Precision Cancer Theranostic Platform by In Situ Polymerization in

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Precision Cancer Theranostic Platform by In Situ Polymerization in Perylene Diimide-Hybridized Hollow Mesoporous Organosilica Nanoparticles Zhen Yang, Wenpei Fan, Jianhua Zou, Wei Tang, Ling Li, Liangcan He, Zheyu Shen, Zhantong Wang, Orit Jacobson, Maria A. Aronova, Pengfei Rong, Jibin Song, Wei Wang, and Xiaoyuan Chen J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06086 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of the American Chemical Society

Precision Cancer Theranostic Platform by In Situ Polymerization in Perylene Diimide-Hybridized Hollow Mesoporous Organosilica Nanoparticles Zhen Yang#†‖, Wenpei Fan,†‖ Jianhua Zou,† Wei Tang,† Ling Li,† Liangcan He,† Zheyu Shen,† Zhantong Wang,† Orit Jacobson,† Maria A. Aronova,∆ Pengfei Rong,*#§ Jibin Song,Φ Wei Wang,*#§ Xiaoyuan Chen*† #Cell

Transplantation and Gene Therapy Institute, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China. §Engineering and Technology Research Center for Xenotransplantation of Human Province, Changsha, Hunan, China. †Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA. MOE Key Laboratory for Analytical Science of Food Safety and Biology College of Chemistry, Fuzhou University, Fuzhou 350108, China. Φ

Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA. ∆

KEYWORDS (Mesoporous organosilica nanoparticles, organic semiconducting molecules, cancer imaging, theranostics)

ABSTRACT: Phototheranostics refers to advanced photonics-mediated theranostic methods for cancer and includes imaging-guided photothermal/chemo therapy, photothermal/photodynamic therapy, and photodynamic/chemo therapy, which are expected to provide a paradigm of modern precision medicine. In this regard, various phototheranostic drug delivery systems with excellent photonic performance, controlled drug delivery/release, and precise photo-imaging guidance have been developed. In this study, we reported a special “in situ framework growth” method to synthesize novel phototheranostic hollow mesoporous nanoparticles by ingenious hybridization of perylene diimide (PDI) within the framework of small-sized hollow mesoporous organosilica (HMO). The marriage of PDI and HMO endowed the phototheranostic silica nanoparticles (HMPDINs) with largely amplified fluorescence and photoacoustic signals, which can be used for enhanced fluorescence and photoacoustic imaging. The organosilica shell can be chemically chelated with isotope 64Cu for positron emission tomography imaging. Moreover, in situ polymer growth was introduced in the hollow structure of the HMPDINs to produce thermosensitive polymer (TP) in the cavity of HMPDINs to increase the loading capacity and prevent unexpected leakage of the hydrophobic drug SN38. Furthermore, the framework-hybridized PDI generated heat under near-infrared laser irradiation to trigger the deformation of TP for controlled drug release in the tumor region. The fabricated hybrid nanomedicine with organic–inorganic characteristic not only increases the cancer theranostic efficacy but also offers an attractive solution for designing powerful theranostic platforms.

1. INTRODUCTION Mesoporous organosilica nanoparticles (MONs) have attracted extensive interests for broad-spectrum applications in energy,1 catalysis,2 biomedical engineering,3 and cancer theranostics.4 MONs exhibit the collective merits, such as mechanical and chemical stability, excellent biocompatibility, and stimuliresponsive biodegradability, of organic and inorganic materials;5 as such, MONs may stand out as an excellent drug delivery system (DDS) for efficient tumor-targeted delivery of cancer drugs.6-7 Most effective cancer drugs, such as paclitaxel (PTX) 8, camptothecin (CPT),9 and 7-ethyl-10-hydroxycamptothecin (SN38),10-12 are hydrophobic and are difficult to administer intravenously. In MON DDS, the hydrophilic nature of the pores seriously restricts the loading capacity of

hydrophobic drugs and lead to easy leakage.13-14 As such, significant improvements have been applied to MONs so they can be suitable for hydrophobic drug delivery. Emerging hollow-structured MONs (denoted as HMONs) possess an internal cavity and increased loading capacity of hydrophobic drugs.15-17 However, most HMONs have a large size (more than 200 nm), which leads to short blood circulation and poor tumor accumulation. Moreover, hydrophobic drugs can easily leak from HMONs because of their weak adsorption interactions.13 To optimize drug delivery, small HMONs (less than 50 nm) should be developed for enhanced EPR effect and controlled drug release for on-demand chemotherapy.18-19 However, the small interior of small HMONs cannot hold sufficient amount of drugs. A DDS made of polymer platform

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is widely used for effective hydrophobic drug delivery;20-26 thermosensitive polymers (TP),27 such as poly(N-isopropyl acrylamide),28-29 poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA),30-31 and pluronic F-127,32-33 are capable of controllable thermal-triggered drug release due to thermal induced phase separation during a temperature shift around the lower critical solution temperature (LCST).34 Thus, an in situ polymerization method should be explored for grafting polymers within the cavity of the HMONs to increase the drug loading capacity, achieving thermal-responsive drug release, and, consequently, preventing premature leakage of hydrophobic drugs. In this regard, high-performance photothermal conversion materials should be identified and “merged” with HMONs to achieve photo-responsive remote control of drug release. Semiconducting perylene diimide (PDI)-based nanomaterials have been widely used in biophotonics, especially in photoacoustic imaging (PA) and photothermal therapy (PTT), owing to their excellent thermal stability, high photothermal conversion, and easy modification.35-40 Engineered NIR PDI nano-formulations can be used for in vivo PA imaging but are unsuitable for near-infrared (NIR) fluorescence (FL) imaging due to their aggregation-caused quenching effect.41 Silica-based materials synthesized by simply coating a silica shell onto the surface of semiconducting nanoparticles can effectively enhance the photothermal, PA, and FL performance of the semiconducting materials due to advanced water isolation and thermal resistance properties of the shell.42-44 As such, constructing a hybrid nanoplatform of PDI and HMONs (HMPDINs) is highly desirable to achieve advanced phototheranostics of FL recovery and PA enhancement. In this study, we report a special “in situ framework growth” approach to prepare HMPDINs through the co-hydrolysis of PEG2000-PDI-silane and organosilica/silica precursors during the synthesis of small-sized HMONs. This method allows for in situ growth of PDI within the framework rather than postconjugation on the surface such that the mesopores of the assynthesized HMPDINs are not blocked for the entry of the drugs. The silica framework will greatly reduce the exposure of PDI to the complicated outer environment to remarkably enhance the PA and photothermal performance of PDI. In particular, the NIR FL of PDI is recovered due to its decreased aggregation and homogeneous distribution within the silica framework, a feature known as the “silica shell shielding” effect.45 The HMPDINs exhibit 211.5-fold higher NIR FL emission over the PDI aggregates in water; this characteristic enables FL imaging together with PA/ positron emission tomography (PET) imaging to track the in vivo delivery of HMPDINs. A method of in situ polymer growth is introduced to endow HMPDINs with increased drug loading efficiency and photothermal-controlled drug release. The hollow-structured HMPDIN acts as a nanoreactor for polymerization of the monomers N-isopropyl acrylamide, 2-(dimethylamino)ethyl methacrylate, and PEG. The formed TP are trapped within the hollow cavity of the HMPDINs to form HMPDINs@TP. The hydrophobic drug SN38 was selected for loading into the HMPDINs@TP (designed as HMPDINs@TP-SN38). The HMPDINs@TP-SN38 could be an excellent phototheranostic nanoplatform for on-demand synergistic photothermal/chemo therapy under precise FL/PA/PET trimodal imaging guidance (Scheme 1). 2. EXPERIMENTAL SECTION

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2.1. Materials and Reagents. Tetraethylorthosilicate (TEOS), ammonia solution (25-28%), and triethanolamine (TEA) were bought from Sinopharm Chemical Reagent Co. Bis(3triethoxysilylproyl)disulfide (BTES), cetanecyltrimethylammonium chloride (CTAC), 3,4,9,10perylenetetracarboxylic dianhydride, (3aminopropyl)triethoxysilane (APTES), N,Ndimethylformamide (DMF), 2-(dimethylamino)ethyl methacrylate(DMAEMA), poly(ethylene glycol) methyl ether methacrylate (mPEGMA, Mn, 300) and N-isopropylacrylamide (NIPAM). 2,2’-azobis(2-methylpropionitrile) (AIBN) were obtained from Sigma-Aldrich. Biomedical agents were obtained from Thermo Fisher Scientific, Waltham, MA. All chemicals were used without further purification. 2.2. Synthesis of PDI hybridized organic–inorganic HMPDINs by a “in situ growth” approach. TEA aqueous solution (0.8 g, 10 wt%) and CTAC aqueous solution (20 g, 10 wt%) were mixed and stirred in an oil bath at 80 °C. After 1 h of stirring, TEOS (1 mL) was added dropwise into the solution, which was subjected to hydrolysis/condensation reaction for 1 h to obtain MSN core. PEG2000-PDI-COOH was synthesized by the reaction of PEG2000-PDI-OH and excess succinic anhydride.46 PEG2000-PDI-silane was synthesized by amidation of PEG2000-PDI-COOH with APTES in DMF without further purification for next step. The PEG2000-PDI-silane DMF solution (1 mL, 30 wt%), TEOS (0.5 mL), and BTES (1 mL) were premixed and added into the MSNs solution. The solution showed green color without any sediment. After 4 h of reaction, the core/shell structured MSNs@MPDINs product was collected by centrifugation and washed with pure ethanol for three times. The structure-directing agent CTAC was removed by dissolving MSNs@MPDINs in a mixture of ethanol and concentrated HCl (37%) (Vethanol:VHCl = 10:1) under vigorous stirring for 12 h at 78 °C. The solvent was removed by centrifugation. The CTAC-extracted MSNs@MPDINs product was dispersed into ethanol (20 mL) and washed three times with ethanol. Finally, the MSNs@MPDINs solution (5 mL) was taken out and dispersed into pure water (100 mL). Ammonia solution (2 mL) was added into the aqueous solution at 95 °C for 3 h to etch the MSNs of MSNs@MPDINs. The hollowmesoporous structured HMPDINs were finally obtained by centrifugation and washing with water three times. 2.3 Synthesis of thermo-sensitive polymer loaded HMPDINs (HMPDINs@TP) by polymerization in situ. Typically, HMPDINs (100 mg) and AIBN (9 mg, 0.036 mmol) were dissolved in 1 mL DMF solution in a 5 mL Schlenk tube. The mixture was added with freshly distilled DMAEMA (134.5 mg, 0.885 mmol), mPEGMA (0.265 g, 0.883 mmol), and NIPAM (0.5 g, 4.42 mmol). O2 in the solution was degassed by argon blowing for 30 min. After vigorous stirring overnight at room temperature, the suspension was heated up to 65 °C and allowed to react for 1 h. The polymer containing silica was collected by precipitation in ample alcohol and centrifuged three times to completely remove the untrapped polymer. The obtained product was denoted as HMPDINs@TP and re-dispersed in water or dried under vacuum for future experiments. When the polymerization time was extended to 2 h, the polymer grew outside HMPDINs. 2.4 Preparation of SN38-loaded HMPDINs@TP (HMPDINs@TP-SN38). HMPDINs@TP (20 mg) was dispersed into the THF (1 mL) solution containing dissolved SN38 (10 mg). H2O (5 ml) was added dropwise into the solution


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under ultrasonication for 10 min. The mixture was dialyzed in PBS buffer for 48 h to remove excess free SN38. The resulting HMPDINs@TP-SN38 was concentrated by centrifugation and dissolved in PBS solution. The dried HMPDINs@TP-SN38 was redissolved into DMSO and centrifuged to test the drug loading content. The concentration of SN38 loaded into HMPDINs@TP was quantified by recording absorbance at 350 nm. The drug release experiment was conducted by testing the absorbance of released SN38 in PBS with 0.5% tween. 2.5 Cell uptake analysis. U87MG cancer cells were cultured in Dulbecco’s modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a mixture atmosphere of 5% CO2 and 95% humidified air at 37 ℃. The cells were seeded in confocal imaging chambers and co-incubated with HMPDINs@TP-SN38 (HMPDINs, 50 µg/mL) for various durations. The cells were fixed, stained with Hoechst, and subjected to confocal Laser fluorescence microscopy (CLSM) (ZEISS, LSM 780, Germany) analysis. The cells were harvested, rinsed with cold PBS, and re-suspended in PBS for flow cytometry analysis. 2.6 In Vitro Photothermal-Chemo Therapy. The toxicity of free SN38, HMPDINs-SN38, and HMPDINs@TP-SN38 was compared. U87MG cells were seeded in 96-well plates (5000 cells in 200 µL DMEM per well) for 24 h. Free SN38, HMPDINs-SN38, and HMPDINs@TP-SN38 (final SN38 concentration 0.09375, 0.1875, 0.375, 0.75, 1.5, 3, 6, and 12 µM) were added to the culture medium of the cells. After incubation for 48 h, MTT assay was conducted to assess cell viability. SN38 (3 µM) was used for comparison of the toxicity of nano-formulations with or without laser. After U87MG cells were seeded for 24 h, the cells were incubated with DMEM, HMPDINs@TP, free SN38, and HMPDINs@TP-SN38 for 24 h. The cells were then treated with or without laser irradiation of 0.5 W/cm2 for 10 min. After laser irradiation, the cells were incubated for another 24 h for the MTT assay and live/dead cell double staining assay. 2.7 In Vivo FL Imaging. All in vivo procedures were conducted in accordance with the protocol approved by the National Institutes of Health Clinical Center Animal Care and Use Committee. FL imaging was performed using a Cri Maestro whole-body animal imaging system (Caliper Life Sciences, Hopkinton, MA). Xenograft U87MG-bearing nude mice were injected intravenously with HMPDINs@TP (100 µg per mouse; n = 5). FL images of the mice were collected at different time points post-injection (1, 4, 24 and 48 h). 2.8 In Vivo PA Imaging. PA imaging of the tumor region was performed by Visual Sonic Vevo 2100 LAZR system to obtain images at 680 nm. Xenograft U87MG-bearing nude mice were placed in the imaging system and administered with anesthesia. HMPDINs@TP (100 µg per mouse; n = 5) were administered through intravenous injection. The representative PA images of the mice were collected at different time points post-injection (1, 4, 24 and 48 h). 2.9 In Vivo PET Imaging. Radioactive 64Cu@HMPDINs@TP were synthesized by chemical modification of isotope 64Cu on the surface of HMPDINs@TP by chelation of the thiol groups. U87MG tumor-bearing nude mice (n = 5) were injected intravenously with 100 μCi of 64Cu@HMPDINs@TP in PBS. PET scanning was conducted at different time points postinjection on a Siemens Inveon microPET scanner (Siemens Healthcare GmbH, Germany). Quantitative PET intensity data were obtained by drawing the regions of interest on the main

organs and tumors with decay-corrected whole-body coronal images. The percentage injected dose per gram of tissue (%ID/g) was calculated according to the measured values. 2.10 In Vivo Photothermal-Chemo Therapy. U87MG xenografted nude mice were systemically administered with PBS (n = 10), HMPDINs@TP (2 mg per mouse; n = 10), and HMPDINs@TP-SN38 (200 µg per mouse; n = 10) via tail vein injection. At 24 h post-injection, each group was divided into two. The tumor regions of living mice were exposed or unexposed to 671 nm laser of 0.5 W/cm2. IR thermal camera was used to record the temperature change in the mouse tumor during irradiation. The average tumor size was calculated as follows: volume = (tumor length) × (tumor width)2/2.

Scheme 1: Blood transport and tumor accumulation of the HMPDI@TP-SN38 and its synergistic PTT/chemo therapy for cancer. 3. RESULTS AND DISCUSSION Herein, the tertiary amine-based PDI acted as a NIR chromophore to synthesize the asymmetric silicon precursor PEG2000-PDI-silane. The silane in PEG2000-PDI-silane molecule was necessary for the growth of PDI within the silica framework through the co-hydrolysis with other silica precursors. The long PEG chain rendered PDI soluble in DMF/water during the formation of the hybrid silica nanoparticles. Furthermore, the PEG extended on the surface of HMPDINs endowed them with high dispersity and good biocompatibility. In asymmetric synthesis, PEG2000-PDICOOH was reacted with (3-aminopropyl)triethoxysilane (APTES) to yield the final PEG2000-PDI-silane (Figures S1-S3). The hard template of small mesoporous inorganic SiO2 nanoparticles (MSNs) was synthesized and served as the seed for the growth of PDI-hybridized mesoporous organosilica shell (MSNs@MPDINs) via the silicon hydroxyl cross-linking reaction of the PEG2000-PDI-silane, BTES, and TEOS. In the asformed core@shell structured MSNs@MPDINs, the Si-C bonds within the PDI-based shell exhibited stronger resistance against ammonia etching than the Si-O bonds within the MSN core; as such, the ammonia could selectively etch away the inner MSN core to leave the intact PDI-based shell.47-48 To the best of our knowledge, hollow-structured HMPDINs, where PDI was grown within the organosilica shell, have not been synthesized yet (Figure 1a). Currently available theranostic PDI with NIR excitation shows minimal NIR FL. However, in the present study, the PDI grown within the organosilica shell showed weak π–π stacking and reduced contact to the



complicated outer environment, which was beneficial to recover the strong NIR emission of PDI for in vitro and in vivo

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FL imaging

Figure 1. Design and synthesis of HMPDINs@TP-SN38 for multimodal imaging-guided photo-controlled synergistic photothermal/chemotherapy. (a) Synthesis route of HMPDINs and the mechanism for the recovered FL and enhanced PA properties. (b) Scheme of the polymerization within HMPDINs for photo-controlled drug release. that the polymers grow within the cavity rather than outside the and simultaneously amplify the NIR absorption for enhanced shell. PA imaging and phototherapy. In comparison with large-sized hollow MONs with a huge cavity, the small cavity of sub-50 nm The TEM and SEM images demonstrated the preparation nanoparticles may load lower amounts of hydrophobic drug process of HMPDINs@TP. Uniform small-sized MSNs were SN38. TP containing PNIPAM, PDMAEMA, and PEG were synthesized through hydrolysis of TEOS by using CTAC as the grafted within the cavity of HMPDINs to improve the drug pore-making agent and TEA as the catalyst (Figures 2b1 and loading efficiency.44 In the TP, PNIPAM is a widely reported 2b2). Following the co-hydrolysis of TEOS, BTES, and hydrogel formulation for drug loading in water. The positively PEG2000-PDI-silane, the core–shell structured MSNs@MPDINs charged tertiary amine of PDMAEMA in water could absorb were synthesized with high dispersity (Figures 2c1 and 2c2). the negatively charged SN38, thereby improving the drug Ammonia selectively etched away the MSNs core to yield loading efficacy.50 Moreover, the drug release could be hollow-structured HMPDINs owing to the higher stability of triggered by remotely controlling the shrinkage of the thermalthe MPDINs shell than the MSNs core (Figures 2d1 and 2d2). responsive polymers upon light irradiation (Figure 1b). The in Each PEG2000-PDI-silane contained PEG2000, which endowed situ growth of the TP into HMPDINs was introduced. In brief, the obtained HMPDINs with high dispersity/stability in HMPDINs, PNIPAM, PDMAEMA, and PEG were dispersed in aqueous solutions. The subsequent polymer growth in the anhydrous DMF, and AIBN acted as a radical initiator to cavity of HMPDINs maintained the spherical morphology and activate the polymerization at 65 ℃ (Figures S4 and S5).51 After mesoporous structure of HMPDINs (Figures 2e1 and 2e2). The 1 h of the reaction, the polymerization was terminated by adding size distribution of nanoparticles also confirmed this synthetic alcohol. Free polymers outside of HMPDINs were removed by process (Figure S7). The aqueous HMPDINs solution exhibited centrifugation. TP could be trapped within the HMPDINs to a green color and could be centrifuged to the bottom of the form HMPDIN@TP because the polymer size was larger than centrifuge tube, thereby validating that the PDI was firmly the pore size of the HMPDINs (Figure 2a). After more than 2 h “merged” with the HMONs (Figure 2e2 inset). After loading of polymerization, the polymers grew outside the HMPDINs SN38, the HMPDINs@TP still showed a spherical morphology and formed a polymer shell on the HMPDINs (Figure S6); as (Figure S8). The energy-dispersive X-ray spectroscopy (EDS) such, controlling the polymerization time is crucial to ensure and elemental mapping images showed that the HMPDINs mainly contained C, Si, S, and O (Figures 2f and 2g). The size


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distribution of the HMPDINs@TP was narrow and centered at 40 nm; hence, they are suitable for further in vitro and in vivo

studies (Figure 2h).

Figure 2. Synthesis and characterization of HMPDIN@TP. (a) Scheme of polymers grafted into HMPDINs by radical polymerization. (b–e) TEM and SEM images of (b1 and b2) MSNs core, (c1 and c2) MSNs@MPDINs, (d1 and d2) HMPDINs, and (e1 and e2) HMPDIN@TP. (f) Elemental mapping images of HMPDINs. (g) EDS spectrum of HMPDINs. (h) Size distribution of HMPDINs@TP calculated from figure 2e1. Organic self-assembled PDI nanoparticles (PDINs) in water stacking and intermolecular interaction of PDI in HMPDINs were also prepared and compared with HMPDINs to decreased; and second, the silica framework shielded the PDI demonstrate the enhanced phototheranostic properties achieved from FL quenching by water interaction. The PDI weight through the growth of PDI within the silica framework. The PDI percentage in HMPDINs was measured to be approximately molecule demonstrated aggregation-caused quenching (Figure 20% by testing the UV-Vis spectra of HMPDINs and PDI in S9). The aqueous solutions of HMPDINs and PDINs exhibited DMF, according to the standard curve of PDI in DMF (Figure a green color (Figure 3a). However, the FL of PDI in HMPDINs S10). The PA spectra of HMPDINs and PDINs were acquired showed stronger intensity than that in PDINs. The HMPDINs at the same mass concentration of PDI by signal collection showed a red shifted absorption pattern with the maximum ranging from 680 nm to 900 nm (Figure 3d). The HMPDINs value at 700 nm and 211.5-fold FL intensity enhancement and PDINs showed attenuation of PA amplitudes from 680 nm compared with that of the PDINs (Figures 3b and 3c). The FL to 900 nm, consistent with their absorption spectra. When recovery of PDI could be explained by the following: first, π–π energy transferred from the laser to the PA agents, the excited



photonics underwent FL (singlet-singlet relaxation S-path), phosphorescence (triplet–triplet relaxation T-path) emission,

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and PA emission (non-radiative relaxation), consistent with the law of

Figure 3. Characterization of HMPDINs. (a) PBS solutions of HMPDINs and PDINs in tubes and their FL imaging signal. (b) UVVis and FL emission spectra of HMPDINs and PDINs. (c) FL emission intensities of PDI in silica and water. ***p < 0.001. (d) PA spectra of HMPDINs and PDINs in PBS (pH = 7.4). (e) PA amplitudes at 700 nm as a function of the concentrations of PDINs and HMPDINs. (f) PA images of PDINs and HMPDINs at different concentrations. (g) Photothermal images and (h) temperature increase curves of the aqueous solution of HMPDINs (0.1 mg PDI/mL) upon laser irradiation with varied power densities. (i) Temperatures of the solutions of PDINs and HMPDINs upon repeated 600 s laser irradiation and 600 s cooling down. energy conservation.52 As the PDI chromophore is not calculated to be 45% (Figure S12). These results demonstrated phosphorescent, the PA mechanism indicates that thermal that the HMPDINs could be an excellent phototheranostic deactivation competes with FL emission in determining platform. acoustic generation upon excitation. Despite the enhanced FL Compared with traditional inorganic HMSNs, organic emission of PDI in HMPDINs, they still showed ~1.2 times HMPDINs exhibit advantages, such as decreased hemolytic higher PA intensity than the PDINs (Figures 3e and 3f). There effects against red blood cells (RBCs), and thus have high are two reasons for the results: one is the thermal resistance biosafety during longtime blood circulation (Figure S13). The effect of the silica framework surrounding the PDI molecules, hemolytic percentage of HMPDIN-treated RBCs was only 9.57 the other is that PDI has a higher absorption at 700 nm in silica ± 3.23% at 250 µg/mL, whereas almost all RBCs were than self-assembly in water, due to the weak aggregation of PDI hemolyzed after inoculation with the same concentration of (Figure S11). HMPDINs also showed excellent photothermal conventional HMSNs. Thus, the introduction of the PEG2000properties, as tested by different laser power densities (Figures PDI-silane during the synthesis of HMPDINs provides PEGs 3g and 3h) and faster temperature increase as compared with covered on the surface of HMPDINs, thereby effectively PDINs under 671 nm laser irradiation (Figure 3i). Also, the improving their biocompatibility. repeated measurements of the laser irradiation demonstrated The well-defined mesoporous structure, large surface area, excellent photostability of the PDI based nanoparticles. The and high pore volume of the HMPDINs were characterized by photothermal conversion efficiency of HMPDINs was


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typical N2 adsorption–desorption technique. The isotherms of HMPDINs and HMPDINs@TP exhibited Langmuir IV hysteresis. The BET surface area was changed from 326.86 to 275.17 m2/g (Figure 4a), demonstrating that the HMPDINs@TP still

Figure 4. (a) N2 adsorption–desorption isotherm and (b) corresponding pore-size distribution of HMPDINs and HMPDINs@TP. (c) TG curves of HMPDINs and HMPDINs@TP. (d) Transmittance curves of TP. (e) Loading contents of SN38 in HMPDINs and HMPDINs@TP. ***p < 0.001. (f) SN38 release curves from HMPDINs and from HMPDINs@TP with or without laser irradiation (arrows illustrate the irradiation time points). exhibited well-defined mesoporous structures. We further calculated the pore size and surface area of HMPDINs before and after polymerization with thermo-sensitive monomers to ensure that the HMPDINs@TP could still load drugs. The average pore size of HMPDINs@TP was 3.4 nm (Figure 4b), indicating that the thermal polymer in the cavity did not block the pores and facilitating the entry of the drugs. The successful grafting of the thermo-sensitive polymers into the cavity of HMPDINs was also confirmed by TG curves, and the polymer content was calculated to be 13.7% in HMPDINs@TP (Figure 4c). A different ratio of monomers in TP influenced the TP’s temperature response (Figure 4d). We finally selected the NIPAM: DMAEMA: PEG molar ratio of 5: 1: 1 for polymerization in HMPDINs because the deformation temperature starting from 37 °C. The HMPDINs@TP was confirmed to have higher SN38 loading efficacy than HMPDINs. As predicted, the SN38 loading content of HMPDINs@TP (13.45±3.23) was 2.47-fold than that of HMPDINs (5.43±2.34) because of charge attraction and polymer intertwining interaction between the polymer and SN38 (Figure 4e). The in vitro release profiles of SN38 from HMPDINs-SN38 and HMPDINs@TP-SN38 with or without 671 nm laser irritation were tested to demonstrate the concept of photo-controlled SN38 release from HMPDINs@TP-SN38 (Figure 4f). HMPDINs-SN38 showed fast SN38 release at 37 °C and up to 90% SN38 was released within 24 h, as



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Figure 5. (a) CLSM images of U87MG cells after co-incubation with HMPDINs@TP-SN38 for 0.5 and 2 h. Red fluorescence: PDI in HMPDINs@TP-SN38; blue fluorescence: DAPI, green florescence; SN38. Scale bar = 20 μm. (b) Cellular Uptake of HMPDINs@TP by flow cytometry assay (the test time point is 0.5 h (gray), 1 h (orange), 3 h (green), 6 h (red)). (c) Relative cell viabilities of U87MG cells after co-incubation with different formulations at varied SN38 concentrations. (d) Relative cell viabilities of U87MG cells after co-incubation with different formulations with or without 671 nm laser irradiation. **p < 0.01, ***p < 0.001. (e) CLSM images of U87MG cells subjected to different treatments according to the live and dead cell assays (Green fluorescence: calcein AM staining living cells and red fluorescence: PI staining dead cells. Scale bar = 60 μm). HMPDIFs@TP-SN38 in vitro. CLSM imaging was conducted SN38 had low solubility in water and could leak out from the to investigate the cellular uptake and photo-controlled drug silica shell. However, HMPDINs@TP-SN38 exhibited slower release properties of HMPDINs@TP-SN38. After incubation SN38 release. Only approximately 33% SN38 was released with HMPDINs@TP- SN38, cellular internalization and drug from HMPDINs@TP-SN38 without 671 nm laser irradiation release of HMPDINs@TP-SN38 were confirmed by the red within 24 h. Hence, the polymers in HMPDINs prevented SN38 signal from HMPDINs and the green signal from SN38. The leakage. After 671 nm laser irradiation, the SN38 release was U87MG cells exhibited an increased uptake of HMPDINs@TPaccelerated, indicating that the generated heat from the laserSN38 from 0.5 to 2 h, as indicated by red signals. After the 671 irradiated HMPDINs@TP caused the volume phase transition nm laser irritation, SN38 was released and arrived at the cell of the polymer to collapse in size and release SN38 molecules nucleus, as determined by the green signals isolated from the more quickly than that without laser treatment. These findings red signals (Figure 5a). The z-stack images of cancer cells also demonstrated the potential of HMPDINs@TP-SN38 for photoshowed the increased intensity of drug internalization and controlled synergistic PTT/chemotherapy. The U87MG cell release (Figure S14). line was chosen to study the therapeutic effect of

Figure 6. In vivo FL images (a) and PA images (c) of U87MG tumor-bearing mice at various time points post-injection of HMPDINs@TP and the corresponding quantification of FL intensities of the tumor site at 700–900 nm (b) and PA intensities (d) at


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760 nm of the tumor as a function of post-injection time (n = 5, mean ± SD) (arrow points at the tumor site). (e) Scheme of the isotope 64Cu labeling of HMPDINs@TP. (f) PET imaging for the whole-body tracking of 64Cu-labelled HMPDINs@TP (arrow points at the tumor site). (g, h) Tumor (g) and liver (h) uptakes of 64Cu-labeled HMPDINs@TP over time, as quantified by PET scans at 1, 4, 24, and 48 h postinjection (n = 5, mean ± SD). (i) Biodistribution of 64Cu-labeled HMPDINs@TP in the tumor and main organs of U87MG tumor-bearing mice at 48 h postinjection. results, the chemotherapy (free SN38) and photothermal The cellular uptake of HMPDINs@TP was demonstrated by therapy (HMPDINs@TP+laser) treated cells showed 58.52% flow cytometry analysis (Figure 5b). The HMPDINs@TP itself and 46.26% cell viability, respectively. However, the cell exhibited negligible cytotoxicity (Figure S15). viability after incubation with HMPDINs@TP-SN38 plus laser HMPDINs@TP-SN38, HMPDINs-SN38, and free SN38 were was only 24.61%, which demonstrated the synergy of incubated with U87MG cells to demonstrate that SN38 in combination of the thermal and chemo therapies in the HMPDINs@TP-SN38 could induce few side effects on the treatment of U87MG cancer cells. In the experiment of livecells. Moreover, the corresponding MTT assay result showed dead assay, the strong red fluorescence from the nucleus in the that U87MG cells treated with HMPDINs@TP-SN38 showed groups of DMEM, DMEM plus laser, free SN38, free SN38 only 30% apoptosis at SN38 concentration of 12 µM; such rate plus laser, HMPDINs@TP, HMPDINs@TP-SN38, and was achieved by HMPDINs-SN38 in a low concentration of treatment of HMPDINs@TP-SN38 plus laser irradiation 0.1875 µM (Figure 5c). The combination of HMPDINs@TPindicated remarkable cell death. However, the strong green SN38 therapy and laser irradiation exhibited the largest toxicity fluorescence and less red fluorescence in the control to U87MG cells than the control groups, as shown by the MTT HMPDINs@TP plus laser with U87MG cells indicated that the results (Figure 5d) and CLSM imaging (Figure 5e). Due to the cells kept an intact physiological state. photothermal effect of HMPDINs upon laser irradiation, the particles themselves caused toxicity with the laser. In the MTT

Figure 7. (a) Thermal images and (b) the corresponding tumor temperature change curves of U87MG tumor-bearing mice after treatment with PBS and HMPDINs@TP upon 671 nm laser irradiation. (c) Tumor growth curves, (d) survival curves, and (e) body



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weight of the mice after different treatments. ***p < 0.001. (f) H&E and (g) TUNEL-stained images of tumor sections subjected to different treatments. treatment groups, the tumor cells subjected to the synergistic We subsequently investigated the capability of the PTT/chemotherapy suffered from the largest extent of apoptosis HMPDINs@TP for in vivo multimodal imaging. The and necrosis (Figure 7f), as confirmed by the confocal imaging biocompatibility of HMPDINs@TP was first evaluated to of TUNEL-stained tumor tissues (Figure 7g). The normal H&Eguarantee its safety for in vivo biomedical application (Figure staining images of major organs indicated the low toxicity of all S16). Healthy balb/c mice were intravenously injected with the treatments to mice (Figure S17). These results validated the HMPDINs@TP. The group receiving PBS was set as the efficacy of the photo/chemo synergistic cancer treatment. control group. The blood indices in the HMPDINs@TP group Moreover, this theranostics provides on-demand synergistic showed no significant changes compared with those in the therapy with negligible side effects. control group. Basing on the FL recovery of PDI in HMPDINs, we evaluated the tumor accumulation of HMPDINs by near4. CONCLUSION infrared FL imaging. U87MG tumor-bearing mice were treated A novel PDI-organosilica nanoplatform (HMPDINs) was by intravenous injection of HMPDINs for FL imaging. The FL successfully constructed through in situ growth of PDI within images of the tumor showed increased intensities after the organosilica framework during the synthesis of HMONs. HMPDINs@TP injection and exhibited the highest FL signal at Silica acts as a solvent medium to effectively disperse PDI in 24 h post-injection (Figure 6a). The relative tumor uptake was the HMONs, thereby recovering the FL signal and amplifying determined by the region of interest (ROI) analysis at 4, 24 and the PA signal of PDI owing to the “silica shell shielding” effect. 48 h time points; the intensities in the tumor region were HMPDINs@TP after 64Cu labeling can achieve precise approximately 2.46-, 3.31-, and 2.46-fold higher than that at 1 FL/PA/PET tri-modal imaging-guided cancer therapy. A novel h post-injection, respectively (Figure 6b). The detailed mouse method of post-in-situ polymer growth method was tumor profile can be illustrated by the PA imaging after successfully developed in the cavity of the nanosized treatment of HMPDINs@TP (Figure 6c). The amplified PA HMPDINs for the loading and controlled release of SN38. This signals in the tumor region at different post-injection time study is the first report that introduces in situ polymerization points were consistent with the FL imaging results (Figure 6d). chemistry for grafting thermosensitive polymers into a hollow 64 The silica-based nanoparticle chelated isotope Cu through the cavity to increase the loading efficiency of hydrophobic drugs. surface-decorated thiol group for PET imaging (Figure 6e);53 as Furthermore, drug release can be controlled by laser stimulation such, the biodistribution of HMPDINs@TP could be quantified. because of the photothermal properties of the HMPDINs. The 64 The Cu-labeled HMPDINs@TP showed the most abundant synergistic therapeutic effect of PTT and SN38 is indicated by tumor accumulation and the highest tumor contrast at 24 h postthe suppression of cancer cell apoptosis and tumor growth. The injection (Figure 6f). Region of interest analysis revealed that marriage of organic and inorganic materials improves the 64 the tumor uptake values of Cu-labeled HMPDINs@TP were multifunction and efficacy of the theranostics and paves a new 2.71 ± 0.58, 4.25 ± 0.74, 5.70 ± 1.66 and 4.94 ± 1.78 %D/g at way for effective precise multi-imaging guided cancer therapy. 1, 4, 24 and 48 h, respectively (Figure 6g). The liver uptake of 64Cu-labeled HMPDINs@TP decreased at 48 h (Figure 6h). The ASSOCIATED CONTENT biodistribution in the tumor and main organs at 48 h was Supporting Information. measuerd by direct tissue sampling and gamma-counting Additional characterization data for HMPDINs@TP-SN38 and in (Figure 6i). The results indicated that the HMPDINs@TP could vitro and in vivo bioapplication of HMPDINs@TP-SN38, be cleared through the hepatobiliary system. including Figures S1−S17 (PDF). The multimodal imaging results motivated us to evaluate the synergistic therapeutic effect of PTT/chemotherapy of AUTHOR INFORMATION HMPDINs@TP-SN38 in vivo. After systemic administration of Corresponding Author HMPDINs@TP-SN38 and PBS into U87MG tumor-bearing mice, thermal imaging was performed under 671 nm laser *[email protected] irritation for 5 min. The HMPDINs@TP-SN38 treated mice *[email protected] showed considerable temperature increase to 55 °C, but the *[email protected] PBS-treated mice showed only 5 °C increment (Figures 7a and Author Contributions 7b). The tumor mice were randomly divided into six groups, ‖Z.Y. and W.F. contributed equally to this manuscript. which were treated with PBS, PBS plus laser, HMPDINs@TP, HMPDINs@TP plus laser, HMPDINs@TP-SN38, and ACKNOWLEDGMENT HMPDINs@TP-SN38 plus laser. The mice treated with PBS, PBS plus laser, and HMPDINS@TP showed fast growth. PTT This work was partially supported by the Intramural Research (HMPDINs@TP plus laser) or chemotherapy (HMPDINs@TPProgram (IRP) of the NIBIB, NIH, and the National Natural SN38) alone was unable to effectively inhibit tumor growth in Science Foundation of China (No. 81771827, 81471715, 21874024). We thank Dr. Vincent Schram in the NICHD mice. However, synergistic PTT/chemotherapy via Microscopy Imaging Core for technical support. HMPDINs@TP-SN38 plus laser resulted in tumor ablation without recurrence (Figure 7c). The therapeutic effect of the REFERENCES treatments corresponded to the survival of mice (Figure 7d). The synergistic therapy based on HMPDINs@TP-SN38+laser 1. Mizoshita, N.; Ikai, M.; Tani, T.; Inagaki, S. Hole-transporting led to prolonged mouse survival rate. All treated mice had Periodic Mesostructured Organosilica. J. Am. Chem. Soc. 2009, 131, 14225-14227. normal body weight, indicating that these treatments are 2. Lin, H.; Chen, Y.; Shi, J. 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