ for 12 h under vacuum (10-3 Torr). Pore size distribution was calculated by applying the Barrett-Joyner-Halenda (BJH) method to the adsorption branch of nitrogen isotherms. Brunauer-Emmett-Teller (BET) surface areas were determined by a linear region of the BET equation on the basis of IUPAC recommendations. Fluorescence measurements of different samples were detected by a F-7000 Spectrophotometer (Hitachi, JP). Ultraviolet-visible (UV-Vis) spectroscopic measurements were conducted on a UV-2600 spectrophotometer (Shimadzu, JP). Fourier transform infrared (FTIR) spectra was recorded on a TENSOR 27 spectrometer (Bruker, Germany). The images were captured using a Fluoview FV500-IX70 confocal microscopy (Olympus, JP). Methyl thiazolyl tetrazolium (MTT) assays were obtained from an Infinite M1000 Pro multimode plate reader


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(Tecan, Switzerland). Photodynamic activity evaluations were induced by a 660 nm laser (HiTech Optoelectronics Co. Ltd, China). Photothermal heating were induced by a 780 nm laser with adjustable power in the range of 0 to 1.6 W (SFOLT Co. Ltd, China). The infrared thermal images were captured by an E60 thermovision camera (FLIR, US). Synthesis of HMSNs. Zeolitic imidazolate framework-8 (ZIF-8) was prepared by following a previously published protocol.46 Specifically, 297.4 mg of 2-methylimidazole (25 mM) and 82.1 mg of Zn(NO3)2·6H2O (25 mM) were successively dispersed into 40 mL of methanol. The resulting solution was mildly stirred at room temperature for 30 min, and the final product of ZIF-8 was obtained. To obtain the mesoporous silica nanoparticles coated ZIF-8 (ZIF-8@MSNs), the as-prepared ZIF-8 nanocrystals were dispersed in 20 mL ddH2O. Then 1 g of CTAC and 400 µL of sodium hydroxide solution (0.1 M) were dropped sequentially into the above mixture under stirring. Next, 150 µL of TEOS was added in six equal increments separated by 30 min intervals under mild stirring at room temperature, and the resulting product of ZIF-8@MSNs was obtained. For the fabrication of hollow mesoporous silica nanoparticals (HMSNs), hydrochloric acid (0.1 M, 30 mL) was added to remove the core template (ZIF-8) and surfactant template (CTAC). A few minutes later, the color of the mixture gradually changed from murky to transparent. The as-prepared HMSNs was obtained by centrifugation (13 000 rpm, 10 min) and extensively rinsed in ethanol and ddH2O, and then dried in a freezing vacuum dryer. Synthesis of DOX@DNA-ICG@HMSNs. 1 mg of HMSNs was re-dispersion into 1 mL ddH2O, and then 1.5 mg ICG was added. After reacting at 50 ℃ for 6 h, ICG loaded HMSNs (ICG@HMSNs) was collected. To remove physisorbed unloaded ICG molecules from the exterior surface of the products, we rinsed the obtained materials with ddH2O at least three times. Next, 0.1 g of above-synthesized ICG@HMSNs and 0.1 mL of 3-chloropropyltrimethoxysilane


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were added into 20 mL of anhydrous toluene. After refluxing for 20 h, the materials were collected and rinsed with ddH2O several times to obtain 3-chloropropyl-functionalized ICG@HMSNs (Cl-ICG@HMSNs). Subsequently, 200 mg of the as-prepared Cl-ICG@HMSNs was added into saturated NaN3 (20 mL). The mixture reacted at 90 ℃ for 12 h and centrifuged and rinsed extensively with ethanol solution and ddH2O to obtain azide-activated ICG@HMSNs (N3-ICG@HMSNs). After centrifugation/water rinsing/redispersion cycles, 2 mg of N3ICG@HMSNs nanoparticles were dissolved in 200 µL of alkyne-functionalize DNA solution (namely DNA1) (65.0 µM). Afterward, 2 µL of the tris-(benzyltriazolylmethylamine) ligand (DMSO/tBuOH = 3:1, 0.1 M) and 1 µL of CuBr solution (DMSO/tBuOH = 3:1, 0.1 M) were dropped sequentially into the solution. After stirring under room temperature for 12 h, the singlestranded

DNA-functionalized

ICG@HMSNs

(ssDNA-ICG@HMSNs)

was

collected.

Simultaneously, the partly-complementary DNA (namely DNA2) was loaded to form scaffolds for DOX intercalation. Specifically, 2 mg of ssDNA-ICG@HMSNs was re-dispersed in 200 µL of aqueous solution containing DNA2 (65.0 µM). After heating the sample to 95 °C for 5 min, 200 µL of DOX (1 mM) was dispersed, then incubated for 30 min at 4°C. Finally, DOX-inserted DNA-ICG@HMSNs (DOX@DNA-ICG@HMSNs) was separated using centrifugation and rinsed thoroughly with ddH2O to remove unloaded DOX, then dried under vacuum. To calculate the DOX loading efficiency, the precipitate was obtained by centrifugation and dissolved in ddH2O solution. Then, the residual amount of DOX was recorded by UV-vis spectrophotometer at 480 nm. Singlet oxygen detection of ICG@HMSNs. 1,3-Diphenylisobenzofuran (DPBF) was applied as a probe to determine reactive oxygen species by recording the UV-vis absorption spectra. Specifically, 15 µL of an ethanol solution with DPBF (2 mg mL−1) was mixed with 1.5 mL of


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water, free ICG (96 µg mL−1) or ICG@HMSNs solution (200 µg mL−1). The solutions were stirred for 100 min in the dark to reach the adsorption-desorption equilibrium state before the measurement. Afterwards, the solutions continuously exposed to 660 nm lasers (10 mW cm−2) for 21 min. Meanwhile, 200 µL samples were taken out for UV-vis spectroscopic measurements at different times points (0, 3, 6, 9, 12, 15, 18 and 21 min). Photothermal performance of DOX@DNA-ICG@HMSNs. The photothermal performance of DOX@DNA-ICG@HMSNs under various power densities of NIR laser was first studied. 1 mL of DOX@DNA-ICG@HMSNs (0.4 mg mL-1) was exposed to 780 nm near-infrared (NIR) laser for 10 min under different power densities (0.8, 1.1, 1.5, 1.9, 2.2 and 2.9 W cm-2). The temperatures were measured every 1 min. Further studies were conducted to research the photothermal efficiencies of DOX@DNA-ICG@HMSNs under different samples concentrations. Specifically, samples with various concentrations of DOX@DNA-ICG@HMSNs (0, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg mL-1) were illuminated with 780 nm laser (2.2 W cm-2, 10 min) and the temperatures were also recorded every 1 min. To study the photostability, the temperatures of DOX@DNA-ICG@HMSNs suspension (0.4 mg mL-1) were recorded during five heating and cooling cycles, where free ICG (40 µg mL-1) was employed as control. NIR laser-controlled drug release in buffer. NIR laser controlled-release study was carried out at a shaking table at 37 °C. Similarly, 1 mg DOX@DNA-ICG@HMSNs was added to 1 mL ddH2O in a 1.5 mL eppendorf centrifuge test tube. At designed time points (every 60 min over a total period of 300 min), the samples were exposed to 780 nm laser (2.2 W cm-2, 10 min), and 200 µL of the release media was taken out for measurement. The concentration of DOX released in the supernatant of release solutions after centrifugation (13 000 rpm, 10 min) was measured using a fluorospectrophotometer (λex=480 nm, λem=560 nm). Following determination by the


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fluorospectrophotometer, the supernatant solutions were taken back to the release buffer and then shaken at room temperature for another 50 min. The release efficiency of DOX was ascertained as follows: the percentage of drug released %=

weight of released drug ×100% total weight of loaded drug

To investigate the NIR responsibility, the release curves with or without NIR laser were compared. All experiments were replicated three times. NIR laser-controlled drug release in cells. To demonstrate the NIR responsibility of DOX@DNA-ICG@HMSNs in vitro, HeLa cells were first plated into coverglass bottom confocal dishes and then cultured in 2 mL DMEM for 24 h. Following removal of the culture medium, fresh culture medium containing 120 µg mL-1 of DOX@DNA-ICG@HMSNs was added. Cells were maintained in an incubator for 2 h and then rinsed in washing buffer for removing the residual nanoparticles which had not uptake by the cells. Cells were exposed to 780 nm laser at a power density of 2.2 W cm-2 for 10 min. Subsequently, the obtained cells were stained by the vital fluorescent dye Heochst-33342. Finally, the cells images were captured by using a confocal microscopy (100× oil-immersion objective lens). Cells without exposure to NIR laser were used as a control. Cell uptake experiments. HeLa cells were co-incubated with DOX@DNA-ICG@HMSNs (120 µg mL-1) in coverglass bottom confocal dishes for different times (0.5, 1, 3 and 7 h). Afterwards, the cells were rinsed twice with wash buffer, suspended in fresh culture medium and exposed to 780 nm laser at a power density of 2.2 W cm-2 for 10 min. After additional incubation for 0.5 (the first group) or 1 h (the second to fourth group), samples were incubated with Heochst-33342 solution (10 mg mL-1) for 10 min. Finally, the cellular uptake of nanoparticles was imaged by a confocal microscopy.


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In vitro cytotoxicity. The cytotoxicity of different nanoparticles was measured by the MTT assay. HeLa cells (1×104 cells per well) were plated at a density of 1×104 cells per well into the 96-well plates. Then the cells were cultured in a humidified incubator at 37 °C with 5% CO2 for 24 h. Subsequently, the cells were respectively treated with HMSNs, ICG@HMSNs, ssDNAICG@HMSNs for 24 h under different concentration (0 to 240 µg mL-1). Following removal of the supernatants, the cells were incubated with 200 µL of fresh culture medium containing MTT reagent (0.5 mg mL-1) for another 4 h. Afterwards, the MTT solution was carefully removed and 150 µL of DMSO was added into each well and then shaken for 10 min. Finally, the optical density (OD) was acquired by multi-detection microplate reader at 490 nm. The viability of cells was ascertained as follows: Viability % =

OD!"#$!#% − OD'($)* × 100% OD+,)!",((#% − OD'($)*

Where ODtreated was obtained from the cells with different treatments. ODcontrolled was obtained from the cells without any treatment. ODblank was obtained from the well plates without cells or nanoparticles. Additional investigation was carried out to determine the influence of the 780 nm light on the viability of cells. Specifically, HeLa cells were seeded onto a 96-well plate at a density of 1 × 104 cells per well and subsequently cultured based on the above-mentioned protocol. Then the cells were exposed to a 780 nm laser for 10 min at different power densities (0.8, 1.1, 1.5, 1.9, 2.2 and 2.9 W cm-2). Viability of HeLa cells was further ascertained using the MTT test as described above. Hemolysis assay. Mouse blood was centrifuged at 2000 rpm for 5 min and rinsed at least five times with PBS to give pure erythrocytes. Afterwards, 300 µL of erythrocytes solution was mixed with 300 µL of water (negative control), PBS (positive control) or ssDNA-ICG@HMSNs


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at different concentrations. The sample was cocultured for 12 h at room temperature. After spinning down the erythrocytes, the supernatants were obtained, and the absorbance at 540 nm was recorded using a UV-vis spectrophotometer. The hemolysis ratio was ascertained based on the following formula: Hemolysis ratio = /

/0120 3/41560781 9:270781 3/41560781

×100%

Where Itest was obtained from the absorbance of erythrocytes with various concentrations of ssDNA-ICG@HMSNs. Inegative was obtained from the absorbance of erythrocytes with PBS. Ipositive was obtained from the absorbance of the complete hemolysis in water. In vitro synergistic chemo-photothermal therapy. To study the synergistic therapy efficiency of DOX@DNA-ICG@HMSNs, HeLa cells were first seeded in a 96-well plate at a density of 1×104 cells per well in 200 µL of DMEM medium containing 12% fetal bovine serum (FBS), 1% L-glutamine and 1% streptomycin-penicillin for 24 h. 20 µL of PBS, free DOX, ssDNA-ICG@HMSNs or DOX@DNA-ICG@HMSNs at different concentrations were added. The cells were maintained at 37 °C for 12 h and the culture medium was refreshed with new medium. Subsequently, the cells were illuminated with or without NIR laser (10 min, 2.2 W cm2

), whereas untreated cells were used as controls. The cells were kept at 37 °C in a humidified

incubator with 5% CO2 for another 12 h. For confocal microscopic analysis, all the cells were stained for 30 min with PI (10 mM) and Calcein-AM (10 mM) to identify dead and living cells, respectively. After washing twice with washing buffer, the fluorescence pictures of dead and living cells were acquired by confocal microscope (40× objective lens). Moreover, the cell viability of HeLa cells with different treatments were then tested by MTT assay as described above.


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In vivo chemo-photothermal combined therapy. Nude mice bearing tumor xenografts were generated by subcutaneously injecting 200 µL of PBS containing 1× 106 human cervical cancer HeLa cells into the flank region of each BALB/c nude mouse. Seven days later, the mice with tumor nodules exceeding 100 mm3 in volume were randomly divided into five groups: (i) intravenous (i. v.) injection of PBS + NIR laser, (ii) i. v. injection of free DOX, (iii) i. v. injection of DOX@DNA-ICG@HMSNs, (iv) i. v. injection of ssDNA-ICG@HMSNs + NIR laser, (v) i. v. injection of DOX@DNA-ICG@HMSNs + NIR laser. The nanoparticles were administered to the animals in a dosage of 0.4 mg kg-1 by DOX or 9 mg kg-1 by SiO2. For the PTT treatment, the mice were illuminated by the 780 nm laser (5 min, 2.2 W cm-2) after 12 h of i. v. injection. The body weight variation was recorded every two days to evaluate the potential systematic adverse effects. After fourteen days, the mice were sacrificed, and the tumors were surgically excised for photographs.

RESULTS AND DISCUSSION Characterization of the synthesized HMSNs and ICG loading. Following the design above, zeolitic imidazolate framework-8 (ZIF-8) nanocrystals were first prepared through a rapid room temperature synthetic route. As demonstrated in Figure 1A, transmission electron microscopy (TEM) observations of ZIF-8 showed that the nanocrystals had regular dodecahedral morphology, high monodispersity and homogeneous size of 285 ± 15 nm. The elemental composition of Zn by X-ray energy dispersive spectroscopy (EDS) (Figure 1D), indicated the successful synthesis of ZIF-8 nanocrystals. Powder X-ray diffraction (XRD) measurements revealed that ZIF-8 exhibit diffraction patterns identical to that of the cubic sodalite-related structure (Figure S1). Subsequently, mesoporous silica shells were coated on ZIF-8 nanocrystals


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to form MSNs coated ZIF-8 (ZIF-8@MSNs) using a modified sol-gel method at room temperature. In this process, CTAC was strongly absorbed on an individual ZIF-8 nanocrystal and served as a template for the fabrication of the mesoporous silica layer via base-catalyzed hydrolysis of TEOS and subsequent condensation of silica onto the surface of the CTAC molecules. TEM images (Figure 1B) directly showed the structure of core@shell nanoparticles with a size of 291 ± 16 nm. The elemental composition of nanoparticles by EDS had changed into Zn and Si (Figure 1E), which belong to the ZIF-8 core and MSNs shell, respectively. The change in elemental composition proved the successful preparation of ZIF-8@MSNs. The assynthesized shell had a considerable number of pores and could allow interaction between the encapsulated ZIF-8 nanocrystals and the surrounding environment, which facilitated the removal of the initial ZIF-8 core. TEM images given in Figure 1C was shown that the internal ZIF-8 nanocrystal could decompose in a dilute aqueous HCl solution to obtain uniform hollow mesoporous silica nanoparticles (HMSNs) (size: 277 ± 25 nm), which could be welldistinguished from the large contrasts between the core and the shell. As displayed in Figure 1F insert, the silica shell was estimated to have a homogeneous thickness of 9.2 ± 0.5 nm. The EDS assay showed that the HMSNs had the elemental composition of Si and O (Figure 1F), indicating the successful preparation of HMSNs. Of particular note, a great number of Cu and C in all of these tests could be ascribed to carbon support films. Zeta potential and dynamic light scattering (DLS) were respectively applied to prove the successful preparation of HMSNs. In Table S1, the hydrodynamic size of the ZIF-8 was 294.2 nm with a poly-dispersity index (PDI) of 0.028. As expected, the hydrodynamic size of the ZIF-8@MSNs nanoparticles increased to 307.9 nm (PDI: 0.218) post mesoporous silica shell coating. Whereas the hydrodynamic size of HMSNs slightly decreased to 298.5 nm (PDI: 0.164) after etching of the ZIF-8 core. At the same


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time, the change in zeta potential between ZIF-8, ZIF-8@MSNs and HMSNs also demonstrated this synthesis process (Figure 2A). Naked ZIF-8 had a surface charge of +51.6 mV. Whereas ZIF-8@MSNs altered the zeta potential by down to -12.9 mV due to the highly anionic MSNs coating, and HMSNs had a charge of -14.8 mV.

Figure 1. TEM images and TEM-EDS spectrum for different samples: (A and D) ZIF-8, (B and E) ZIF-8@MSNs and (C and F) HMSNs. (Inserts: TEM images with higher magnification.)

The mesoporous channels of as-obtained HMSNs were further confirmed by XRD patters evaluation. As illustrated in Figure 2B, the small-angle XRD line profiles of HMSNs showed a


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sharp peak at a diffraction angle (2θ) of about 0.5°, confirming the successful preparation of the disordered mesoporous silica structures. The wide-angle XRD spectra of HMSNs exhibited a single broad peak (2θ = 20-30°) (Figure 2C), attributed to the amorphous mesoporous silica matrix. The nitrogen adsorption-desorption isotherm could be classified as a type-IV isotherm (Figure 2D), demonstrating the mesoporous channels. Meanwhile, the HMSNs samples were also characterized by the surface area, pore volume and pore diameter in Table S2 and Figure 2D insert. According to the Barrett-Joyner-Halenda (BJH) model, the pore size of HMSNs was found to be approximately 1.43 nm. The small mesoporous channels not only facilitated the removal of the sacrificial template to obtain HMSNs, but also gave HMSNs the properties of limiting the escape of loaded cargo from drug delivery system (DDS). Moreover, the surface area and pore volume, obtained by the application of the Brunauer-Emmett-Teller (BET) model, were 705.4 m2 g-1 and 0.36 cm3 g-1, respectively. B)

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Figure 2. Characterization of DOX@DNA-ICG@HMSNs. (A) Zeta potential of different nanoparticles in aqueous solutions. (B) Small-angle XRD patters and (C) Wide-angle XRD patters of HMSNs and DOX@DNA-ICG@HMSNs. (D) N2 adsorption-desorption isotherms of as-obtained HMSNs and DOX@DNA-ICG@HMSNs. (Inserts: Corresponding pore diameter distributions.)

After proving the successful synthesis of HMSNs, we believed that the as-prepared HMSNs with a huge cavity showed apparent advantages for indocyanine green (ICG) carrier. ICG was not only the sole near-infrared (NIR) fluorescent pigment approved by the U.S. Food and Drug Administration (FDA) for use in human,47 but also showed high-efficiency for transforming laser energy into thermal energy, which was fascinating for photothermal therapy (PTT).48 Unfortunately, application of ICG was limited by its numerous disadvantageous properties, such as concentration-dependent aggregation and poor aqueous stability in vitro. Moreover, ICG molecules were easily bound to nonspecific plasma proteins, resulting in quick elimination from the body.49 With the hope to overcome those existing challenges, using a HMSNs-based DDS to load ICG could greatly increase the stability, loading capability and circulation time. As ICG had unique optical properties, the UV-vis absorbance spectrum of free ICG and ICG loaded HMSNs (ICG@HMSNs) in aqueous solution was measured. As displayed in Figure S2, the absorbance spectra of ICG@HMSNs (λmax=896 nm) demonstrated an obvious red-shift in contrast to that of free ICG (λmax=780 nm), which might be ascribed to a great deal of ICG aggregates in HMSNs.50 Remarkably, the loading capacity of ICG was ascertained to be 930 mg g-1 SiO2, which was more than 30 times compared to that in our previous reported MCM-41-type MSNs (HMSNs: 930 mg g-1 SiO2 versus MCM-41-type MSNs: 30.1 mg g-1 SiO2).14 The reason for extremely high DOX loading efficiency could be ascribed to the large hollow core of HMSNs. The photodynamic efficacy of ICG in ICG@HMSNs nanoparticles was measured through the use of


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1,3-diphenylisobenzofuran (DPBF) as a sensor to determine reactive oxygen species (ROS). DPBF could quickly react with the produced ROS, and further generated colourless odibenzoylbenzene, accompanied by the decline in the absorption peak at 410 nm. As shown in Figure S3, when water was illuminated with a 660 nm light, negligible change of the absorption spectrum was detected due to few ROS generation. Contrarily, when ICG@HMSNs was illuminated for 21 min, the absorption peak at 410 nm was decreased by ~5%, which was almost the same as that in free ICG (~7% decline), demonstrating the photodynamic activity of ICG@HMSNs. These measurements also revealed that the coating of HMSNs on ICG was a physical process without destroying the chemical structure of ICG. Characterization of the synthesized DOX@DNA-ICG@HMSNs. The outstanding properties of the HMSNs motivated us to apply them to construct on-demand DDS. Toward this end, ICG@HMSNs was first modified with the chlorine group to fabricate Cl-ICG@HMSNs. Afterwards, the Cl-ICG@HMSNs was mixed with the solution of NaN3 to obtain N3ICG@HMSNs. Then, an alkyne-tethered single stranded DNA (ssDNA, namely DNA1) was tethered to the surface of fresh synthesized N3-ICG@HMSNs through the click-chemistry reactions between alkyne and azide. Fourier transform infrared (FTIR) spectroscopy was used to illustrate the successful synthesis of ssDNA functionalized ICG@HMSNs (ssDNAICG@HMSNs). As demonstrated in Figure S4, the sample Cl-ICG@HMSNs only displayed silica framework vibrations, while the sample N3-ICG@HMSNs showed characteristic azide stretch signal at 2110 cm-1. After DNA1 attachment, the absorption band of ssDNAICG@HMSNs at 2110 cm-1 displayed a strong decrease in intensity. This study has demonstrated that DNA1 was indeed linked to N3-ICG@HMSNs through covalent bond. According to UV-vis spectroscopic measurements, the amount of tethered DNA1 was


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approximately 690 µmol g−1 SiO2. Afterwards, the double-stranded DNA structures were assembled through the hybridization of ssDNA-ICG@HMSNs and the partly-complementary DNA (namely DNA2). Meanwhile, the resulting CG-rich double-stranded DNA formed scaffolds for anticancer drug doxorubicin (DOX) intercalation. The final product was named DOX-inserted DNA-ICG@HMSNs (DOX@DNA-ICG@HMSNs). The as-synthesized DOX@DNA-ICG@HMSNs was measured by the fluorescence spectrometer (Figure S5). Upon DNA intercalation, the fluorescence of loaded DOX could be quenched in contrast to free DOX. The DOX-loading efficiency of DOX@DNA-ICG@HMSNs was determined to be approximately 48 mg g-1 SiO2 by using a standard curve approach. Having confirmed the successful loading of DOX into DOX@DNA-ICG@HMSNs, we next investigated the colloidal stability of DOX@DNA-ICG@HMSNs in phosphate buffered saline (PBS), Dulbecco's Modified Eagles Medium (DMEM) and fetal bovine serum (FBS). As presented in Figure S6, DOX@DNA-ICG@HMSNs were observed with negligible agglomeration under all the above-mentioned conditions. Furthermore, their homogeneous dispersion was sustained more than 24 h. The remarkable dispersion stability under physiological solutions suggested that DOX@DNA-ICG@HMSNs could be used as auspicious delivery vehicles in vivo. In addition, the hydrodynamic size was also measured by DLS (Table S1). Owing to the functionalization of DNA1, the average size of ssDNA-ICG@HMSNs has changed to 347.0 nm (PDI: 0.154), which was signally larger than that of HMSNs (298.5 nm). While the hydrodynamic diameter of DOX@DNA-ICG@HMSNs (347.1 nm, PDI: 0.179) was similar to that of ssDNAICG@HMSNs. Additionally, zeta potentials were found to be consistent, For ICG@HMSNs, ssDNA-ICG@HMSNs and DOX@DNA-ICG@HMSNs, the surface charges were -13.2, -16.0 and -17.9 mV, respectively. The mesoporous structure of the synthesized DOX@DNA-


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ICG@HMSNs was confirmed by small-angle and wide-angle XRD measurements (Figure 2B and Figure 2C). Moreover, the nitrogen adsorption-desorption isotherm of DOX@DNAICG@HMSNs also displayed a characteristic type IV curve (Figure 2D). The surface area, pore volume and pore size of DOX@DNA-ICG@HMSNs were 656.8 m2 g-1, 0.23 cm3 g-1 and 1.43 nm (Table S2), respectively. These results strongly proved that: (i) ICG was indeed embedded into DOX@DNA-ICG@HMSNs core and has not affected the surface properties and shape of DOX@DNA-ICG@HMSNs. (ii) the mesopores structures of DOX@DNA-ICG@HMSNs have not been destroyed by the related functionalization. As a NIR laser sensitive nanoplatform, it was necessary to research the photothermal transduction efficiency of DOX@DNA-ICG@HMSNs. Afterwards, the temperature elevation of DOX@DNA-ICG@HMSNs solution under NIR laser exposure was researched. In Figure 3A, the solution temperature gradually raised with increased power density of 780 nm laser. And the temperature could reach 30.9, 33.2, 35.5, 39.8, 43.0 and 51.0 ℃ after irradiation with different laser power density (0.8, 1.1, 1.5, 1.9, 2.2 and 2.9 W cm-2, respectively). These data have proved that DOX@DNA-ICG@HMSNs could efficiently transduce laser energy to thermal energy, and there was a significant positive correlation between the photothermal effect produced by ICG and the laser power density. To select a maximal laser power density with negligible side effects, another study was subsequently designed to assess the effect of laser power density on cell viability. It was found in Figure 3B the majority of cells still survived after exposure to NIR laser of various power densities (0.8, 1.1, 1.5, 1.9 and 2.2 W cm-2, respectively). Nevertheless, only 80.74% of cells survived upon illumination with NIR laser at 2.9 W cm-2. These data indicated that the NIR laser at 2.9 W cm-2 exhibited photoinduced cytotoxicity to a certain degree. Based on these results, we finally used 2.2 W cm-2 as the optimal laser power density in the


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following experiments. To further study the efficiency of using DOX@DNA-ICG@HMSNs for PTT, different concentrations of DOX@DNA-ICG@HMSNs samples were illuminated by a 780 nm light (2.2 W cm-2). Figure 3C demonstrated that the temperature variation of the H2O sample slightly changed upon NIR laser exposure (from 25.0 to 28.6 ℃). Nevertheless, the temperature of the DOX@DNA-ICG@HMSNs samples displayed a significant time- and concentrationdependent temperature rise. And the temperature could reach 29.1, 31.2, 32.7, 36.7, 44.3 and 45.0 ℃ at different concentrations of DOX@DNA-ICG@HMSNs (0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg mL-1, respectively). The thermal images also revealed a direct visual observation for the temperature variation. As displayed in Figure 3D, the temperatures monitored by an infrared thermal camera at various irradiation times and concentrations were similar to the temperatures which were measured using an on-line temperature acquisition device. In addition, we designed an experiment to investigate the photostability of DOX@DNA-ICG@HMSNs upon NIR laser exposure, where free ICG was used as control. As displayed in Figure S7A, the photothermal conversion capability of DOX@DNA-ICG@HMSNs suspension had negligible variation during five heating and cooling cycles upon 780 nm laser illumination (2.2 W cm−2, 10 min). In contrast, photothermal performance of free ICG decreased significantly in the second cycle, and gradually weakened within five rounds. Also, there are no significant changes in morphology of DOX@DNA-ICG@HMSNs (Figure S7B) after NIR laser irradiation for 30 min. To this end, the excellent photothermal properties of DOX@DNA-ICG@HMSNs have been well-verified.


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Figure 3. Investigation of photothermal transduction efficiency. (A) The photothermal heating curves of DOX@DNA-ICG@HMSNs (0.4 mg mL-1) with 780 nm NIR laser illumination at various power densities. (B) The influence of NIR laser power density (0.8-2.9 W cm-2) on viability of HeLa cells. (C) The photothermal heating curves of DOX@DNA-ICG@HMSNs with different concentrations (0.025-0.8 mg mL-1) upon 780 nm laser exposure at 2.2 W cm-2. (D) The thermal imaging photographs of different concentrations of DOX@DNA-ICG@HMSNs upon 780 nm laser illumination (10 min, 2.2 W cm-2).

NIR-triggered release behavior of DOX@DNA-ICG@HMSNs in buffer. The desired property of DOX@DNA-ICG@HMSNs was that it could unload its cargo in a controlled manner under NIR laser irradiation, which was caused by light induced hyperthermia. Then, the drug release experiments in buffer were carried out with or without NIR laser exposure. As displayed


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in Figure 4, DOX release from DOX@DNA-ICG@HMSNs was negligible without NIR exposure (at most 16% during 5 h), indicating that DOX@DNA-ICG@HMSNs was robust, and the premature release of the drug was successfully prevented. Remarkably, interesting burst-like release by DOX@DNA-ICG@HMSNs was significantly augmented, e.g. from 12.47% to 22.01% at the 1st h, under 780 nm laser illumination (10 min, 2.2 W cm-2). The release of drugs was practically switched off when the NIR laser illumination was retreated. The same phenomena were found after repeated laser pulses at the 2nd, 3rd, 4th and 5th h, in which the release rate of drugs was boosted from 28.95% to 35.66%, from 39.73% to 41.74%, from 44.71% to 45.88% and from 46.91% to 47.64%, respectively. Consequently, it should be pretty obvious that the DOX release from DOX@DNA-ICG@HMSNs could be repeatedly triggered by NIR laser.

Figure 4. NIR-triggered release behavior of DOX@DNA-ICG@HMSNs. DOX@DNAICG@HMSNs (0.1 mg mL-1) dispersion was illuminated with or without NIR laser (2.2 W cm-1, 780 nm) at the 1st, 2nd, 3rd, 4th and 5th h, respectively. In vitro NIR-triggered drug release and cellular uptake of DOX@DNA-ICG@HMSNs. In order to further study whether the NIR laser-responsive function of DOX@DNA-ICG@HMSNs was maintained in cells, the release behavior in the living cells was observed using the confocal


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laser scanning microscopy (CLSM). As illustrated in Figure 5, without the application of an NIR laser stimulus, the fluorescence of DOX could be initially quenched due to intercalation into the DNA double helices. Only negligible red fluorescence from DOX was observed after incubation for 2 h. This data demonstrated that DOX@DNA-ICG@HMSNs as an endocellular DDS could effectively hold DOX before NIR laser exposure. Upon 780 nm laser illumination (10min, 2.2 W cm-2), the red fluorescence of DOX, belonging to the released DOX of internalized DOX@DNAICG@HMSNs, was obviously visible in the cytoplasm of HeLa cells. The photothermal heating of DOX@DNA-ICG@HMSNs caused the denaturation of DNA helices, leading to the inserted DOX being released into the cells. These data mentioned above have indicated that the controlled-release performance of DOX@DNA-ICG@HMSNs was NIR laser-dependent. Hoechst-33342

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Figure 5. The confocal microscopy images of controlled release performance of DOX@DNAICG@HMSNs (120 µg mL-1) with or without 780 nm light exposure (10 min, 2.2 W cm-2) after incubating with HeLa cells for 2 h. Nucleus was stained with Hoechest-33342 (shown as the blue color).


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It is well known that the effective cytotoxicity of DOX is relied on its nuclear localization. Therefore, we had a further investigation on the cellular uptake and nuclear import of DOX@DNA-ICG@HMSNs. In Figure 6, when NIR laser denatured the DNA helices of DOX@DNA-ICG@HMSNs, the DOX was released from DOX@DNA-ICG@HMSNs in cytoplasm and then translocated into cell nucleus, leading to the visible overlapped fluorescent between Hoechst-33342 and DOX in cell nucleus (4 h incubation time). Moreover, when increasing the incubation time of DOX@DNA-ICG@HMSNs with HeLa cells, more drug vehicles could internalize into cells and more DOX has been released. These released DOX also could be then located in cell nucleus, leading to a visibly enhanced overlapped fluorescence between Hoechst-33342 and DOX (8 h incubation time). From these results, it was easily concluded that the DOX@DNA-ICG@HMSNs was efficiently internalized into HeLa cells, and promptly translocated into the nucleus upon NIR laser irradiation. Hoechst-33342

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Figure 6. The cellular uptake in vitro. The CLSM images displayed cellular localization of DOX after different incubation time (1, 2, 4 and 8 h) with DOX@DNA-ICG@HMSNs (120 µg mL-1). Blue represented the fluorescence of Hoechest-33342, red represented the fluorescence of DOX.

In vitro synergistic therapy. Biocompatibility was key concern for the biomedical applications of drug carriers. Therefore, before we moved on to further in vitro synergistic therapy experiments, the intrinsic toxicity of HMSNs, ICG@HMSNs and ssDNA-ICG@HMSNs to HeLa cells was first studied. As Figure 7A showed, the HMSNs, ICG@HMSNs and ssDNAICG@HMSNs exhibited minimal cytotoxicity to cells at concentrations ranging from 0 to 240 µg mL-1. Nearly 99.57%, 98.65% and 98.54% of the cells were alive at a maximum concentration of 240 µg mL-1. The concentration of DOX@DNA-ICG@HMSNs applied were lower than 240 µg mL-1 in the following experiments, so we could assume that the drug delivery vehicle had no obvious short-term toxicity to HeLa cells. We have further investigated the biocompatibility of ssDNA-ICG@HMSNs through hemolysis assay. The concentration of ssDNA-ICG@HMSNs used in the hemolysis experiment (2400 µg mL-1) was ten times higher than that in cell viability assay (240 µg mL-1). As illustrated in Figure S8, there was no obvious hemolysis (

DNA-Functionalized Hollow Mesoporous Silica Nanoparticles with

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