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Controlled Release and Delivery Systems

Photothermal-triggered Controlled Drug Release From Mesoporous Silica nanoparticles Based On Base-pairing Rules Xiaoting Li, Xinhui Wang, Mingli Hua, Haohan Yu, Shaohua Wei, Ao Wang, and Jiahong Zhou ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00478 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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ACS Biomaterials Science & Engineering

Photothermal-triggered Controlled Drug Release From Mesoporous Silica nanoparticles Based On Base-pairing Rules Xiaoting Li,† Xinhui Wang,† Mingli Hua,† Haohan Yu,† Shaohua Wei,† Ao Wang, ‡,* Jiahong Zhou†,* †

College of Life Sciences, Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu

Collaborative Innovation Centre of Biomedical Functional Materials, Key Laboratory of Applied Photochemistry, Nanjing Normal University, Wenyuan Road, Nanjing, Jiangsu 210023, China ‡

Key Lab of Biomass Energy and Material, Jiangsu Province, National Engineering Lab. For

Biomass Chemical Utilization, Key and Open Lab. of Forest Chemical Engineering, SFA, Institute of Chemical Industry of Forest Products, Chinese Academy of Forestry, Suojin 5th Village, Nanjing, Jiangsu 210042, China *Corresponding Author, E-mail: [email protected] (A Wang); [email protected] (JH Zhou). KEYWORDS: photothermal controlled drug release, gate keeper, poly adenine, base-pairing rules, anti-tumor immunity

ACS Paragon Plus Environment

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ABSTRACT: Base-pairing is stable under physiological temperature but broke by heating, which is the basic mechanism for nucleic acid amplification in biology. In this manuscript, a simple controlled drug release system was prepared based on this rule and its in vivo activity was studied. Poly adenine (poly A), the tail of the synthesized RNA chain, was exploited as gatekeeper of thymine (T) modified MSN (MSN-T) based on the simple A-T base-pairing rules. The gate keeper could maintain stable to avoid drug (chemotherapy and photothermal therapy drugs) release during delivery process but be effectively removed to trigger drug release by photothermal effect at tumor tissue by near infrared (NIR) laser irradiation. In vitro and in vivo experiment all indicated that the prepared nanomedicine could effectively suppress tumor growth and activate anti-tumor immunity.

INTRODUCTION MSN have abundant of empty channels, which could encapsulate a large amount of drugs, relatively. In addition, their tunable size of pores with a narrow distribution, thermal stability and nice chemical, and satisfied biocompatibility make them suitable for drug delivery applications.1,2 The surface functionalized MSN by stimuli-responsive caps or gatekeepers groups, such as nanoparticles, polymers and biomolecules, can control release of various cargos under the proper stimulus.3-5 Thus, MSN is a promising platform for various biotechnological and biomedical applications.6,7 Especially, to develop such smart drug delivery systems with highly specific controlled drug release at tumor position has attracted tremendous attention in cancer treatment.8,9 Base-pairing rules are the observed pairings of bases when strands of DNA, RNA, or both, pair with each other through adenine (A) with thymine (T) in an A-T pairing and cytosine (C) with guanine (G) in a C-G pairing. Such pairing are stable under physiological temperature but could be broken by heating, which is the basic mechanism for polymerase chain reaction (PCR) of selected sections of DNA or RNA amplification. Many previous researches used nucleic acid as


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controlled drug release gatekeepers for MSN10-13 and some of their drug release was triggered by heating induced base complementary pairing damage.14,15 These gatekeepers all maintain stable to avoid drug release but obtained satisfied controlled release efficacy after suitable triggering. However, most of these systems need special elaborate nucleic acid sequences design to ensure the specific enzyme cutting sites or structure, such as G-quadruplex or T-Hg2+-T structure.16 In addition, few in vivo experiment were carried out to verify such gatekeepers based on base-pairing rules can maintain stable in delivery process in blood but effective break in tumor tissue by suitable triggering. Thus, in this manuscript, a very simple controlled release system for MSN based on base-pairing rules was designed and prepared in this manuscript (Scheme 1). Doxorubicin (DOX, a chemotherapy drug) and indocyanine green (ICG, a photothermal therapy drug) were coencapsulated inside MSN-T to obtain DOX&ICG@MSN-T. After that, commercial poly A, a string of adenine bases, which is the tail of the synthesized RNA chain during post-transcriptional RNA processing, was mixed with DOX&ICG@MSN-T under physiological temperature. Quite simply, poly A could cap the pores of MSN quickly through A-T pairing (DOX&ICG@MSNT@poly A) to avoid drug release during nanomedicine delivery process in blood since the pairing is stable under physiological temperature. After DOX&ICG@MSN-T@poly A arriving tumor tissue, 808 nm NIR laser will be used to irradiate tumor tissue. The photothemal conversion ability of ICG could heat DOX&ICG@MSN-T@poly A to induce A-T pairing breaking and trigger DOX release to show chemotherapy activity to cancer. Except trigger drug release, the photothemal conversion effect of ICG could also induce cell death by PTT process.17 In vitro and vivo experiment all indicated that DOX&ICG@MSN-T@poly A could effectively suppress tumor growth.


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In addition, many reports indicated that the adenosine receptor would be unregulated since the hypoxia microenvironment of solid tumor. Adenosine and its receptor signaling has been reported to play an important role in antitumor immunity.18-21 Therefore, poly A modification on the surface of the nanomedicine might provide tumor specific accumulation function and induce anti-tumor immunity response.22 Interestingly, in vivo experiment indicated that NIR light mediated anticancer process of DOX&ICG@MSN-T@poly A could active anti-tumor immunity to slightly suppress the untreated tumor growth. Therefore, such positive immunity response provide this nanomedicine great potential to be used in chemo-PTT-immunity combination therapy in the future.

Scheme 1. Schemetic presentation of DOX&ICG@MSN-T@poly A design, preparation, triggered drug release, anticancer process and immunity activation mechanism. METHODS


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Materials. Tetraethyl orthosilicate (TEOS) was obtained from TCI. [3-(2-aminoethyl) aminopropyl] triethoxysilane (APTES) and Thymine-1-acetic acid were purchased from J&K chemical Co., Ltd. Cetyltrimethylammonium bromide (CTAB) and sodium hydroxide (NaOH) were from Shanghai Lingfeng Chemical Reagent Co., Ltd. N-(3-Dimethylaminopropyl)-N′ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were obtained from Aladdin. Polyadenylic acid potassium salt (poly A, SKU: P9403), which is prepared from adenosine diphosphate with polynucleotide phosphorylase, was from Sigma-Aldrich. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Sigma-Aldrich. Doxorubicin hydrochloride (DOX) was obtained from Hualan Chemistry Technology Co., Ltd. Indocyanine green (ICG) was purchased from Beyotime. Dulbecco’s minimum essential medium (DMEM) was purchased from Gibco. Fetal bovine serum (FBS) was from Sijiqing. Hematoxylin and eosin (H&E) was purchased from Shanghai Yeasen Biotechnology Co. Ltd. The water used throughout the experiments was double distilled water. All the chemical agents were of analytical grade and used without further treatment. Characterizations. High-resolution TEM (HRTEM) images were made on a JOEL JEM-2100F High resolution Transmission Electron Microscope operated at an accelerating voltage of 200 kV. Ultraviolet-Visible (UV-Vis) absorption spectra were measured with a Varian Cary 5000 spectrophotometer. Fluorescence spectra were recorded on a Cary Eclipse fluorometer. Fluorescence lifetimes were recorded on a FM-4P-TCSPC fluorescence spetrofluorometers with an excitation wavelength of 450 nm. The Fourier transform infrared (FTIR) spectra were determined by a Nicolet Nexus 670 FTIR infrared spectrometer. Fluorescence microscopy images were observed on a confocal laser scanning microscopy (CLSM, Nikon Eclipse Ti). Multifunctional microplate reader (Tecan Spark 10 M) was used to determine the absorbance of


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formazan (MTT assay). Morphology of cells were monitored by using a Nikon Ti Fluorescence microscope. The size distributions and zeta potentials were measured on a zeta potential analyzer (Zetasizer Nano ZS90, Malvern). An 808 nm Laser was used as the light source. MSN preparation. MSN were synthesized via the typical method with the template (CTAB), the catalyst (NaOH) and the silica precursor (TEOS). CTAB (0.5 g) was dissolved in distilled water (240 mL) by magnetic stirring for 1 h. After that, NaOH (1.75 mL) was added into above solution under stirring and heating to 80 °C for 1 h. Then, TEOS (2.5 mL) was dropwise added into above solution. The mixture was continuously stirred for 2 h. After that, the precipitate was gathered by centrifugation. The precipitate was washed with distilled water and methanol. The MSN (0.7 g) was dispersed in a mixture of methanol (70 mL) and concentrated HCl (0.70 mL, 37.2%) and refluxed for 12 h to remove the CTAB. The result MSN was centrifuged and washed with distilled water for 6 times. Thymine modified MSN (MSN-T) preparation. MSN (0.5 g) and ATPES (0.50 mL) were mixed in anhydrous toluene (50 mL). Then, the mixture was refluxed at 80 °C under the N2 atmosphere. After 24 h, the sample was centrifuged and washed with distilled water and methanol for 3 times. 0.26 mg of thymine-1-acetic acid (pH was adjusted to neutral) was reacted with EDC (48 mg) and NHS (18 mg) in 1 mL of distilled water, which was stirred for 1h at room temperature. Then, the obtained amino group modified MSN (10 mg) was added into the mixture.23 The mixture was continuously stirred for 12 h. The result MSN-T was centrifuged and washed using distilled water for 6 times. DOX&ICG@MSN-T@poly A. DOX (0.6 mg), ICG (1 mg) and MSN-T (2 mg) were mixed in distilled water (1 mL) and stirred for 12 h. The result DOX and ICG co-loaded MSN-T was


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obtained by centrifugation (9600 rpm/min) for 5 min and washed by distilled water. The result DOX&ICG@MSN-T and poly A (2 mg) were dispersed in distilled water (1 mL) and continuously stirred for 2 h. Finally, the result DOX&ICG@MSN-T@poly A was obtained by centrifugation (9600 rpm/min) for 5 min and washed by distilled water. According to the difference of ultraviolet absorption before and after loading, the payload of DOX and ICG could be calculated. Photothermal triggered drug release. The DOX release from DOX&ICG@MSN-T or DOX&ICG@MSN-T@poly A (with or without 808 nm light irradiation) were carried out by the following protocol in 3 groups. In group 1 (DOX&ICG@MSN-T@poly A + 808 nm light irradiation), the freeze-dried powder of DOX&ICG@MSN-T@poly A was put on the bottom of centrifuge tube. After that, 1 mL H2O was slightly added into the tube. DOX&ICG@MSNT@poly A on the bottom was irradiated by 808 nm laser for 1 min (1.2 W/cm2) to promote MSN pore opening and drug release by light-heat conversion of ICG. The temperature increasing during the light irradiation process was recorded using FLIR thermal imager. Then, the sample was shake to promote the released DOX into H2O. The tube was centrifuged. The released DOX in the supernate was detected by UV-Vis spectra and quantified according to the following equation: amount of DOX released

Drug relsase percent (%) = amount of DOX encapsulated in nanoparticles × 100%

(1)

Above experiment was repeated for 11 times and the total released DOX in each cycle was calculated. In group 2 (DOX&ICG@MSN-T@poly A) and group 3 (DOX&ICG@MSN-T), similar experiment was carried out without light irradiation. Photothermal conversion property. Saline and DOX&ICG@MSN-T@poly A ([ICG] = 0.3 mmol/L) aqueous solution were stored in Eppendorf tubes. They were irradiated with an 808 nm


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laser (2.0 W/cm2) for 4 min. The temperature changing during irradiation process was recorded by FLIR thermal imager. The female BALB/c mice were obtained from Comparative Medicine Centre of Yangzhou University. 4T1 cells (3 × 106) suspended in 200 μL of culture medium (DMEM) were subcutaneously injected into the back of mouse. The mice were treated by drug when the volume of tumor reached about 100 mm3. In vivo photothermal conversion property of DOX&ICG@MSN-T@poly A was carried out as following process. 200 μL of DOX&ICG@MSN-T@poly A ([ICG] = 5.8 μmol/L) was injected into the 4T1 tumor-bearing mice through tail vein injection. The mice, injected by saline, was set as control. 6 h later, the tumor position of the mice was irradiated by 808 nm laser (0.6 W/cm2, 15 min). The tumor temperature changing during irradiation process was recorded by FLIR thermal imager. In vitro cellular uptake and anticancer activity. Cellular uptake of DOX&ICG@MSN-T@poly A was carried out using confocal laser scanning microscopy, which can verify the distribution and diversification of DOX&ICG@MSN-T@poly A inside the cells. First, A549 cells were seeded on glass-bottom Petri dishes in incubator (5% CO2, 37 °C). After incubation for 12 h, the cells were treated with free DOX and DOX&ICG@MSN-T@poly ([DOX] = 5.1 μmol/L) for 2 h. After that, cells were irradiated by 808 nm laser (1.2 W/cm2) for 1.5 min. Then, the culture medium was removed. The cells were washed by PBS for 3 times and imaged under confocal laser scanning microscope. The cellular uptake efficiency of drugs was also studied. A549 cells were cultured in a 24-well plate in an incubator (5% CO2, 37 °C) for 12 h. After washing with PBS, the cells were treated with free DOX and DOX&ICG@MSN-T@poly ([DOX] = 5.1 μmol/L) for 4 h. Subsequently, the


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drug-containing medium was transferred into a 96 well plate. Then, the cells were irradiated by 808 nm laser (1.2 W/cm2, 1.5 min) and the concentration of drug not taken up were measured by Multifunctional microplate reader. The cellular uptake percent was calculated according to the standard curves. For the dark toxicity test, A549 cells were seeded into 96 well plates at a density of 1× 106 cells per well in incubator (5% CO2, 37 °C) for 24 h. Then, the cells were treated with DOX, DOX@MSN-T@poly A, ICG@MSN-T@poly A, MSN-T@poly A and DOX&ICG@MSNT@poly A ([DOX] = 5.1 μmol/L, [ICG] = 5.8 μmol/L; the concentrations of DOX and ICG were consistent with the concentrations of DOX and ICG in the complex). After 24 h, cell survival percent was measured by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) colorimetric assay. For the chemo-photothermal cytotoxicity test, after treated by above drugs for 4 h under same experiments condition, the cells were exposed to 808 nm laser (1.2 W/cm2) for 1.5 min. After that, the cells were incubated for 24 h. Then, cell viability was measured by MTT assay. In vivo anticancer activity and immune activation related cytokine assays. In vivo anticancer experiments were carried out as following protocols. 4T1 cells were bilateral subcutaneously injected into right (treated side) and left (untreated side) of all mice. The differences of tumor suppression efficacy between the treated and untreated side could prove the photothermally controlled drug release and tumor therapy. Mice were divided into five groups: saline (group 1), free DOX (group 2), DOX@MSN-T@poly A (group 3), ICG@MSN-T@poly A (group 4) and DOX&ICG@MSN-T@poly A (group 5). The mice bearing 4T1 tumors were treated by drug when the volume of tumor reached about 100 mm3. Various above drugs were tail vein injected. After 6 h, the right tumor of all mice were irradiated by 808 nm laser (0.5 W/cm2) for 6 min. And no light


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irradiation treatment was carried out to the left tumor. The tumor sizes (left and right) and body weight of mice were measured every day in 14 days. The tumor volume was calculated following the formula: volume = width2 × length/2. Finally, the mice were sacrificed and their organs (tumor, spleen, kidneys, heart, lung, liver and brain) slices were stained by H&E following the standard protocol. To evaluate the immune activation effect, 4T1 tumor bearing mice was tail vein injection treated by DOX, ICG@MSN-T@poly A and DOX&ICG@MSN-T@poly at day 1, 3 and 5. The 808 nm light irradiation was conducted at 6 h after injection. At day 5 after irradiation, mice were sacrificed and tumors were gathered for the immunological evaluations. Briefly, the tissues of tumor were cut into small pieces with ophthalmic scissors, which was put into a glass homogenizer containing PBS (pH = 7.4). Then, the single cell suspension was prepared by gentle grinding. The supernatant of tumors could be collected and centrifuged and then stored at -80°C until use. Finally, the supernatant of tumors were used to determine IL-10 and IL-12B levels using commercially available ELISA kit. Cytokine levels (IL-10 and IL-12B) of tissue homogenates were determined by Mouse ELISA kits purchased from Biolegend, Inc. and Cloud-Clone Corp. Plates were read at 450 nm by microplate reader. Detection limits were 2.7 pg/mL for IL-10 and 15.6 pg/mL for IL12B. Assays were performed according to the manufacturer’s instructions. RESULTS AND DISCUSSION Morphology and structure of DOX/ICG -Loaded Nanoparticles. The size and morphology of the MSN-T and DOX&ICG@MSN-T@poly A were characterized by high-resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS). The prepared MSN-T had an average hydrodynamic diameter of approximately 98.1 nm (Figure 1A and C).


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After drug loading and surface modification by poly A, a thin layer of polymers could be observed clearly and the hydrodynamic diameter slightly increased to 101.2 nm (Figure 1B and D). Furthermore, as shown in Figure 1E, the mapping pattern of DOX&ICG@MSN-T@poly A (Si from MSN; Cl from DOXHCl; P from poly A; S from ICG) further proved that DOX and ICG were successfully encapsulated inside MSN-T and poly A was capped on the surface of DOX&ICG@MSN-T. Fourier-transform infrared (FTIR) spectra of MSN-T, poly A and DOX&ICG@MSN-T@poly A were exhibited in Figure 1F. According to the principle of complementary base pairing, the bonding between A and T was mediated by the hydrogen bonds between -NH (or -C=O) of T and -NH2 (or -N=) of A (Figure 1H). The -NH and -C=O peaks of T were located at 3444 and 1628 cm-1, separately. The -NH2 peak of poly A was located at 3412 cm-1. After forming A=T, these peaks moved to 3433 and 1645 cm-1, separately. This phenomenon proved that there are strong interactions between poly A and MSN-T through between -NH (or -C=O) of T and -NH2 (or -N=). Further evidence was from the zeta potential changing along with drug loading and poly A encapsulation. As shown in Figure 1G, the zeta potential of MSN-T, DOX, ICG and poly A were +16.9, +21.2, -16.3 and -37.7 mV. The zeta potential of DOX@MSN-T was +39.3 mV, this change was mainly due to DOX loaded in the pores of MSN. The zeta potential of DOX&ICG@MSN-T was +30.1 mV, which was lower than that of DOX@MSN-T since the negative charged ICG loading. After capping by poly A, the zeta potential was reduced to -16.6 mV, which further proved that the negative charged poly A was successfully capping on the surface of DOX&ICG@MSNT. All of above phenomenon verified that DOX&ICG@MSN-T@poly A was successfully prepared.24


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Figure 1. (A) HRTEM image of MSN-T; (B) HRTEM image of DOX&ICG@MSN-T@poly A; (C) DLS pattern of MSN-T; (D) DLS pattern of DOX&ICG@MSN-T@poly A; (E) Mapping pattern of DOX&ICG@MSN-T@poly A; (F) FTIR spectra of MSN-T, poly A and DOX&ICG@MSN-T@poly A; (G) Zeta potential changing during drug loading and poly A capping process; (H) The principle of complementary base pairing between A and T.


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Photothermal conversion effect of DOX&ICG@MSN-T@poly A. 808 nm laser was used as the light source to measure the photothermal conversion efficiency in of DOX&ICG@MSNT@poly A aqueous solution. As shown in Figure 2A and B, the temperature of DOX&ICG@MSN-T@poly A aqueous solution was raised gradually under laser irradiation and the temperature increased by about 31.6 °C within 4 min, demonstrating its excellent photothermal conversion effect. 25,26

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Figure 2. The thermal image (A) and the increased temperature (Tt-T0, Tt is the measured temperature of the samples post various time irradiation, T0 is the original measured temperature, B) in saline and DOX&ICG@MSN-T@poly A aqueous solutions with the prolonged 808 nm laser irradiation time. The thermal image (C) and the increased temperature (D) at tumor position of saline and DOX&ICG@MSN-T@poly A tail vein injected tumor bearing mice with the prolonged 808 nm laser irradiation time.


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In vivo photothermal conversion was further investigated after 6 hours of the tail vein injection of DOX&ICG@MSN-T@poly A into the mice with laser irradiation at tumor site (808 nm, 0.6 W/cm2). As shown in Figure 2C and D, the temperature of the tumor sites increased gradually with the prolonged irradiation time. In contrast, only a slight temperature change was detected in saline injection group. These results imply that the efficient photothermal conversion effect of DOX&ICG@MSN-T@poly A could provide enough heat for A=T interaction breaking to trigger drug release and provide synergistic PTT activity for cancer treatment. Photothermal triggered drug release. The DOX release behavior of DOX&ICG@MSN-T@poly A was studied by UV-Vis absorbance spectra, fluorescence spectra and fluorescence lifetime measurement. As shown in Figure 3A, without poly A protection, DOX exhibited a relatively fast release from MSN. Almost no DOX released from MSN in DOX&ICG@MSN-T@poly A without light irradiation because of the gate keeper function of poly A. On the contrary, after exposed to 808 nm light, the loaded DOX could be released effectively. In every irradiation cycle, the drug was irradiated by 808 nm laser for 1 min (1.2 W/cm2), and the temperature increasing during this process was recorded using FLIR thermal imager. During 1 min irradiation time, the temperature of DOX&ICG@MSN-T@poly A gradually increased from 37.1 to 50.8 ℃, which indicated that the intrinsic dehybridization temperature and its influence on the pore opening and the consequential drug release (Figure S1). These results indicate that the complementary base pairing between poly A and MSN-T could be break and poly A capping could be removed by photothermal effect. Successful drug loading and efficient photothermal triggered release were further studied using fluorescence spectra, fluorescence lifetime measurement. As shown in Figure 3B, the fluorescence intensity of DOX was sharply reduced after encapsulated inside DOX&ICG@MSN-T@poly A.


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This fluorescence absence of DOX could be due to the concentration quenching derived from selfaggregation of drugs in a small space of the MSN pores. Similar as fluorescence intensity, the fluorescence lifetime of DOX was sharply decreased from 1.03 to 0.62 ns (Figure 3C).27 After irradiated by 808 nm light, the fluorescence intensity and fluorescence lifetime of DOX&ICG@MSN-T@poly A was obviously recovered, which indicated that poly A gate keeper removing and DOX releasing by photothermal effect.

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Figure 3. (A) Release profiles of DOX from DOX&ICG@MSN-T, DOX&ICG@MSN-T@poly A and DOX&ICG@MSN-T@poly A with 808 nm light irradiation; (B) Fluorescence spectra of DOX, DOX&ICG@MSN-T@poly A and DOX&ICG@MSN-T@poly A with 808 nm light irradiation;

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In vitro and in vivo anticancer activity assay. As shown in Figure 4A, after irradiated 1.5 min by 808 nm laser, DOX&ICG@MSN-T@poly A treated cells showed similar fluorescence signal pattern with free DOX, which indicated that DOX&ICG@MSN-T@poly A could be effectively entrapped by cancer cells and DOX could be effectively released from MSN after poly A removing. As shown in Figure 4B, the cellular targeting efficiency was further detected by incubating A549 cells with DOX and DOX&ICG@MSN-T@poly A, respectively. To evaluate the in vitro anticancer activity of DOX&ICG@MSN-T@poly A, the dark and light cytotoxicity of DOX, DOX@MSN-T@poly A, ICG@MSN-T@poly A, DOX&ICG@MSNT@poly A and MSN-T@poly A ([DOX] = 5.1 μmol/L; [ICG] = 5.8 μmol/L; MSN-T = 2 mg/mL; poly A = 2 mg/mL) were studied. Except DOX, no obvious cytotoxicity was detected in other drugs treated cells, which indicate the excellent biocompatibility of our nanomedicine and little drug release without light irradiation (Figure 4C). On the contrary, after the laser irradiation (808 nm), all drugs (except MSN-T@poly A and DOX@MSN-T@poly A) showed obvious cytotoxicity to cancer cells and compared with other groups, DOX&ICG@MSN-T@poly A has the best anticancer activity. Most importantly, no obvious anticancer activity of DOX@MSNT@poly A was detected after light irradiation, which indicated that the photothermal effect from ICG is essential for DOX release (Figure 4D). Such results proved the photothermally controlled drug release and tumor therapy. 35 30

Cellular uptake percent (%)

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**

##

***

60

40

***

20

20

0

0 o Co n tr

l

A A o lyA o lyA DOX p o ly p o ly -T@p -T@p N-T@ N-T@ MSN MSN @MS @MS ICG@ DOX &ICG DOX

o Co n tr

l

A A o lyA o lyA DOX p o ly p o ly -T@p -T@p N-T@ N-T@ MSN MSN @MS @MS ICG@ DOX &ICG DOX

Figure 4. Confocal fluorescence microscopy images (A) and cellular uptake percent (B) of A549 cells incubated with DOX&ICG@MSN-T@poly A (with 808 nm laser irradiation (1.2 W/cm2) for 1.5 min; Bar = 10 μmol/L); (C) Dark cell toxicity comparison of DOX, DOX@MSN-T@poly A, ICG@MSN-T@poly A, DOX&ICG@MSN-T@poly A and MSN-T@poly A ([DOX] = 5.1 μmol/L; [ICG] = 5.8 μmol/L; MSN-T = 2 mg/mL; poly A = 2 mg/mL) (data are expressed as means ± SD; *P < 0.05, **P < 0.01 and ***P< 0.001 drug treated cells versus control); (D) Light toxicity

comparison

of

DOX,

DOX@MSN-T@poly

A,

ICG@MSN-T@poly

A,

DOX&ICG@MSN-T@poly A and MSN-T@poly A ([DOX] = 5.1 μmol/L; [ICG] = 5.8 μmol/L; MSN-T = 2 mg/mL; poly A = 2 mg/mL) (data are expressed as means ± SD; **P < 0.01 and ***P < 0.001 drug treated cells versus control; ## P < 0.01 DOX&ICG@MSN-T@poly A treated cells versus other drugs treated cells). The in vivo tumor accumulation ability of DOX&ICG@MSN-T@poly A was studied with the tumor-bearing mice model and compared with free ICG. In vivo fluorescence imaging was used to track DOX&ICG@MSN-T@poly A in 4T1 tumor-bearing BALB/c mice after intravenous injection of free ICG and DOX&ICG@MSN-T@poly A (dose of ICG = 8 mg/kg, DOX = 5 mg/kg) (Fig. 5A and B). As shown in Figure 5C and D, the tumor accumulation ability of DOX&ICG@MSN-T@poly A was obvious superior than that of free ICG. Furthermore, the in vivo anticancer activity of DOX&ICG@MSN-T@poly A was studied with the tumor-bearing


17


mice model. In our experiments, mice after various treatments showed no obvious body weight change (Figure S2). H&E staining of various organs (heart, liver, spleen, lung, kidney and brain) slices also showed no apparent pathological changes after all of the treatment except DOX treated one (Figure 5G). These results verified the high biocompatibility of these drugs.

Fluorescence Intensity/108

10

ICG DOX&ICG@MSN-T@polyA 8

B

6

4

2

0

Tumor(left)

Tumor(right)

Fluorescence Intensity/108

10

ICG DOX&ICG@MSN-T@polyA 8

D

6

4

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Tumor 1

E

600

3 Tumor Volumet - Volume0 / mm

3 Tumor Volumet - Volume0 / mm

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Control DOX DOX@MSN-T@polyA ICG@MSN-T@polyA DOX&ICG@MSN-T@polyA

400

200

0 0

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4

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8

10

12

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Tumor 2

F

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Control DOX DOX@MSN-T@polyA ICG@MSN-T@polyA DOX&ICG@MSN-T@polyA

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Figure 5. (A) In vivo ICG fluorescence images of 4T1 tumor-bearing mice injection of free ICG and DOX&ICG@MSN-T@poly A; (B) ICG fluorescence intensity of tumors between mice injection of free ICG and DOX&ICG@MSN-T@poly A; ICG fluorescence image (C) and data (D) of tumor and other organs accumulation comparison between free ICG and DOX&ICG@MSN-T@poly A; (E) Tumor-volume change as a function of treatment time by various drug at the untreated side (without 808 nm irradiation); (F) Tumor-volume change as a function of treatment time by various drug at the treated side (with 808 nm irradiation); (G) H&E stained histological sections of heart, liver, spleen, lung, kidney and tumor from the mice after various drug treatment (Bar = 50 µm).


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Figure 5 E and F showed the volume changing of left (without light irradiation) and right side tumor (with light irradiation). Without light irradiation, weak tumor suppression efficacy was detected in the left side tumor of DOX&ICG@MSN-T@poly A treated mice. On the contrary, the tumor growth of light treated right side was totally suppressed in above DOX&ICG@MSNT@poly A treated mice. The differences of tumor suppression efficacy between the treated and untreated side could prove the photothermally controlled drug release and tumor therapy. In addition, as shown in Figure 5G, the DOX&ICG@MSN-T@poly A and light treated tumor showed small portion of purple blue (nuclei stained by hematoxylin) area, which was obviously smaller than other groups, indicating that the majority of cancer cells have suffered apoptosis or necrosis. Immunity activation assay. Interestingly, during above in vivo treatment, we found that the tumor growth speed of untreated side (without light irradiation) was also partly suppressed comparing with saline treated one (Figure 5E and S3). In addition, part of tumor cells in untreated side also suffered apoptosis or necrosis (Figure 6A). Many reports indicated that the tumor treatment could active antitumor immunity, which could contribute tumor suppression at the untreated side. To verify that the antitumor immunity of above mice was activated, the following experiments were carried out. M1 type macrophage and cytotoxic T lymphocytes (CTL) can promote tumor suppression via activating anti-tumor immunities (Figure 6B).28,29 Firstly, IL-10 (predominant cytokine secreted by M2 macrophages) and IL-12B (predominant cytokine secreted by M1 macrophages) amount in tumor tissue from saline, DOX, ICG@MSN-T@poly A and DOX&ICG@MSN-T@polyA treated mice were analyzed. As shown in Figure 6C and D, obvious IL-10 down-regulation but IL-12B up-regulation were detected, which indicated that M2 type macrophage were polarized to M1 type.


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In addition, as shown in Figure 6E, there were more CTL (CD8+-CD4+) cells were detected in tumor tissue comparing with the control group.30-32 These results all proved that the anticancer anti-tumor immunities was activated after treating by our drugs. And the activation efficiency of DOX&ICG@MSN-T@poly A was superior to other groups, which could also contributed to its satisfied tumor suppression effect. Some reports indicated that chemotherapy and photo-based therapy could trigger anti-tumor immunities activation.33 And some reports also indicated that adenosine is an endogenous modulator of innate immune system with therapeutic potential.34 Therefore, we proposed that the chemotherapy by DOX, PTT by ICG and poly A all contributed to the anti-tumor immunities activation of DOX&ICG@MSN-T@polyA.

12000 400

C #

** 200

* **

100

10000

IL-12B (pg/mL)

300

IL-10 (pg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


D

***

8000 ##

6000

**

4000

###

**

2000 0

0

trol Con

A A poly DOX poly -T@ -T@ MSN MSN @ G@ ICG IC & DOX

trol Con

A A poly poly -T@ -T@ MSN MSN @ @ G ICG &IC DOX DOX


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Figure 6. (A) H&E stained histological sections of untreated side tumor (Bar = 50 µm); (B) The proposed mechanism of anti-tumor immune activation pathway; (C) IL-10 (*P < 0.05 and **P < 0.01 drugs versus control; # P < 0.05 DOX&ICG@MSN-T@poly A versus other drugs) and (D) IL-12B in tumors after various treatments (**P < 0.01 and ***P < 0.001 drugs versus control; ## P < 0.01, ### P < 0.001 DOX&ICG@MSN-T@poly A versus other drugs); (E) Representative FCM data of CTL infiltration in tumors. CONCLUSIONS Precision delivery of anticancer drugs to tumor site is crucial for improving therapeutic efficacy and minimizing adverse effects. Therefore, exploiting simple but effective controlled release system is significant. In this manuscript, a simple controlled release system of MSN was design and prepared based on the base-pairing in biology. In vitro and vivo experiment all indicated that the gate keeper could maintain stable under physiological temperature but efficient release drug triggering by photothermal effect. The prepared nanomedicine could effectively suppress tumor growth and activate anti-tumor immunity, which provide its great potential to be used in chemoPTT-immunity combination therapy in the future. AUTHOR INFORMATION Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This research was financially supported by the National Natural Science Foundation of China (NO. 21671105 and 21571105), the project BK20161554 supported by NSF of Jiangsu Province of China, the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Foundation of Jiangsu Collaborative Innovation Centre of Biomedical Functional Materials (161090H001) and Jiangsu Key Lab of Biomass Energy and Material (JSBEM-S201804). ABBREVIATIONS poly A, poly adenine; MSN, mesoporous silica nanoparticles; PTT, photothermal therapy ; DOX, doxorubicin hydrochloride; ICG, indocyanine green; NIR, near infrared; H&E, hematoxylin and eosin; MTT assay, [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay; ELISA, enzyme linked immunosorbent assay. ASSOCIATED CONTENT Supporting Information Temperature of DOX&ICG@MSN-T@poly A increasing during laser irradiation, body weight change as a function of treatment time of various drugs, and the tumor volume comparison after 14 days treatment by various drugs. REFERENCES


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Photothermal-triggered Controlled Drug Release From Mesoporous Silica nanoparticles Based On Base-pairing Rules Xiaoting Li, Xinhui Wang, Mingli Hua, Haohan Yu, Shaohua Wei, Ao Wang, and Jiahong Zhou


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Photothermal-triggered Controlled Drug Release ... - ACS Publications

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