Rattle-Structured Rough Nanocapsules with in-Situ-Formed Gold

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Rattle-Structured Rough Nanocapsules with In-Situ Formed Gold Nanorod Cores for Complementary Gene/Chemo/Photothermal Therapy Xinyan Chen, Qing Zhang, Jinliang Li, Ming Yang, Nana Zhao, and Fu-Jian Xu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01440 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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Rattle-Structured Rough Nanocapsules with In-Situ Formed Gold Nanorod Cores for Complementary Gene/Chemo/Photothermal Therapy Xinyan Chen, a,b,c,† Qing Zhang, a,b,c,† Jinliang Li,d Ming Yang,d Nana Zhao, a,b,c,* and Fu-Jian Xu a,b,c,* a

Beijing Advanced Innovation Center for Soft Matter Science and Engineering, State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, China b Key Laboratory of Carbon Fiber and Functional Polymers (Beijing University of Chemical Technology), Ministry of Education, Beijing, 100029, China c Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing, 100029, China d Shandong Provincial Key Laboratory of Radiation, Oncology, Cancer Research Center, Shandong Cancer Hospital affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, Shandong 250117, China * To whom correspondence should be addressed Email: [email protected] (F. J. Xu), [email protected] (N. Zhao) †Both authors contributed equally to this work.

Table of contents

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ABSTRACT The morphology of nanoparticles influences their cellular uptake process while rough surface-enhanced affinity renders rough nanoparticles desirable in related biomedical applications. In this work, rattle-structured rough nanocapsules (Au@HSN-PGEA, AHP) composed of in-situ formed gold nanorod (Au NR) cores and polycationic mesoporous silica shells were constructed for trimodal complementary cancer therapy. Taking advantage of surface roughness, near-infrared (NIR)-responsiveness and controlled release manner, AHPs were expected to realize the co-delivery of sorafenib (SF, a hydrophobic anti-proliferative and antiangiogenic drug) and antioncogene p53 for malignant hepatocellular carcinoma (HCC) treatment. The rough surface feature of AHP was investigated for cellular uptake and the subsequent gene transfection. The feasibility of photothermal Au NR cores for NIR-triggered SF release was also testified. Notably, synergistic effects based on photothemal therapy (PTT)-enhanced chemotherapy were achieved. In addition, the good in vivo performance of the proposed multifunctional nanoparticles with rough surfaces was also demonstrated. The current work extends the biomedical applications of the intriguing rough nanoparticles and provides a facile strategy to construct flexible platforms for complementary gene/chemo/photothermal therapy. KEYWORDS: rattle; rough nanocapsule; gold nanorod; drug; complementary therapy.

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Mesoporous silica nanoparticles exhibit promising prospects in diverse biomedical areas with the virtues of high specific surface area, controlled morphology, favorable biocompatibility and facile surface functionalization.1,2 Particularly, they have been intensively explored in the construction of drug/gene delivery systems.3,4 The morphology of nanoparticles is demonstrated to influence cellular uptake process and intracellular delivery efficiency.5,6 Rough surface-enhanced adhesion to facilitate cellular internalization was observed with virus- and rambutan-like nanoparticles.7-9 Rambutan-like silica nanoparticles with spiky surfaces were also proved to possess enhanced plasmid DNA binding capability and better transfection performances compared with analogues with other surface topographies.9 These results suggest the great potential of intriguing rough nanoparticles in biomedical applications. However, the previous work only addressed in vitro testing of the rough nanoparticles. Moreover, the realized functionality seems limited. Therefore, from the perspective of practical applications, it would be desirable to design multifunctional nanoparticles with rough surfaces and evaluate their in vivo performance. Gold nanorods (Au NRs) with favorable optical absorption in the near-infrared (NIR) region are attractive candidates for widespread biomedical applications such as photothermal therapy (PTT) and photoacoustic (PA) imaging.10-12 The emerging nanoplatforms integrating silica nanoparticles and Au NRs are impressive in realizing multifunctional theranostics.13-15 Among these reports, diverse synthetic strategies were

utilized

to

fabricate

core-shell,

yolk-shell,

and

Janus

Au-SiO2

hetero-nanoparticles. Nevertheless, the synthesis of Au-SiO2 hetero-nanoparticles with the feature of rough surfaces is still lacking. Furthermore, hetero-nanoparticles from hollow silica nanoparticles (HSN) as nanocapsules are superior for drug delivery over the solid counterparts since the hollow cavity could improve the loading capacity. 3


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Meanwhile, the hollow cavity of HSN also could serve as a chemical reactor to encapsulate movable cores.16 In this regard, “ship-in-a-bottle” strategy could be readily exploited to grow and entrap Au NRs within the interior cavity of HSN. Most of the encapsulated cores in the nanoreactors utilizing this strategy are spherical and the morphology control over the post-synthesized cores is rarely achieved.16-18 Although Au NR cores were formed in the silica capsules of micron size range,19,20 the generation of Au NRs in nanocapsules still remains challenging. If Au NRs could be encapsulated in rough HSN, the resultant nanorattles could maintain both the exterior morphology of HSN and characteristics of the Au NR cores.21 The interior cavity could also provide enough space for the drug loading while the outer shell could be engineered through facile surface functionalization. Sorafenib (SF), a hydrophobic anti-proliferative and antiangiogenic drug, has been approved by U.S. Food and Drug Administration (FDA) for malignant hepatocellular carcinoma (HCC) treatment.22-25 The poor solubility of SF greatly hindered its therapeutic efficacy. In order to increase the bioavailability and avoid adverse effects, the development of advanced SF delivery system is pressing. A variety of nanocarriers have been utilized to load SF through hydrophobic interaction, physical adsorption, hydrogen bonding, and π-π interaction.23-25 Although the co-delivery of multiple drugs as well as imaging-guided therapy have been realized by these delivery systems,24-28 there is hardly any design for nanocarriers with rough surfaces for enhanced SF delivery to HCC cells. Recently, light-responsive drug delivery system based on mesoporous silica nanoparticles for controllable drug release is attractive to realize highly specific therapeutic effectiveness, which could be applied in the precisely controlled SF release.29,30 Moreover, the nanoplatforms with imaging-guided multimodal synergistic cancer therapy is worth for further exploration to conquer the 4


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complexity and heterogeneity of tumors.31 As the complementary treatments of chemotherapy, gene therapy (GT) displays great potential for cancer treatment through effective delivery of genes to regulate cellular processes.32 In addition, PTT provides alternative modality of NIR light-based phototherapy, which is practical and less toxic.33,34 It could be ideal to produce rough hetero-nanoparticles of Au NR cores and silica capsules for trimodal gene/chemo/photothermal therapy. Herein, we constructed rattle-structured rough nanocapsules (Au@HSN-PGEA, AHP) composed of in-situ formed Au NR cores and polycationic mesoporous silica shells for trimodal complementary cancer therapy (Figure 1). The design concept is to combine the advantages of surface roughness, NIR-responsiveness and controlled release manner for HCC treatment. HSN with rough surfaces were firstly synthesized as nanoreactors. Then Au NR cores were in-situ synthesized inside the cavity of HSN utilizing “ship-in-a-bottle” approach, resulting in rough Au@HSN nanorattles. The outer shell of the nanorattles were functionalized with a superior polycation, CD-PGEA (two armed ethanolamine-functionalized poly(glycidyl methacrylate) with one β-cyclodextrin core)13,35,36 to carry genes for GT. The interior space around Au NR cores was reserved for SF loading. The rough surface feature of AHP is expected to facilitate cellular uptake. In this regard, the irradiation of NIR light could not only induce PTT, but also trigger the drug release by opening the polycation cloak.13,36,37 The feasibility of multimodal gene/chemo/photothermal complementary HCC treatment with PA imaging was investigated in details in vitro and in vivo.

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RESULTS AND DISCUSSION Synthesis and Characterization of Rattle-Structured Nanohybrids. The synthetic procedures of rattle-structured rough nanocapsules (Au@HSN-PGEA, AHP) composed of Au NR core, rough silica shell and polycationic cloak are illustrated in Figure

1.

First,

rough

HSNs were

prepared through

the formation of

resorcinol-formaldehyde resin/silica nanocomposites and the subsequent removal of the organic composition.9,38 As shown in transmission electron microscopy (TEM) image (Figure 2a), HSNs with the overall sizes of 120-160 nm were obtained, which are smaller than the previously investigated rough HSNs.9,38 TEM and scanning electron microscopy (SEM) images of higher magnification (inset in Figure 2a; Figure S1, Supporting Information) clearly reveal the rough feature of HSN. The mean diameter of the inner cavity is ~90 nm and spiky shell is ~25 nm thick. The mesopores on the shells are appropriate for reactants to get through and confine the reactions in the cavity.38 Therefore, Au NR cores were formed in situ within the interior cavity of rough HSN taking advantage of a seed-mediated growth strategy. Au seeds were first prepared in the hollow cavity of HSN nanocapsules through the reduction of chloroauric acid with sodium borohydride in the presence of cetyltrimethylammonium bromide (CTAB), where the seeds outside HSN were washed away. Thereafter, Au seeds-encapsulated HSNs were dispersed in the growth solution, where Au NRs formed in the hollow cavity of HSN. As shown in Figure 2a, rattle-structured rough Au@HSN with entrapped Au NR cores (~30 nm in length and ~10 nm in diameter) were achieved. Compared with traditional Au nanoparticles encapsulated in nanocapsules, the synthetic method we employed here ensured that the morphology of both exterior rough silica surface and interior rod-shaped Au cores of the Au@HSN nanocapsules were maintained. The rough surface is expected to increase endocytosis 6


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efficiency while the Au NR cores are supposed to provide photothermal effect. Rattle-structured smooth Au@HSN (sm-Au@HSN) with comparable sizes of 130-170 nm with rough Au@HSN was also prepared employing in-situ growth strategy, where Au NR cores were formed in the hollow cavity of smooth HSN (sm-HSN) (Figure S2, Supporting Information). The outer surface of the Au@HSN was then functionalized for controlled drug encapsulation and gene condensation. 3-aminopropyl triethoxysilane (APTES) was first utilized to modify the surfaces with terminal amine groups, which then reacted with adamantanecarboxylic acid to introduce adamantine (Ad) groups and form Au@HSN-Ad. In the absence of drug, Au@HSN-Ad was assembled with CD-PGEA to produce flexible nanohybrids of Au@HSN-PGEA (AHP) through host-guest interactions. In the presence of drug and after the loading of SF, SF-Au@HSN-Ad was assembled with CD-PGEA to produce SF-Au@HSN-PGEA (SAHP) (Figure 1). The superior polycation CD-PGEA could not only carry genes but also constitute supramolecular nanovalves.13,36 In this work, CD-PGEA was prepared from CD-PGMA (Mn = 1.5 × 104, polymer dispersity index (PDI) = 1.4) through atom transfer radical polymerization (ATRP) and the subsequent ring-opening reaction. Detailed characterization of the polymer including 1H-NMR spectra (Figure S3, Supporting Information) and gel-permeation chromatography (GPC, Table S1, Supporting Information) is shown in Supporting Information. The rational design guarantees that SF could be preserved in the cavity until NIR irradiation is performed. The successfully surface functionalization processes of Au@HSN without SF loading were verified by X-ray photoelectron spectroscopy (XPS; Figure S4, Supporting Information). The larger particle size of AHP (~270 nm) over Au@HSN (~150 nm) acquired from atomic force microscopy imaging (AFM, Figure 2b) 7


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confirmed the successful attachment of CD-PGEA polymer brushes on the surface of Au@HSN. Thermal gravimetric analysis (TGA, Figure 2c) suggests that the weight ratios of CD-PGEA in AHP and SAHP could be calculated to be around 52% and 53%, which are comparable. Rattle-structured smooth nanocapsules (sm-AHP) and SF-loaded smooth nanocapsules (sm-SAHP) were obtained in the same way after the assembly of CD-PGEA on the surface of sm-Au@HSN. To track the change in the porous structure during surface functionalization processes, nitrogen adsorption-desorption measurements were carried out to analyze the Brunauer-Emmett-Teller (BET) surface area and pore size of Au@HSN-based nanoparticles. As shown in Figure S5 and Table S2 (Supporting Information), after the attachment of CD-PGEA, the BET surface area of AHP decreased obviously compared with that of Au@HSN, due to the filling up of polymers in the void space between spikes on the rough surface.9 The broad pore size distribution of Au@HSN may be caused by the wide distribution of porous spaces between spikes, in accordance with the previous reports on silica nanoparticles with spiky surfaces.7,38 In addition, the BET surface area of SAHP were comparable to that of AHP, implying the negligible effect of SF loading on the porous structure and surface properties of AHP. Gene Transfection Performance of AHP. AHP was expected to condense and deliver genes due to the polycationic component CD-PGEA. To investigate the interaction between AHP and pDNA, N/P ratio was first defined as the molar ratio of nitrogen (N) in CD-PGEA to phosphate (P) in pDNA. The particle size of AHP/pDNA complexes was observed to be ~220 nm judging from AFM imaging (Figure 2b), which is smaller than the AHP counterparts (~270 nm). The intuitive AFM images imply the shrinkage of polymer chains after the interaction with pDNA. 8


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The pDNA condensation capability of AHP was then characterized by agarose gel electrophoresis. Figure 2d displays the gel retardation results of AHP/pDNA and CD-PGEA/pDNA. AHP demonstrated better pDNA condensation capability than CD-PGEA (within the N/P ratio of 1.5 and 2, respectively), confirming the successful assembly of CD-PGEA and the enhanced performance. Hydrodynamic particle sizes and surface potentials measured by dynamic light scattering were also used to characterize pDNA condensing ability of AHP. As shown in Figure 2e, the hydrodynamic particle sizes of AHP/pDNA complexes were within 350 nm, which are all smaller than the pristine AHP due to the electrostatic interaction between AHP and pDNA. The hydrodynamic particle size may be bigger than the actual size of the complexes due to the existence of an electric dipole layer of water, which is considered to be suitable for in vivo study.39 All the complexes kept positive surface charge (~30 mV), which would facilitate the affinity to negatively charged cell membranes. The good pDNA condensation capability revealed by the above biophysical properties of AHP demonstrates their great potential for promising gene delivery. Before the utilization for biomedical applications, it is essential to evaluate the cytotoxicity of AHP. As shown in Figure 3a, the cell viability of AHP/pDNA complexes was considerably lower than the branched PEI (25 kDa, “gold-standard” nonviral gene carriers)35,36 in both HEK293 and hepatoma HepG2 cell lines at various N/P ratios. Although the cell viability of AHP was observed to moderately decrease with N/P ratio (or concentration of AHP), the cell viabilities of HepG2 and HEK293 cells could still be above ~60% even at the high N/P ratio of 30, suggesting their compromised cytotoxicity as safe carriers. To assess the gene transfection efficiency of AHP/pDNA complexes, luciferase 9


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was first employed as the reporter gene in both hepatoma HepG2 and HEK293 cell lines. As displays in Figure 3b, the optimal transfection efficiencies of AHP occurred at the N/P ratio of 20, which might be caused by the mutual influences of pDNA condensation

capability

and

cytotoxicity.

Notably,

AHP/pDNA

complexes

demonstrated significantly higher optimal transfection efficiencies in both cell lines than PEI (25 kDa) and CD-PGEA at their optimal N/P ratios (10 and 20, respectively),35,36 verifying the potential of AHP for prominent gene transfection. It is worth mentioning that AHP/pDNA complexes showed higher transfection efficiencies than sm-AHP/pDNA complexes in both cell lines at all N/P ratios, which confirmed the positive impact of rough surface on gene transfection. To visualize gene transfection of AHP in the selected HepG2 cells, enhanced green fluorescent protein (EGFP) gene expression was performed. Typical fluorescence images of EGFP gene expression mediated by AHP at the optimal N/P ratio of 20 as well as the control CD-PGEA and PEI at their optimal N/P ratios are shown in Figure 3c. AHP demonstrated superior gene transfection performance with the highest percentage of 37% EGFP-positive cells, in contrast with CD-PGEA (19%) and PEI (16%), in agreement with the results of luciferase transfection assay (Figure 3b). The favourable pDNA condensing capability and gene transfection efficacy of AHP are speculated to correlate with their rough surfaces, where the surface spikes were reported to possess special protection feature to bind DNA chains via multivalent interactions.9 In addition, the rough surface-enhanced cellular uptake should also be taken into account. In order to verify the hypothesis, we further assess the cellular internalization of AHP/pDNA, sm-AHP/pDNA and CD-PGEA/pDNA complexes in HepG2 cells at the optimal N/P ratio of 20. As indicated in Figure 3d, fluorescence microscopy images were analyzed after their incubation with HepG2 cells for 4 h. 10


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YOYO-1

labeled

pDNA

represented

green

fluorescence,

and

4′,6-diamidino-2-phenylindole (DAPI)-labeled nucleus appeared in blue. The HepG2 cells treated with AHP/pDNA complexes exhibited much stronger pDNA signals than sm-AHP/pDNA or CD-PGEA/pDNA complexes, representing more complexes internalized in the cells since free pDNA without protection will be soon degraded by nucleases. Then flow cytometry assay was further performed to quantitatively analyze the cellular uptake. AHP/pDNA, sm-AHP/pDNA and CD-PGEA/pDNA complexes demonstrated the internalization ratios of 85.7%, 76.3% and 70.3%, respectively, which reveals the same trend of fluorescent intensity. These results are in accordance with the previously reported rough surface enhanced internalization,7-9 which will further help understand the contribution of surface morphology to gene transfection performance. It has been proved that cationic polymer coated rough silica nanoparticles could maintain spiky nanotopography and enable strong binding affinity with pDNA which were entangled and hidden inside the spikes on the surface to facilitate the cellular uptake process.9 Different from the liposome-like nanovesicles that present appropriate targeting ligands on the surface to interact with membrane receptors,40 AHP with rough surfaces might also take featured pathways to enter cells, as reported in the case of the interaction between rough virus-like silica nanoparticles and cancer cells.7 Photothermal Effect of AHP and NIR-Responsive Drug Release. As Au NR cores were entrapped in the interior cavity of rattle-structured Au@HSN and AHP, they are supposed to demonstrate surface plasmon resonance (SPR) absorption. Therefore, the optical properties of Au@HSN and AHP were first studied by UV-vis spectroscopy. As shown in Figure 4a, a longitudinal SPR peak of as-prepared Au@HSN was observed at the wavelength of 725 nm, while an obvious red shift of 11


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20 nm occurred in the longitudinal SPR peak of AHP, which is caused by the refractive index change when the polymer was assembled on the surface of Au@HSN. The desirable absorbance of AHP in the NIR region motivated us to explore their photothermal effect. The temperature curves of AHP solutions upon irradiation at 808 nm were recorded. It is found that the temperature of AHP suspensions rose rapidly after 5 min irradiation while deionized water hardly show any change (Figure 4b). In addition, the temperature variation evidently depended on the concentration of AHP solutions. For instance, temperature elevation of 20.2 oC was observed for the suspension of 200 µg/mL while a larger value of 46.3 oC was monitored for suspension of 800 µg/mL. The final temperature after irradiation was high enough to kill cancer cells. The live/dead staining images of HepG2 cells provided intuitive evidences. As shown in Figure 4c, HepG2 cells treated with AHP under irradiation for 5 min were stained with ﬂuorescein diacetate (FDA) and propidium iodide (PI), where the live cells were visualized in green and dead cells in red. Above 40% cell apoptosis was observed while most cells under irradiation in the absence of AHP survived. These preliminary in vitro photothermal results encouraged us to further validate the photothermal performances of AHP hybrids in vivo. Hepatoma-bearing nude mice were injected with AHP solution and phosphate buffer saline (PBS, control) respectively, and then the tumors were exposed to 808 nm laser (2 W/cm2) for 5 min. The laser power employed herein followed the previous work to validate the photothermal performance of AHP.11,13,39,41,42 During this period, infrared thermal images and temperature variations of the tumors were recorded every 30 s. As demonstrated in Figure 4d, the tumors administrated with PBS showed negligible color change or temperature increase. In contrast, apparent color change was found after 1 min irradiation in the presence of AHP. The temperature of tumors increased 12


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to be ~53

o

C after 5 min irradiation, confirming the feasibility of AHP as

photothermal agents for PTT in vivo. According to the design concept of the rough rattle-structured AHP, CD-PGEA assembled on the surface could be considered as cloak and nanovavles for controlled drug release. Meanwhile, the photothermal effect of AHP could be utilized to trigger the detachment of CD-PGEA13,39 and the resultant cascaded SF release from the hollow cavity of AHP. SF was first loaded to the cavity of Au@HSN through the mesopores on the spiky silica shells. After the assembly of CD-PGEA gatekeepers on the rough surface, SAHP with SF encapsulated in the cavity were fabricated. In the absence of NIR laser, the release profile of SF were basically flat and the total released SF was less than 5% within 250 min (Figure 4e). This phenomenon exactly proved the shielding effect of CD-PGEA, which in turn explains the SF release behaviors when interval NIR laser irradiation was performed. When NIR laser was applied, dramatic SF release was recorded due to the detachment of CD-PGEA induced by the photothermal effect of AHP. When NIR irradiation was withdrawn, the release of SF slowed down evidently. The NIR-responsive release of SF will facilitate the HCC treatment at specific tumor site with enhance therapeutic efficacy and minimized adverse effects. The antitumor effect of SAHP and sm-SAHP was assessed by MTT assay in HepG2 cells (Figures 4f, S6, Supporting Information), where the cells were treated with free SF, SAHP/pDNA, sm-SAHP/pDNA, as well as SAHP/pDNA and sm-SAHP/pDNA with NIR irradiation. Free SF showed moderate killing effect and the cell viability was ~63% (Figure S7, Supporting Information). Without NIR irradiation, the SAHP/pDNA with equivalent amount of SF loading exhibited cell viability of ~74%. In this case, the antitumor effect of SF was blocked by the 13


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gatekeepers, in consistent with the release profiles of SAHP (Figure 4e). When NIR laser irradiation was applied, the cell viability treated with SAHP/pDNA was considerably lower (~28%) than the former two groups, which is attributed to both hyperthermia and NIR-triggered SF release. In contrast, the cell viability mediated by sm-SAHP/pDNA under irradiation was observed to be ~39% (Figure S6, Supporting Information), verifying the superiority of the rough nanotopography. In addition, the above antitumor activities of SAHP/pDNA with/without NIR irradiation were confirmed by fluorescent images of live/dead staining (Figures 4h,i). It is worth mentioning that synergistic antitumor effect of SF was achieved (degree of synergy (S) > 0) during the process of NIR irradiation, which was determined by the Bliss Independence Model.43 The effective cooperation between PTT and chemotherapy resulted in improved cancer cell-killing effect of SF, where the NIR irradiation might trigger the rapid SF release and diffusion.44 The multimodal synergistic therapy not only possesses collective advantages of several therapeutic modalities, but also avoids excessive adverse effects since better antitumor efficacy could realized with reduced dose of therapeutic agents. Complementary Gene/Chemo/Photothermal Therapy In Vitro and In Vivo. The rattle-structured rough AHP with photothermal Au NRs cores was exhibited to encapsulate antitumor drug SF in the cavity and condense pDNA on the outer surfaces, which is anticipated for complementary gene/chemo/photothermal therapy. The efficacy of combined therapy was first assessed by the preliminary testing in vitro. In this work, the commonly used antioncogene, p53, was employed to suppress the proliferation of tumor cells for GT. We first tested the stability of SAHP/p53 complexes in medium with 10% fetal bovine serum (FBS). The particle size of the complexes at the N/P ratio of 20 was found to keep constant during the incubation 14


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(Figure S8), confirming the serum stability of the complexes. As shown in Figure 4f, the HepG2 cells treated with AHP/p53 complexes showed cell viability of ~60%, confirming the feasibility of AHP as gene carriers for GT. Meanwhile, the cell viability mediated by sm-AHP/p53 was ~69% (Figure S6, Supporting Information), confirming the better gene delivery capability of rough nanocapsules. The complementary groups of AHP/p53 and SAHP/p53 with NIR irradiation were also investigated and the tri-modal complementary gene/chemo/photothermal therapy exhibited the lowest cell viability of ~22%. The corresponding live/dead staining images are shown in Figures 4g,j. Furthermore, synergistic chemotherapy and bimodal gene/photothermal therapy (S > 0) were found for enhanced antitumor effect. It is supposed that PTT could improve the cellular uptake of drug and genes through the NIR responsiveness,31 which may contribute to the prominent effect of SAHP/p53 nanoplatform. For the comparison, sm-SAHP/p53 mediated tri-modal complementary therapy produced the cell viability of ~28%, in agreement with superiority of the spiky surface. Multimodal imaging integrated with therapy could provide comprehensive and valuable information for the therapeutic response to assess and adjust the treatment. Dual-modal PA/CT imaging could combine the advantage of high spatial/density resolution and high sensitivity. The feasibility of the rattle-structured rough AHP as PA and CT imaging contrast agents in vitro and in vivo was then testified. The intensity of PA signals increased with the concentration of AHP while the enhanced brightness was detected in the corresponding PA images (Figure 5a), which arises from photothermal Au NR cores. After the intratumoral injection of AHP, the brilliant PA signals acquired from the tumour region verified the penetration of NIR light and the potential of AHP for PA imaging in vivo (Figure 5b), confirming the adequate 15


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accumulation of AHP around the tumour. In addition, the X-ray attenuation intensity increased linearly with the concentration of AHP solution and enhanced CT contrast signals in the tumour region were observed after the injection of AHP (Figure S9, Supporting Information), in accordance with the results of PA imaging. In this regard, intriguing dual-modal PA/CT imaging-guided complementary therapy could be achieved to monitor accumulation of AHP and the therapeutic effects. Encouraged by the promising antitumor results of complementary gene/chemo/ photothermal therapy in vitro, we further studied the in vivo performance of the versatile SAHP/p53 nanoplatform via intratumoral injection. The hepatoma-bearing nude mice were randomly divided into five groups with four mice in each group for different administrations: PBS as control group, AHP with NIR irradiation as PTT group, AHP/p53 as GT group, SAHP with NIR irradiation as bimodal chemo/photothermal therapy group, and SAHP/p53 with NIR irradiation as trimodal gene/chemo/photothermal therapy group. The volumes of tumors from different groups were measured every two days to acquire tumor growth curves (Figure 6a). Since the tumors of mice in the trimodal gene/chemo/photothermal therapy group seemed almost vanished after 8 days, the in vivo antitumor experiments were terminated accordingly. All the hepatoma-bearing mice were sacrificed and the tumors were weighed and imaged (Figure 6a). The tumors in the control group were found to grow rapidly during the period, which manifested the typical growth behavior of malignant hepatoma. In comparison, the tumor growth in the GT group was obviously suppressed to a certain extent, in accordance with the high transfection efficiency of AHP (Figure 3b) and the antitumor function of p53 (Figure 4f,g). However, the average volume of tumors after the treatment was still four times larger than the initial value, implying the insufficiency of single-modal GT. Similarly, when 16


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only PTT was applied, the tumor volume decreased first and then increased soon after two days, suggesting the proliferation of survival tumor cells after NIR irradiation. This situation was improved after bimodal chemo/photothermal therapy was applied, where the volume of tumors was much smaller due to the synergistic effects of PTT and hyperthermia-enhanced SF release (Figure 4f). In this case, obvious recurrence of tumors was still observed after four days. It is impressive that the tumors in the trimodal gene/chemo/photothermal therapy group were totally suppressed and significantly smaller than any other groups. The continuous inhibition of tumor growth indicates the superiority of complementary trimodal therapy. During the cascaded processes, synergistic integration of GT and bimodal chemo/photothermal therapy was achieved (S > 0), which might contribute to the satisfactory therapeutic efficacy in vivo. As Au NR cores of the rattle-structured AHP worked for PTT, the hyperthermia accompanied triggered the disassembly of the CD-PGEA to facilitate SF and p53 release for GT and chemotherapy. The expression of antioncogene p53 (protein, P53) was visualized by immunohistochemical staining of the dissected tumor tissue (Figure 6b). Hardly any p53 protein-positive area was observed in the samples of the control group, PTT group

or

chemo/photothermal

therapy

group.

In

contrast,

the

GT

and

gene/chemo/photothermal therapy groups exhibited obvious P53 protein expression in brown. To evaluate the therapeutic effects of different treatments, the cell states in the tumor tissues were analyzed with hematoxylin-eosin (H&E) staining. While hepatoma cells in the control group seemed normal, cell apoptosis was observed after different treatments. As shown in Figure 6c, more or less shrinkage and fragmentation of nuclei occurred. There was also loss of hepatoma cells in some region. When trimodal gene/chemo/photothermal therapy was carried out, the hepatoma cells experienced the 17


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most severe apoptosis among all the groups. The above histological results further verified the profound therapeutic efficacy of NIR-responsive complementary trimodal therapy. To testify the morphology effect on complementary therapy in vivo, we also conducted

bimodal

chemo/photothermal

therapy,

and

trimodal

gene/chemo/photothermal therapy with sm-SAHP on hepatoma-bearing nude mice. Compared with the PBS group, the tumor growth in bimodal therapy group mediated by sm-SAHP was suppressed to some extent while trimodal therapy demonstrated the best effect on tumor inhibition (Figures S10a-c, Supporting Information). On the other hand, judging from the relative volume and weight of the resultant tumors from all the groups (Figures S10d,e), the trimodal complementary therapy group of SAHP exhibited significantly smaller tumors than the other groups, including the trimodal therapy group of sm-SAHP. These results further validate the former conclusions from the in vitro therapeutic data that the rattle-structured rough nanocapsules are superior over the smooth counterparts. The in vivo cytotoxicity of the SAHP/p53 platform was assessed by histological analysis of the major organs from different treatments. As displayed in Figure 6d, negligible abnormality was observed in the H&E staining images of the slices of heart, liver, spleen, lung, and kidney. Moreover, the body weight of the mice was tracked during the period of treatments and no noticeable weight loss was found in all the groups (Figure S11, Supporting Information). These results suggest excellent biocompatibility and compromised cytotoxicity of the developed platform.

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CONCLUSIONS In summary, rattle-structured rough nanocapsules (Au@HSN-PGEA, AHP) composed of in-situ formed gold nanorod (Au NR) cores and polycationic mesoporous silica shells were successfully fabricated. The “ship-in-a-bottle” synthetic strategy was applied to generate Au NR cores inside the cavity of hollow silica nanoparticles (HSN) to preserve the rough surface feature and the interior space for drug loading. Polycationic CD-PGEA was assembled on the surface of Au@HSN to carry genes. Meanwhile, CD-PGEA was utilized as gatekeeper to realize NIR-triggered drug release. The rough surface-enhanced cellular internalization was utilized to facilitate gene transfection performance. Taking advantage of the inherent photothermal characteristic of Au NR cores, PTT and synergistic effects arising from PTT enhanced-chemotherapy were achieved. The proposed rattle-structured rough nanocapsules were proven to be satisfactory platforms for complementary gene/chemo/photothermal therapy of malignant hepatocellular carcinoma. In addition, PA imaging was testified for imaging-guided therapy. This work presents a rational design and modal system of multifunctional rough nanoparticles for impressive therapeutic effectiveness.

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EXPERIMENTAL SECTION Synthesis of Rough AHP. Rough HSNs were first synthesized by a one-pot approach to prepare phenolic resin/silica nanocomposites through controlled hydrolysis of tetraethyl orthosilicate (TEOS).9 The resultant HSNs were obtained after calcination at 550 oC for 2 h. Then the Au NR cores were synthesized in the hollow cavity

of

rough

HSN

following

a

seed-mediated

growth

strategy.

Au

seeds-encapsulated HSNs were synthesized by dispersing HSNs in the solution of cetyltrimethylammonium bromide (CTAB) and HAuCl4. Au seeds were formed subsequently by the reduction of HAuCl4 with NaBH4. Au@HSN with Au NRs inside the interior cavity was finally achieved by the addition of Au seeds-encapsulated HSN in the growth solution, where gold Au NRs were formed through the reduction of HAuCl4 with ascorbic acid in the presence of CTAB. AHP was then fabricated by surface functionalization of Au@HSN. Polycationic CD-PGEA was first synthesized through ATRP and the subsequent ring-opening reaction reported in our previous work.35,36 CD-Br was synthesized as ATRP initiator to prepare CD-PGMA. Ethanolamine (EA) was then added to open the epoxy rings, resulting in CD-PGEA. The surface of Au@HSN was modified by 3-aminopropyl triethoxysilane (APTES) to produce Au@HSN-NH2 with terminal amino groups. Au@HSN-Ad was then prepared through the reaction of amino groups and carboxylic groups of adamantanecarboxylic acid (Ad-COOH). After the self-assembly of Au@HSN-Ad with CD-PGEA, AHP (Au@HSN-PEGA) was finally achieved. The detailed procedures are described in Supporting Information. In Vitro Cytotoxicity, Gene Transfection and Cellular Uptake. The cytotoxicity of AHP/pDNA was evaluated by MTT assay in HepG2 and HEK293 cell lines. The procedures followed our work reported previously.6 Transfection assays were carried 20


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ACS Nano

out taking plasmid pRL-CMV encoding Renilla luciferase and pEGFP-N1 encoding enhanced green fluorescent protein as a reporter genes.6 Flow cytometry was utilized to evaluate the cellular internalization.45 The detailed procedures are shown in Supporting Information. Photothermal Effect of AHP. The photothermal effects of AHP solutions with various concentrations and AHP-accumulated tumor sites of the tumor-bearing mice were studied under an 808 nm laser at a power density of 2 W/cm2. Temperature variation was recorded by an IR thermal camera. The detailed procedures could be found in Supporting Information. SF Loading and NIR-Triggered SF Release. SAHP and sm-SAHP were prepared in the same way and SAHP was taken as the example. The drug loading of SF within the Au@HSN nanorattles with CD-PGEA as gatekeepers was performed following our previous procedures.36 After surface modification with Ad-COOH, Au@HSN-Ad was dispersed in SF solution in DMSO for 24 h at room temperature. SF-loaded Au@HSN-Ad (SF-Au@HSN-Ad) was collected by centrifugation and then dispersed in ethanol. The mixture was then added dropwise to CD-PGEA aqueous solution, which was stirred at room temperature for 24 h to produce SF-loaded AHP (SAHP). The loading capacity was determined by a high-performance liquid chromatography (HPLC). To investigate NIR-triggered drug release, SAHP aqueous solution was agitated at 37 oC. NIR irradiation was carried out at pre-determined intervals (2 W/cm2, 5 min). The detailed procedures are described in Supporting Information. Complementary Gene/Chemo/Photothermal Therapy In Vitro and In Vivo. MTT assay was utilized to evaluate the cell viability mediated by p53 gene transfection in HepG2 cell line, following the previous reported procedures.11 AHP and sm-AHP mediated complementary were performed respectively with the same 21


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procedure. Here, we take the AHP as the example. AHP/p53, AHP/p53+NIR, SAHP/p53+NIR, and naked p53 groups were investigated in vitro. For tumor therapy, five groups of tumor-bearing BALB/C nude mice were treated with PBS, AHP+NIR, AHP/p53, SAHP+NIR, and SAHP/p53+NIR, respectively. The detailed procedures are described in Supporting Information. PA and CT Imaging of AHP In Vitro and In Vivo. PA images of AHP solutions with various concentrations were obtained using a multispectral optoacoustic tomography imaging system. CT scans were acquired using a single photon emission computed tomograghy (SPECT/CT) scanning system. Tumor-bearing mice injected with AHP solutions were imaged before and 10 min after the injection. The detailed procedures are shown in Supporting Information.

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ACS Nano

ACKNOWLEDGEMENTS This work was supported by the National Key Research and Development Program of China (grant number 2016YFC1100404), National Natural Science Foundation of China (grant numbers 51773013, 51373017, 51733001 and 51473014), the Fundamental Research Funds for the Central Universities (Project No. BHYC1705A).

SUPPORTING INFORMATION AVAILABLE Experimental details and additional characterization such as SEM images of rough Au@HSN, TEM images of sm-HSN and sm-Au@HSN, 1H NMR spectra, XPS spectra, nitrogen sorption-desorption isotherms and pore size distribution curves, fluorescence image of FDA-PI stained HepG2 cells treated by free SF, and body weight evolution of tumor-bearing mice can be found in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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Growth and Metastasis of Hepatocellular Carcinoma by Co-Delivery of Ursolic Acid and

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Figure 1. Schematic illustration of the preparation of SAHP and the resultant responsive drug/gene codelivery process.

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ACS Nano

Figure 2. (a) TEM images of HSN and Au@HSN. Insets show the corresponding TEM images of higher magnification, scale bar: 50 nm. (b) AFM images of Au@HSN, AHP and AHP/pDNA (N/P = 20). (c) Thermogravimetric analysis of Au@HSN, Au@HSN-Ad, AHP and SAHP. (d) Electrophoretic mobility retardation assay of pDNA complexes with AHP and CD-PGEA under various N/P ratios. (e) Particle sizes and zeta potentials of the AHP/pDNA complexes at various N/P ratios compared with those of the pristine AHP (mean ±SD, n = 3).

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Figure 3. (a) MTT assay of HEK293 and HepG2 cell lines treated with AHP/pDNA and PEI/pDNA complexes at various N/P ratios. (b) Luciferase gene transfection assay of the AHP/pDNA and sm-AHP/pDNA complexes at different N/P ratios in comparison with those mediated by PEI (25 kDa, at the optimal N/P ratio of 10) and CD-PGEA (at the optimal N/P ratio of 20) in HEK293 and HepG2 cell lines. (c) Representative fluorescent images of pEGFP expression mediated by AHP, CD-PGEA, and PEI at their optimal N/P ratios in HepG2 cells. (d) Fluorescence microscopy and merged images, as well as flow cytometry analysis of HepG2 cells treated with YOYO-1-labeled AHP/pDNA, sm-AHP/pDNA and CD-PGEA/pDNA for 4 h. Scale bar: 50 µm. 32


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Figure 4. (a) UV-vis absorption spectra of Au@HSN and AHP. (b) Temperature elevation of water and AHP solutions upon irradiation at 808 nm (2 W/cm2). (c) Fluorescent images of FDA-PI stained HepG2 cells treated with/without (control) AHP (33 µg/mL) upon laser irradiation. (d) Photothermal images and temperature plot of tumor-bearing mice with the injection of PBS and Au@HSN-PGEA (3.77 mg/mL, 100 µL) after irradiation with an 808 nm laser. (e) Cumulative release profile of SF from SAHP with NIR irradiation at pre-determined intervals. (f) MTT assay of HepG2 viabilities treated with free SF, SAHP/pDNA, AHP/pDNA+NIR, SAHP/pDNA+NIR, AHP/p53, AHP/p53+NIR and SAHP/p53+NIR at the N/P ratio of 20. Corresponding fluorescent images of FDA-PI stained HepG2 cells treated with AHP/p53 (g), AHP (inset in g), SAHP/pDNA (h), SAHP/pDNA+NIR (i), and SAHP/p53+NIR (j) at the N/P ratio of 20. Scale bar: 100 µm.

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Figure 5. (a) PA intensity and corresponding PA images of AHP solution with different concentrations. (b) PA images of the tumor bearing mouse before and after the injection with AHP (3.77 mg/mL, 100 µL). Tumor regions are highlighted by the white dashed circles.

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Figure 6. (a) Time-dependent growth curves, average tumor weights and representative photographs of excised tumors of mice after the treatment with PBS, AHP+NIR, AHP/p53, SAHP+NIR, and SAHP/p53+NIR, respectively. (b) Immunohistochemical and (c) H&E stained images of the dissected tumors after various treatments of 8 d. (d) Histology analysis of major organs (hear, liver, spleen, kidney, and lung) of different groups of mice. Scale bar: 50 µm.

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Rattle-Structured Rough Nanocapsules with in-Situ-Formed Gold

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