Light-Induced Hydrogel Based on Tumor-Targeting Mesoporous Silica

Jun 6, 2016 - School of Chemistry, Australian Centre for NanoMedicine, and ARC Centre ... Multiple-Responsive Mesoporous Silica Nanoparticles for High...
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A Light-Induced Hydrogel Based on Tumor-targeting Mesoporous Silica Nanoparticles as a Theranostic Platform for Sustained Cancer Treatment Xin Chen, Zhongning Liu, Stephen G. Parker, Xiaojin Zhang, J. Justin Gooding, Yanyan Ru, Yuhong Liu, and YongSheng Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02562 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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A Light-Induced Hydrogel Based on Tumor-targeting Mesoporous Silica Nanoparticles as a Theranostic Platform for Sustained Cancer Treatment Xin Chen*‡, Zhongning Liu‡, Stephen G. Parker, Xiaojin Zhang*, J. Justin Gooding, Yanyan Ru, Yuhong Liu, Yongsheng Zhou* Yanyan Ru, Prof. Yuhong Liu and Prof. Xin Chen [‡] [*] (Corresponding-Author) School of Chemical Engineering and Technology, Institute of Polymer Science in Chemical Engineering, Shanxi Key Laboratory of Energy Chemical Process Intensification, Xi’an Jiao Tong University, Xi’an, 710049, PR China E-mail: [email protected] Dr. Zhongning Liu [‡], Prof. Yongsheng Zhou [*] (Corresponding-Author) Department of Prosthodontics, Peking University School and Hospital of Stomatology; National Engineering Laboratory for Digital and Material Technology of Stomatology; Beijing Key Laboratory of Digital Stomatology, Beijing 100081, PR China E-mail: [email protected] Dr. Stephen G. Parker, Prof. J. Justin Gooding School of Chemistry, Australian Centre for NanoMedicine and ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney 2052, Australia Prof. Xiaojin Zhang [*] (Corresponding-Author) Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, PR China E-mail: [email protected] [‡] These authors contributed equally to this work [*] Corresponding-Authors

Abstract: Herein, we report a facile fabrication of a polymer (azobenzene and α-cyclodextrin-functionalized hyaluronic acid) and gold nano-bipyramids (AuNBs) conjugated mesoporous silica nanoparticles (MSNs) to be used as an injectable drug delivery system for sustained cancer treatment. Owing to the specific affinity between the hyaluronic acid (HA) on MSNs and the CD44 antigen over-expressed on tumor cells, the MSNs can selectively attach to tumor cells. The nanocomposite material then exploits thermo-responsive interactions between α-cyclodextrin and azobenzene, and the photothermal properties of gold nano-bipyramids, to in situ self-assemble into a hydrogel under near-infrared (NIR) radiation. Upon gelation, the drug (doxorubicin) loaded MSNs carriers were enclosed in the HA network of the hydrogel. Whilst further degradation of the HA in the hydrogel, due to the upregulation of hyaluronidase (HAase) around the tumor tissue, will result in the release of MSNs from the hydrogel which can then be taken by tumor cells and deliver their drug to the

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cell nuclei. This design is able to provide a microenvironment with rich anticancer drugs in, and around, the tumor tissue for time periods long enough to prevent the recrudescence of the disease. The extra efficacy that this strategy affords builds upon the capabilities of conventional therapies. Keywords: Light responsiveness, hydrogel-nanoparticles transformation, mesoporous silica nanocarriers, biodegradable platform, sustained cancer treatment 1. Introduction Owing to the remarkable physicochemical properties, nanocarriers with the capacity of stimulus-responsiveness has become an effective approach to achieve localized chemotherapy, offering potential solutions for many of the current challenges in cancer treatments1-5. Among these drug carriers explored, mesoporous (pore diameter 2–50 nm) silica nanoparticles (MSNs) have been considered to be an ideal candidate for controlled delivery of anti-cancer drug, due to their high surface area, size-adjustable pore size, well-defined pore structures, excellent biocompatibility and, especially, their readily functionalized surface 6. Various functionalized MSN vehicles triggered by a single stimulus including redox 7, 8, pH 9, 10, temperature 11, 12, enzymes 13, 14 and photo-irradiation15, 16 have been successfully used for different anti-cancer applications. More recently dual stimuli 4, 5 responsive MSNs have been developed in an attempt to achieve even better selectivity for cancer cells. To perform the targeted drug delivery, current MSN delivery vehicles are all designed to achieve endocytosis into tumor cells, followed by the immediate stimulus-triggered bursts of drug release which normally only takes hours to complete. This strategy results in a high drug concentration in tumor cells for a short period of time, which is likely to leave some residual tumor cells in deep tumor tissue and finally cause tumor recurrence even after the treatment17. The combination of MSNs drug carriers with responsive sol-gel transformed system to localize these MSNs only upon application of specific stimuli, may be a solution to long term delivery of a drug where needed. Such a strategy may further avoid tumor recurrence. Due to the three-dimensional cross-linked network and high water content in most hydrogels, the drug-loaded nanocarriers are unable to migrate away from the hydrogel, but the release of small molecule drugs from the hydrogel has no hindrance 18-20 . Since most of the current applications of hydrogels relate to gaining an understanding of how cells behave within the extra-cellular matrix, they are designed to gelate immediately after they are exposed to the external physical or chemical stimuli 21-23 so as to avoid free diffusion of the materials encapsulated within them. Therefore, in order to further their capabilities in anticancer treatments, the improvement of their mobility and associated tumor targeting abilities, without limiting their ability to contain the drug payload, is required. To realize such stimuli responsive gelation of MSNs for drug delivery we introduce azobenzene and α-cyclodextrin (α-CD) functionalized hyaluronic acid (HA) on the

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surface of MSNs to give a switchable nanoparticles-hydrogel system as an injectable tumor-targeting drug delivery system. The hyaluronic acid moiety is designed to provide the tumor targeting, due to its specific interaction with CD44 24-26, which is a major receptor for HA over-expressed in many solid tumor cells 27, resulting in the specific identification of tumor cells. The transient and responsive interactions between the α-CD and azobenzene governs the hydrogel self-assembly, allowing the MSNs drug carriers to perform mobility and fixity before and after stimulus introduction, respectively. Azobenzene can be switched from its α-CD repulsive cis-isomer to α-CD matched trans-isomer28, 29 using either visible light in the range of 435 nm or heat. Neither external application of visible light nor heat is practical for therapy, due to the shallow penetration of visible light in tissue30 and because heating needs to be localized. In contrast, near-infrared light (NIR) is a widely used extrinsic stimulus because of its living tissue penetration31, 32. NIR radiation can also be used for localized heating of plasmonic gold nanostructures in photothermal contrast agents 33, drug release 34 and therapy 35. Here, the photothermal properties of gold nano-bipyramids are used in combinations with NIR radiation to thermally transform the azobenzene into the trans isomer to trigger hydrogel formation at the desired time. To achieve all the desired properties in a single particle system we described above, herein, we report the fabrication of triple-layer core-shell nanocomposites (HA-MSNs-AuNBs) composed of gold nano-bipyramids (core), mesoporous silica (middle layer as a drug container), and azobenzene and α-cyclodextrin-functionalized hyaluronic acid (shell). These nanocomposites are able to move freely in blood circulation with nanoparticle performance while specifically accumulate around tumor tissue due to their targeting property, whereupon in situ formation of a hydrogel consist of these HA-MSNs-AuNBs in 2 min is initiated with localized NIR irradiation. This NIR based switching of the particles into gels enables the individual HA-MSNs-AuNBs to be transported by the blood circulatory system and then form hydrogels around tumor tissue after the introduction of the stimulus. In co-operation with the enzymatic degradation of the HA network in hydrogel, which could be triggered by the hyaluronidase (HAase) around tumor tissue 36-38 to perform the release of the payload and payload contained MSN vehicles, the switchable nanoparticles-hydrogel system is able to present great potential for targeting and sustained tumor treatments (Figure 1). 2. Results and Discussion The HA-MSNs-AuNBs were fabricated by a stepwise combination of gold nano-bipyramids (AuNBs) synthesis, mesoporous silica coating on gold nano-bipyramids (MSNs-AuNBs) and surface functionalization of the as-synthesized MSNs-AuNBs using azobenzene and α-cyclodextrin-functionalized hyaluronic acid. The preparation of AuNBs and MSNs-AuNBs were all according to reported protocols with some modifications 39, 40 (see the Supporting information). Briefly, the

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AuNBs were first synthesized through the seed-mediated growth process using as-prepared CTAB-stabilized spherical gold nanoparticles (AuNP, 2 nm) as seeds. Then the appropriate amount of cetyl-trimethyl ammonium bromide (CTAB) was added to form a bilayer around the AuNBs and served as an organic template for the formation of the mesoporous silica layer. For the preparation of the HA-MSNs-AuNBs, core-shell nanocomposites, the as-synthesized MSNs-AuNBs were first functionalized with amino groups using 3-aminopropyltriethoxysilane, following with the loading of doxorubicin (Dox, anti-cancer drug). Then the Dox-loaded MSNs-AuNBs, and commercially available cis-isomer of p-aminoazobenzene, were reacted with NHS/EDC activated α-cyclodextrin functionalized with hyaluronic acid to form a polymer cap restricting the Dox from leaving the MSN pores. The same chemical functionality also introduces the stimuli-responsive interactions (α-CD/azobenzene) and tumor targeting points (HA) on the MSNs-AuNBs. The resultant AuNBs, MSNs-AuNBs and HA-MSNs-AuNBs were first investigated by transmission electron microscopy (TEM). As can be seen from figure 2, the AuNBs present a low polydispersity with a general tip-to-tip distance of 45 nm and a base length of 22 nm (Figure 2a). After silica coating, the mesoporous silica shell has a homogeneous thickness of ∼20 nm with a single AuNB as the core (Figure 2b), offering an opportunity for MSN-AuNBs to be used as a general drug carrier. The introduction of azobenzene and α-cyclodextrin functionalized hyaluronic acid dramatically changed the size of MSN-AuNBs from around 60 to 150 nm (Figure 2c) and reveals that the thickness of the HA polymer shell is about 45 nm. The mesoporous structure of MSNs-AuNBs and the HA shell formation were further characterized by pore distribution measurements (Figure 2d). As can be seen from this figure, the MSNs-AuNBs present an obvious porous structure with an average diameter of about 2.8 nm. After HA covering, the average pore size decreased from 2.8 nm to near to 0 nm, which indicates the HA shell could effectively block pores in HA-MSNs-AuNBs to impede the premature release of encapsulated cargos. Furthermore, the sequential synthesis and functionalization steps for HA-MSNs-AuNBs fabrication were investigated by UV-Vis spectra, dynamic light scattering (DLS), nuclear magnetic resonance (NMR) and infrared (IR) spectra (Figure S1 - Figure S6), which present a strong localized surface plasmon resonance (LSPR) band for AuNBs at 760 nm in the NIR window and the characteristic absorption peak of azobenzene at 330 nm (UV-Vis spectra, Figure S1), uniform size distribution (DLS, Figure S2), as well as characteristic proton signals and covalent bonds vibrations of α-CD and HA (NMR and IR, Figure S3-S5 and Figure S6 ), revealing successful formation of the HA-MSNs-AuNBs triple-layer core-shell nanocomposites. Dox was chosen as a model drug to assess the drug loading and controlled release behavior of HA-MSN-AuNBs. The calculated Dox loading capacity and encapsulation efficiency of NH2-MSN-AuNBs and HA-MSN-AuNBs at different weight ratio of DOX/MSNs were listed in Table 1. The loading capacity of Dox

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increased quickly with the increase of DOX/MSNs ratio and could reach up to 32 wt% and 25.5 wt% for NH2-MSN-AuNBs and HA-MSN-AuNBs, respectively, at DOX/MSNs = 0.5/1. The encapsulation efficiency of NH2-MSN-AuNBs and HA-MSN-AuNBs showed peak values as 99.6 wt % and 92.6 wt %, at DOX/MSNs = 0.3/1. Further rise of the drug concentration leaded to decrease of encapsulation efficiency, duo to the excess drug over the loading content of NH2-MSN-AuNBs and HA-MSN-AuNBs. These results indicate that NH2-MSN-AuNBs and HA-MSN-AuNBs all possess high drug loading efficiency to DOX, and the polymer functionalization has no significant effect to the drug loading. To test the stimuli-responsiveness, the Dox-loaded HA-MSNs-AuNBs solution was treated with various photo-irradiations. As can be seen from Figure 3, NIR irradiation (wavelength 780 nm) on the solution over 2 min could dramatically increase the viscosity of the system to give a hydrogel, while UV light irradiation (wavelength 365 nm) for approximately 20 min could recover the low viscosity state. The transformation between hydrogel and nanoparticles solution could be repeatedly induced by NIR light and UV light irradiation, which was due to 1) the reversible change between trans and cis isomer of azobenzene in the HA-MSNs-AuNBs nanocomposites triggered by different stimuli (Figure S7), and 2) distinct interactions of α-CD units with trans-azobenzene (affinity) and cis-azobenzene (mismatch) in HA-MSNs-AuNBs nanocomposites 28, 41 . The morphology of the hydrogel formed from Dox-loaded HA-MSNs-AuNBs was also investigated by scanning electron microscope (SEM) and fluorescence microscope (Figure S8), which demonstrate a homogeneous distribution of HA-MSNs-AuNBs nanoparticles and Dox within the gel structure, indicating that the hydrogel network is held together by interactions between these nanoparticles and the HA-MSNs-AuNBs nanoparticles/hydrogel are effective for Dox loading. To mimic the environment of healthy and tumor tissue, the stability and controlled release profile of payload (Dox) from a HA-MSNs-AuNBs hydrogel was investigated under simulated physiological conditions (i.e., PBS buffer in the presence or absence of HAase). As can be seen from the figure 3, no morphology change of the HA-MSNs-AuNBs hydrogel could be observed after 2 days incubation in pure PBS buffer. However, obvious degradation happens with the addition of HAase. Complete disintegration of the hydrogel was observed within 7 days. This high stability in the simulated environment of healthy tissue (pure PBS) and slow enzymatic degradability in the simulated environment of tumor tissue (PBS with HAase) would cause specific drug transfer to tumor cells and a sustained drug release profile. The drug release profile was determined by monitoring the absorption of the Dox in the solution at 550 nm in UV-Vis spectra (Figure S9) as a function of time. It can be seen from figure S9 that HAase is the key requirement to trigger the drug release from HA-MSNs-AuNBs hydrogel. Quantification of the final release percentages of Dox from the hydrogel after 7 days incubation in PBS only is less than 3 %. The slight leakage could be attributed to the desorption of a small amount of physically adsorbed Dox. In contrast,

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in the presence of HAase, the release of the Dox dramatically increased over time, with the cumulative release of payload reaching up to 95% over 7 days. Corresponding UV-Vis spectra are displayed in figure S9b. The correlation between enzyme triggered degradation and Dox release was also investigated by monitoring the weight change of the HA-MSNs-AuNBs hydrogel against incubation time in PBS containing HAase, using 2.5 um filter to insulate the degraded and non-degraded hydrogel (Figure S10). As can be seen from the figure, the hydrogel showed a fast weight loss in the first 3 days, and then the biodegradation procedure went gentle, which reached about 80% as a final biodegradation percentage in 7 days. The weight loss of the hydrogel is proportional to the Dox release profile, indicating the drug release profile of our system is mainly determined by the enzyme responsive biodegradation of the HA. Having demonstrated the NIR irradiation and enzyme dual responsive behavior of the HA-MSNs-AuNBs nanoparticles/hydrogel, cellular experiments were performed in order to explore the potential therapeutic application of this nanoparticles/hydrogels switchable system. In this case, sterilized (254 nm UV irradiation for 30 min before cell experiments) Dox-loaded HA-MSNs-AuNBs nanoparticles were exposed to human squamous carcinoma cells (SCC, representative cancer cell) and human keratinocyte cells (HaCaT, representative normal control). To fully mimic the real tissue microenvironment, these two type cells were each cultured to form multi-cellular spheroids (MCS). MCS are effectively in vitro three dimensional tumor models, which are more efficiently mimicking the biological and pathological status of a tumor microenvironment 42. The two kinds of MCS composed of either the cancer cells or the noncancer cells were first centrifuged and cultured onto agarose-coated 24-well plates for 7 days to form MCS, then incubated with Dox-loaded HA-MSNs-AuNBs nanocomposites for 2 h following by 2 min exposure to 1.2 W/cm2 NIR irradiation on the two types of MCS. After a further 2 days incubation, the resulting MCS present a major difference in color with white HaCaT cell spheroids and red (Dox-loaded HA-MSNs-AuNBs hydrogel) SCC cell spheroids (Figure S11), indicating the specific targeting and selective hydrogel formation happened only on simulated tumor tissue (SCC cell spheroids). More details were given by scanning electron microscope (SEM), fluorescence microscope and optical microscope (Figure 4 and Figure S12). It can be seen from Figure 4, that the hydrogel homogeneously formed on the outside of SCC cell spheroids and completely covered the MCS with a thickness on the millimeter size scale. Strong red fluorescence (Dox) was observed both in the hydrogel network and SCC cells, which indicates that the enzymatic degradation of this hydrogel happens within 2 days incubation, leading to the delivery of some Dox and/or Dox loaded HA-MSNs-AuNBs nanoparticles from the hydrogel to SCC tumor cells. As a control group, HaCaT cell spheroids and a Dox-loaded HA-MSNs-AuNBs mixture was also collected and investigated after the same stepwise treatments as the SCC cells group (Figure S12). As can be seen from Figure S12, there is no visible hydrogel around the HaCaT cells spheroids, due to the

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repulsive interaction between the negatively charged HA-MSNs-AuNBs nanoparticles (COO- in HA) and the negatively charged cell membrane 43, 44. The few red fluorescence (Dox) appears in cells located at the edge of the HaCaT cell spheroids could be attributed to the premature release of free Dox, indicating the free Dox is much harder to penetrate the tissue than HA-MSNs-AuNBs nanoparticles. These results illustrated the highly selective, sustained and efficient drug delivery to tumor tissues performed by our HA-MSNs-AuNBs nanoparticles/hydrogel switchable system, which could be an ideal candidate for practical anti-cancer therapy. In vitro cytotoxicity of the HA-MSNs-AuNBs nanoparticles/hydrogel (100 µg/mL), free Dox (0.5 µg/mL) and Dox-loaded HA-MSNs-AuNBs nanoparticles/hydrogel (0.5 µg/mL Dox) were tested on both SCC cell spheroids and HaCaT cell spheroids using the DNA quantification after the similar stepwise treatments of 1) 2 h incubation, 2) 2 min NIR irradiation and 3) 12-96 h incubation. All the samples were sterilized by UV irradiation (254 nm) for 30 min before cell experiments. As shown in Figure 5 and Figure S13, The HA-MSNs-AuNBs nanoparticles/hydrogel showed no obvious cytotoxic effects on the two types of cells at a high concentration (100 µg/mL) up to 96 h incubation. These results demonstrate that the HA-MSNs-AuNBs nanoparticles/hydrogel is well tolerated by a bio-system, which is in good agreement with previous works on MSN and HA 45.The free Dox showed some cytotoxicity in the first 48 h to both normal and tumor cells with the decrease of cell viability from around 90% (12 h incubation) to about 60% (48 h incubation). However, the cytotoxicity gradually weakens after 48 h incubation due to the cell resistance to chemotherapeutic drugs and the low tissue penetration ability of free drugs 46, 47, resulting in the subsequent rise of cell viability to both normal and tumor cells. A similar phenomenon was also observed in the free Dox group with NIR irradiation, which indicates that the NIR irradiation does not affect the function of free Dox. The Dox-loaded HA-MSNs-AuNBs nanoparticles displayed disparate cytotoxicity with and without 2 min NIR irradiation to the simulated tumor tissue (SCC cell spheroids). The group with no NIR irradiation presents a very strong cytotoxicity at the beginning with only 35% cell survival after 48 h incubation, followed by a distinct cell number recovery to 50% at 96 hours. This recovery in cell viability is attributed to the effective drug delivery and fast enzyme triggered drug release performed by HA-MSNs-AuNBs nanoparticles, which could bring a high concentration of anti-cancer drugs to tumor cells in the shallow tissue to rapidly kill a large number of tumor cells, but still leave few residual tumor cells in the deep tissue, resulting in the final recurrence 17. As expected, a stepwise decrease of the cell viability against the time with no rebound was observed in the group of Dox loaded HA-MSNs-AuNBs nanoparticles with NIR irradiation. Compared with the above experiments without NIR irradiation or with only free Dox, the Dox-loaded HA-MSNs-AuNBs nanoparticles after NIR irradiation were more efficient in controlling the growth of simulated tumor tissue in long term, indicating the potential that the HA-MSNs-AuNBs nanoparticle-hydrogel switchable system offered with an effective

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and sustained intracellular DOX delivery property that could be potentially used to avoid the major challenge for cancer therapy, tumor recurrence. The Dox-loaded HA-MSNs-AuNBs nanoparticles with NIR irradiation were also examined in HaCaT cell spheroids as a control group to investigate the selectivity (Figure S13). As can be seen from this figure, very low toxicity with over 80% cell viability was observed after 96 h incubation with the same amount of Dox as the experiment to SCC cell spheroids, indicating that the Dox-loaded HA-MSNs-AuNBs nanoparticle-hydrogel switchable system exhibits a high selectivity towards tumor cells. 3. Conclusion In summary, we have demonstrated, as a proof of the concept, that the use of azobenzene and α-cyclodextrin-functionalized HA as both an enzyme degradable gate cap, targeting agent and responsive cross-linker, in combination with AuNBs as a photothermal converter and MSNs as drug carriers, provides a suitable method for the design of an NIR and enzyme dual responsive nanoparticle-hydrogel switchable system. This switchable system benefits for long term cancer therapy due to the efficient, sustained and selective drug delivery ability which is well presented in this work. The transformation from nanoparticles to the long term drug release platform (hydrogel) is induced by generating an interaction between α-cyclodextrin and trans-azobenzene in HA-MSNs-AuNBs nanoparticles using 2 min NIR irradiation, while the tumor targeting is achieved by the HA modification, resulting in the selective accumulation of these nanoparticles around a tumor tissue prior to NIR irradiation. The final cargo release, was triggered by specific enzymes presenting in the tumor microenvironment. In vitro studies have shown the feasibility of using these injectable hydrogels as a sustained and targeted drug delivery system to remarkably enhance the long term killing efficiency and finally avoid tumor recurrence. The stimuli responsive nature of this HA-MSNs-AuNBs nanoparticles-hydrogel switchable system is designed to not only integrate the advantages of chemotherapy and photo-thermal therapy, but take advantage of the unique physiological environment in/around solid tumors, making it an excellent candidate for therapeutic applications. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.*******. (1) Detailed experimental procedures, including synthesis of the AuNBs, MSN-AuNBs, α-CD-HA and HA-MSN-AuNBs; hydrogel formation and disassembly; drug release and cell experiments. (2) UV-Vis spectra and DLS results of AuNBs, MSN-AuNBs and HA-MSN-AuNBs (Figure S1 and S2). (3) Characterization of α-CD-HA synthesis by 1H NMR spectrums (Figure S3-S5). (4) The FTIR of MSN-AuNBs and HA-MSN-AuNBs (Figure S6). (5) Light-responsiveness of

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HA-MSN-AuNBs (Figure S7). (6) microscopy images of HA-MSNs-AuNBs hydrogel (Figure S8). (7) HAase responsive degradation of HA-MSNs-AuNBs hydrogel (Figure S9 and S10). (8) Biological experiment results (Figure S11-13). (9) DOX loading capacity and encapsulation efficiency of NH2-MSN-AuNBs and HA-MSN-AuNBs (Table S1) Acknowledgements This work was supported by the "Young Talent Support Plan" of Xi'an Jiaotong University (XC), the National Natural Science Foundation of China (Nos. 81400498: ZL), the "Program for New Century Excellent Talents (NCET) in University" from Ministry of Education of China (NCET-11-0026: YZ), the Construction Program for National Key Clinical Specialty from National Health and Family Planning Commission of China to Department of Prosthodontics of PKUSS (YZ). We also acknowledge the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (CE140100036, JJG, SGP) and an ARC Australian Laureate Fellowship (FL150100060, JJG)

Figure 1. A schematic representation of a) Dox-loaded, polymer (azobenzene and α-cyclodextrin functionalized hyaluronic acid) and gold nano-bipyramids conjugated

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mesoporous silica nanoparticles, corresponding stimuli-responsive hydrogel formation and degradation, b) as well as the targeted drug delivery to tumor tissue based on this nanoparticles/hydrogel switchable system. NIR at 780 nm converts cis-azobenzene to the trans form, leading to the formation of MSNs based hydrogel due to the non-covalent interactions between α-cyclodextrin and trans-azobenzene on MSNs. The hyaluronic acid serves as a targeting agent and enzyme degradable gate cap performing the selective tumor accumulation (before hydrogel formation) and responsive drug delivery (after hydrogel formation).

Figure 2. Transmission electron microscopy images of a) gold nano-bipyramids, b) mesoporous silica-coated gold nano-bipyramids (MSNs-AuNBs), and c) polymer (azobenzene and α-cyclodextrin functionalized hyaluronic acid) and gold nano-bipyramids conjugated mesoporous silica nanoparticles (HA-MSNs-AuNBs). d) Pore size distribution of MSNs-AuNBs (black curve) and HA-MSNs-AuNBs (Red curve).

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Figure 3. Photographs of Dox-loaded HA-MSNs-AuNBs nanoparticles solution (a), HA-MSNs-AuNBs nanoparticles based hydrogel with Dox loading before (b) and after 2 h incubation in pure PBS (c), as well as the corresponding hydrogel after 2 days (d) and 7 days (e) incubation in PBS with HAase, presenting the stimuli-responsive transformation between the nanoparticles solution and hydrogel, as well as the stability and enzymatic degradation of the corresponding hydrogel.

Figure 4.Scanning electron microscopy images (a-c), optical microscope images (d-f)

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and fluorescence microscopy images (g-i) of the complex composed by Dox-loaded HA-MSNs-AuNBs nanocomposites and human squamous carcinoma cell spheroids (simulated tumor tissue) after stepwise treatments of 2 h co-culture, 2 min NIR irradiation and 2 days further co-culture. The left column displays overall images, middle column displays magnifying images of the hydrogel section, and right column displays magnifying images of the cell section.

Figure 5.Time-dependent cytotoxicity of HA-MSNs-AuNBs nanoparticles, Dox with and without NIR irradiation, and Dox-loaded HA-MSNs-AuNBs nanoparticles with and without NIR irradiation on the viability of human squamous carcinoma cell spheroids (simulated tumor tissue) with equal concentrations of free Dox (0.5 µg/mL). References (1) Park, J. H.; von Maltzahn, G.; Xu, M. J.; Fogal, V.; Kotamraju, V. R.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Cooperative Nanomaterial System to Sensitize, Target, and Treat tumors, Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 981-986. (2) Sailor, M. J.; Park, J. H. Hybrid Nanoparticles for Detection and Treatment of Cancer, Adv. Mater. 2012, 24, 3779-3802. (3) von Maltzahn, G.; Park, J. H.; Lin, K. Y.; Singh, N.; Schwoppe, C.; Mesters, R.; Berdel, W. E.; Ruoslahti, E.; Sailor, M. J.; Bhatia, S. N. Nanoparticles that Communicate in vivo to Amplify Tumour Targeting, Nat

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Mater. 2011, 10, 545-552. (4) Chen, X.; Soeriyadi, A. H.; Lu, X.; Sagnella, S. M.; Kavallaris, M.; Gooding, J. J. Dual Bioresponsive Mesoporous Silica Nanocarrier as an "AND" Logic Gate for Targeted Drug Delivery Cancer Cells, Adv. Funct. Mater. 2014, 24, 6999-7006. (5) Chen, X.; Cheng, X. Y.; Soeriyadi, A. H.; Sagnella, S. M.; Lu, X.; Scott, J. A.; Lowe, S. B.; Kavallaris, M.; Gooding, J. J. Stimuli-Responsive Functionalized Mesoporous Silica Nanoparticles for Drug Release in Response to Various Biological Stimuli, Biomater. Sci-Uk, 2014, 2, 121-130. (6) Zhao, Y. N.; Vivero-Escoto, J. L.; Slowing, I. I.; Trewyn, B. C.; Lin, V. S. Y. Capped Mesoporous Silica Nanoparticles as Stimuli-Responsive Controlled Release Systems for Intracellular Drug/gene Delivery, Expert Opin. Drug Del. 2010, 7, 1013-1029. (7) Liu, R.; Zhao, X.; Wu, T.; Feng, P. Y.; Tunable Redox-Responsive Hybrid Nanogated Ensembles, J. Am. Chem. Soc. 2008, 130, 14418-14419. (8) Luo, Z.; Cai, K. Y.; Hu, Y.; Zhao, L.; Liu, P.; Duan, L.; Yang, W. H. Mesoporous Silica Nanoparticles End-Capped with Collagen: Redox-Responsive Nanoreservoirs for Targeted Drug Delivery, Angew. Chem., Int. Ed. 2011, 50, 640-643. (9) Park, C.; Oh, K.; Lee, S. C.; Kim, C. Controlled Release of Guest Molecules from Mesoporous Silica Particles Based on a pH-Responsive Polypseudorotaxane Motif, Angew. Chem., Int. Ed. 2007, 46, 1455-1457. (10) Angelos, S.; Yang, Y. W.; Patel, K.; Stoddart, J. F.; Zink, J. I. pH-Responsive Supramolecular Nanovalves Based on Cucurbit[6]uril Pseudorotaxanes, Angew. Chem., Int. Ed. 2008, 47, 2222-2226. (11) Aznar, E.; Mondragon, L.; Ros-Lis, J. V.; Sancenon, F.; Marcos, M. D.; Martinez-Manez, R.; Soto, J.; Perez-Paya, E.; Amoros, P. Finely Tuned Temperature-Controlled Cargo Release Using Paraffin-Capped Mesoporous Silica Nanoparticles, Angew. Chem., Int. Ed. 2011, 50, 11172-11175. (12) Schlossbauer, A.; Warncke, S.; Gramlich, P. M. E.; Kecht, J.; Manetto, A.; Carell, T.; Bein, T. A Programmable DNA-Based Molecular Valve for Colloidal Mesoporous Silica, Angew. Chem., Int. Ed. 2010, 49, 4734-4737. (13) Coll, C.; Mondragon, L.; Martinez-Manez, R.; Sancenon, F.; Marcos, M. D.; Soto, J.; Amoros, P.; Perez-Paya, E. Enzyme-Mediated Controlled Release Systems by Anchoring Peptide Sequences on Mesoporous Silica Supports, Angew. Chem., Int. Ed. 2011, 50, 2138-2140. (14) Bernardos, A.; Aznar, E.; Marcos, M. D.; Martinez-Manez, R.; Sancenon, F.; Soto, J.; Barat, J.M.; Amoros, P. Enzyme-Responsive Controlled Release Using Mesoporous Silica Supports Capped with Lactose, Angew. Chem., Int. Ed. 2009, 48, 5884-5887. (15) Mal, N. K.; Fujiwara, M.; Tanaka, Y. Photocontrolled Reversible Release of Guest Molecules from Coumarin-Modified Mesoporous Silica, Nature, 421 (2003) 350-353. (16) Lin, Q. N.; Huang, Q.; Li, C. Y.; Bao, C. Y.; Liu, Z. Z.; Li, F. Y.; Zhu, L. Y. Anticancer Drug Release from a Mesoporous Silica Based Nanophotocage Regulated by Either a One- or Two-Photon Process, J. Am. Chem. Soc. 2010, 132, 10645-10647. (17) Wolinsky, J. B.; Colson, Y. L.; Grinstaff, M. W. Local Drug Delivery Strategies for Cancer Treatment: Gels, Nanoparticles, Polymeric Films, Rods, and Wafers, J. Controlled Release 2012, 159, 14-26. (18) Baumann, M. D.; Kang, C. E.; Tator, C. H.; Shoichet, M. S. Intrathecal Delivery of a Polymeric Nanocomposite Hydrogel After Spinal Cord Injury, Biomaterials, 2010, 31, 7631-7639. (19) Baumann, M. D.; Kang, C. E.; Stanwick, J. C.; Wang, Y. F.; Kim, H.; Lapitsky, Y.; Shoichet, M. S. An Injectable Drug Delivery Platform for Sustained Combination Therapy, J. Controlled Release 2009, 138, 205-213.

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(20) Jin, N. X.; Morin, E. A.; Henn, D. M.; Cao, Y.; Woodcock, J. W.; Tang, S. C.; He, W.; Zhao, B. Agarose Hydrogels Embedded with pH-Responsive Diblock Copolymer Micelles for Triggered Release of Substances, Biomacromolecules, 2013, 14, 2713-2723. (21) Li, Z.; Wang, F.; Roy, S.; Sen, C. K.; Guan, J. J. Injectable, Highly Flexible, and Thermosensitive Hydrogels Capable of Delivering Superoxide Dismutase, Biomacromolecules, 2009, 10, 3306-3316. (22) Nguyen, K. T.; West, J. L. Photopolymerizable Hydrogels for Tissue Engineering Applications, Biomaterials, 2002, 23, 4307-4314. (23) Kretlow, J. D.; Klouda, L.; Mikos, A. G. Injectable Matrices and Scaffolds for Drug Delivery in Tissue Engineering, Adv. Drug Delivery Rev. 2007, 59, 263-273. (24) Toole, B. P. Hyaluronan: From Extracellular Glue to Pericellular Cue, Nat. Rev. Cancer. 2004, 4, 528-539. (25) Lee, Y. H.; Lee, H.; Kim, Y. B.; Kim, J. Y.; Hyeon, T.; Park, H.; Messersmith, P. B.; Park, T. G. Bioinspired Surface Immobilization of Hyaluronic Acid on Monodisperse Magnetite Nanocrystals for Targeted Cancer Imaging, Adv. Mater. 2008, 20, 4154-4157. (26) Toole, B. P. Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities, Clin. Cancer. Res. 2009, 15, 7462-7468. (27) Aruffo, A.; Stamenkovic, I.; Melnick, M.; Underhill, C. B.; Seed, B. Cd44 Is the Principal Cell-Surface Receptor for Hyaluronate, Cell, 1990, 61, 1303-1313. (28) Chen, X.; Hong, L.; You, X.; Wang, Y. L.; Zou, G.; Su, W.; Zhang, Q. J. Photo-Controlled Molecular Recognition of Alpha-cyclodextrin with Azobenzene Containing Polydiacetylene Vesicles, Chem. Commun. 2009, 11, 1356-1358. (29) Chen, X.; Jiang, H.; Wang, Y. L.; Zou, G.; Zhang, Q. J. Beta-Cyclodextrin-Induced Fluorescence Enhancement of a Thermal-Responsive Azobenzene Modified Polydiacetylene Vesicles for a Temperature Sensor, Mater. Chem. Phys. 2010, 124, 36-40. (30) Yang, X. J.; Liu, X.; Liu, Z.; Pu, F.; Ren, J. S.; Qu, X. G. Near-Infrared Light-Triggered, Targeted Drug Delivery to Cancer Cells by Aptamer Gated Nanovehicles, Adv. Mater. 2012, 24, 2890-2895. (31) Klohs, J.; Wunder, A.; Licha, K. Near-Infrared Fluorescent Probes for Imaging Vascular Pathophysiology, Basic. Res. Cardiol. 2008, 103, 144-151. (32) Zhang, J.; Preda, D. V.; Vasquez, K. O.; Morin, J.; Delaney, J.; Bao, B. N.; Percival, M. D.; Xu, D. G.; McKay, D.; Klimas, M.; Bednar, B.; Sur, C.; Gao, D. Z.; Madden, K.; Yared, W.; Rajopadhye, M.; Peterson, J. D. A Fluorogenic Near-Infrared Imaging Agent for Quantifying Plasma and Local Tissue Renin Activity in vivo and ex vivo, Am. J. Physiol-Renal. 2012, 303, F593-F603. (33) Liu, H. Y.; Chen, D.; Li, L. L.; Liu, T. L.; Tan, L. F.; Wu, X. L.; Tang, F. Q. Multifunctional Gold Nanoshells on Silica Nanorattles: A Platform for the Combination of Photothermal Therapy and Chemotherapy with Low Systemic Toxicity, Angew. Chem., Int. Ed. 2011, 50, 891-895. (34) Yavuz, M. S., Cheng, Y. Y.; Chen, J. Y.; Cobley, C. M.; Zhang, Q.; Rycenga, M.; Xie, J. W.; Kim, C.; Song, K. H.; Schwartz, A. G.; Wang, L. H. V.; Xia, Y. N. Gold Nanocages Covered by Smart Polymers for Controlled Release with Near-Infrared Light, Nat. Mater. 2009, 8, 935-939. (35) Lee, S. M.; Park, H.; Choi, J. W.; Park, Y. N.; Yun, C. O.; Yoo, K. H. Multifunctional Nanoparticles for Targeted Chemophotothermal Treatment of Cancer Cells, Angew. Chem., Int. Ed. 2011, 50, 7581-7586. (36) Balazs, E. A.; Voneuler, J. The Hyaluronidase Content of Necrotic Tumor and Testis Tissue, Cancer Res. 1952, 12, 326-329. (37) Lapcik, L.; Lapcik, L.; De Smedt, S.; Demeester, J.; Chabrecek, P. Hyaluronan: Preparation, structure, properties, and applications, Chem. Rev. 1998, 98, 2663-2684.

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Table Of Contents

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Figure 1. A schematic representation of a) Dox-loaded, polymer (azobenzene and α-cyclodextrin functionalized hyaluronic acid) and gold nano-bipyramids conjugated mesoporous silica nanoparticles, corresponding stimuli-responsive hydrogel formation and degradation, b) as well as the targeted drug delivery to tumor tissue based on this nanoparticles/hydrogel switchable system. NIR at 780 nm converts cis-azobenzene to the trans form, leading to the formation of MSNs based hydrogel due to the non-covalent interactions between α-cyclodextrin and trans-azobenzene on MSNs. The hyaluronic acid serves as a targeting agent and enzyme degradable gate cap performing the selective tumor accumulation (before hydrogel formation) and responsive drug delivery (after hydrogel formation). 185x171mm (300 x 300 DPI)

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Figure 2. Transmission electron microscopy images of a) gold nano-bipyramids, b) mesoporous silica-coated gold nano-bipyramids (MSNs-AuNBs), and c) polymer (azobenzene and α-cyclodextrin functionalized hyaluronic acid) and gold nano-bipyramids conjugated mesoporous silica nanoparticles (HA-MSNs-AuNBs). d) Pore size distribution of MSNs-AuNBs (black curve) and HA-MSNs-AuNBs (Red curve). 100x95mm (300 x 300 DPI)

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Figure 3. Photographs of Dox-loaded HA-MSNs-AuNBs nanoparticles solution (a), HA-MSNs-AuNBs nanoparticles based hydrogel with Dox loading before (b) and after 2 h incubation in pure PBS (c), as well as the corresponding hydrogel after 2 days (d) and 7 days (e) incubation in PBS with HAase, presenting the stimuli-responsive transformation between the nanoparticles solution and hydrogel, as well as the stability and enzymatic degradation of the corresponding hydrogel. 77x46mm (300 x 300 DPI)

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Figure 4.Scanning electron microscopy images (a-c), optical microscope images (d-f) and fluorescence microscopy images (g-i) of the complex composed by Dox-loaded HA-MSNs-AuNBs nanocomposites and human squamous carcinoma cell spheroids (simulated tumor tissue) after stepwise treatments of 2 h coculture, 2 min NIR irradiation and 2 days further co-culture. The left column displays overall images, middle column displays magnifying images of the hydrogel section, and right column displays magnifying images of the cell section.

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Figure 5.Time-dependent cytotoxicity of HA-MSNs-AuNBs nanoparticles, Dox with and without NIR irradiation, and Dox-loaded HA-MSNs-AuNBs nanoparticles with and without NIR irradiation on the viability of human squamous carcinoma cell spheroids (simulated tumor tissue) with equal concentrations of free Dox (0.5 µg/mL). 41x34mm (300 x 300 DPI)

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