Porous Silicon@Au Nanocomposite

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Controlled Release and Delivery Systems

Multifunctional Chitosan/Porous Silicon@Au Nanocomposite Hydrogels for Long-Term and Repeatedly Localized Combinatorial Therapy of Cancer via a Single Injection Bing Xia, Weiwei Zhang, Haibei Tong, Jiachen Li, Zhenyu Chen, and Jisen Shi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01533 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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ACS Biomaterials Science & Engineering

Multifunctional Chitosan/Porous Silicon@Au Nanocomposite Hydrogels

for

Long-Term

and

Repeatedly

Localized

Combinatorial Therapy of Cancer via a Single Injection

Bing Xia,*,†,‡ Weiwei Zhang,‡ Haibei Tong,‡ Jiachen Li,‡ Zhenyu Chen,† and Jisen Shi*,†

†Key

Laboratory of Forest Genetics & Biotechnology (Ministry of Education of China),

Nanjing Forestry University, Nanjing 210037 (P. R. China). ‡College

of Science, Nanjing Forestry University, Nanjing 210037 (P. R. China).

Corresponding authors *E-mail: [email protected]

KEYWORDS: porous silicon nanocomposites, chitosan hydrogels, in situ gelation, biodegradation, chemo-photothermal therapy

ABSTRACT: Considering the future clinical applications of localized cancer therapy, it is of great importance to construct injectable biodegradable nanocomposite hydrogels with combinatorial therapeutic efficacy. Here, porous silicon nanoparticles (PSiNPs) as host matrix were chosen to fabricate PSiNPs@Au nanocomposites via in situ reductive synthesis of gold nanoparticles. And then PSiNPs@Au nanocomposites were further incorporated

into

thermo-sensitive

chitosan

(CS)

ACS Paragon Plus Environment

hydrogels

to

construct

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

CS/PSiNPs@Au nanocomposite hydrogels, which showed in situ gelation at physiological temperature, excellent biodegradability and biocompatibility. Espeically, with the encapsulation of CS hydrogels, PSiNPs@Au nanocomposites had a long-term stable photothermal effect with higher local temperature under near-infrared (NIR) laser irradiation, whether in vitro or in vivo. Besides, assisted with NIR laser irradiation, CS/PSiNPs@Au nanocomposite hydrogels exhibited a long-term sustained release of anticancer drugs (doxorubicin hydrochloride, DOX) in acidic tumor environments. Finally, DOX/CS/PSiNPs@Au precursors were administrated into tumor-bearing mice via a single intratumoral injection, which presented a significant synergistic chemophotothermal therapeutic efficacy under repeated NIR laser irradiation during longterm cancer treatments. Accordingly, we developed a novel stratege to prepare multifunctional CS/PSiNPs@Au nanocomposite hydrogels, and also demonstrated their potential applications on localized cancer therapy in future clinic.

1. INTRODUCTION In clinical cancer treatments, systemic chemotherapy often suffers from its severe adverse effects to others healthy tissues in body, because of its limited specificities to cancer cells and tumor tissues.1,2 To minimize the off-target toxicity of the conventional chemotherapy, intratumoral delivery systems of anticancer drugs based on injectable biodegradable hydrogels have been developed.3-7 Because they can provide a controlled and sustained release at the target tumor site, create much higher local drug concentrations, lower the overall required dose, and direct the biological effect to target


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cancer cells and tumor tissues. Especially, gold nanoparticles (AuNPs) with significant photothermal effect under near-infrared (NIR) irradiation were further encapsulated into injectable biodegradable hydrogels to construct multifunctional nanocomposites hydrogels, which can not only be used to directly hyperthermia ablation of tumors, but also be helpful to trigger and enhance chemotherapy, aiming at the synergistic therapeutics efficacy.8 Based on these multifunctional nanocomposites hydrogels, a new strategy of cancer treatments called as “localized combinatorial therapy” has exhibited some notable progress, including efficiently inhibiting tumor growth, overcoming cancer multidrug resistance, and preventing tumor metastasis/recurrence.914

Compared with others inorganic nanoparticles, the preferential advantages of porous silicon nanoparticles (PSiNPs) are their excellent biocompatibility and biodegradability. They can be readily degraded into non-toxic orthosilicic acid, and then throughly excreted through the urine.15-23 Besides, PSiNPs also have a versatile delivery capability of

various

therapeutics

agents

including

organic

drugs,

macromolecular

siRNA/plasmid DNA/antibody/enzyme, and even functional nanoparticles, which resulted in their wide applications on cancer diagnosis and therapy.24-32 Recently, AuNPs were further attached into PSiNPs to fabricate photothermal PSiNPs@Au nanocomposites via electrostatics interaction, covalent conjugation or in situ growth.3338

Notably, in contrast to AuNPs alone, AuNPs trapped in nanoprous of PSiNPs could

lead to an enhanced photothermal effect due to dipole-dipole coupling and longer retention time at target tumor site. PSiNPs@Au nanocomposites could be used as



hyperthermia agents to destruct cancer cells in vitro and thermal ablate tumor in vivo.33,36 However, to our knowledge, instead of simple AuNPs, the construction of multifunctional PSiNPs@Au nanocomposite hydrogels for localized cancer therapy has not been reported. Herein, chitosan (CS) hydrogels were chosen to prepare CS/PSiNPs@Au nanocomposite hydrogels via the incorporation of PSiNPs@Au nanocomposites, because CS had in situ gelation, excellent biodegradability, low toxicity and immunostimulatory activities in body.39-43 And then the characteristics of CS/PSiNPs@Au nanocomposite hydrogels including in situ gelation, degradation, photothermal effect, and anticancer drug release were systematically studied, whether in vitro or in vivo. Furthermore, CS/PSiNPs@Au nanocomposite hydrogels with loading of anticancer drugs were administrated into tumor-bearing mice via a single intratumoral injection to observe their combined chemo-photothermal therapeutic efficacy under repeated NIR laser irradiation at different time points during long-term cancer treatments.

2. EXPERIMENTAL SECTION 2.1 Materials and instruments. The single side polished and p-type silicon wafers ((100) oriented, boron doped, 8 – 10 Ω cm resistivity) were bought from Hefei Kejing Materials Technology Co. Ltd., China. β-glycerphosphate (β-GP) was purchased from Hefei Bomei Biotechnology Co., Ltd., China. CS (deactetylation degree >99%, Mw = 100 kDa), cholorauic acid (HAuCl4), and doxorubicin hydrochloride (DOX) were bought from Sigma-Aldrich Chemicals, USA. Deionized (DI) water (≥ 18 MΩ cm


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resistivity, Millipore) was adopted to prepare different aqueous solutions in our experiments. Others chemicals were purchased from Sinopharm Chemical Reagent, China. A Lambda 950 spectrophotometer was utilized to record UV-vis adsorption. Zetasizer Nano ZS dynamic-light-scattering (DLS) measurements (Malvern Instruments, UK) was used to test size and zeta potential of samples at 25 °C. Kratos AXIS Ultra DLD system (UK) with a monochromatic Al Kα X-ray beam (1486.6 eV) at 150 W in a residual vacuum of < 4 × 10−9 Pa was adopted to monitor the atomic components and elemental concentrations of samples. X-ray diffraction (XRD) (Ultimal IV, Rigaku, Japan) was used to analyze crystal structures of samples. Transmission Fourier-transform infrared spectroscopy (FTIR, Vertex 80, Bruker, USA) was used to detect the changes of their chemical functional groups. The morphology of PSiNPs-based nanocomposites was observed by scanning electron microscopy (SEM, JEOL JSM-7600F, Japan) with the accelerating voltage of 15 kV, and high-resolution transmission electron microscope (HRTEM, JEOL JEM-1400, Japan) with the accelerating voltage of 120 kV, respectively. Hydrogels samples were observed by SEM images were taken by FEI QUANTA 200 SEM (Netherlands) with the accelerating voltage of 20 kV. 2.2. Preparation of PSiNPs@Au nanocomposites. Silicon wafers were cleaned by 3:1 (v/v) concentrated H2SO4/30% H2O2 after 30-min boiling, and then repeatedly rinsed with DI water. The clean silicon wafers (2×2 cm2) were incubated in an ethanolic HF solution (40% HF/ethanol (1:1 v/v)), electrochemically etched at the current



intensity of 20 mA/cm2 for 45 min, and then sonicated in toluene with high-purity nitrogen purging for 3 h, to prepare hydrogen-terminated PSiNPs. Subsequently, 1.5 mg PSiNPs was stirred in 1 mL HAuCl4 solution (1 mg/mL) at 60 ºC. When the color of reaction solutions turned form yellow to deep purple, they were centrifuged (1.3 × 104 rpm, 10 min), subseqently washed by DI water to obtain PSiNPs@Au nanocomposites. PSiNPs@Au nanocomposites were further sonicated in DI water overnight, followed by filtration with a 0.2 μm membrane to prepare oxidized PSiNPs@Au nanocomposites. 2.3. Preparation of CS/PSiNPs@Au nanocomposite hydrogels. CS powder was dissolved in hydrochloric acid solution (0.1 mol/L) and stirred at room temperature over night to prepare 2% (w/w) CS solution. Subsequently, 100 µL β-GP aqueous solution (4%, w/w) was added to 900 µL CS solution and incubated for 20 min at 37 ºC to prepare CS hydrogels. Besides, 1 mg oxidized PSiNPs@Au nanocomposites (or oxidized PSiNPs) were dispersed into 1 mL CS solution by sonication, respectively. The CS-based hydrogels were fabricated using the same above method of CS hydrogels. To dry-freezing these hydrogels, they were frozen in -20 oC overnight, and then were lyophilized for three days at -50 oC. 2.4. Photothermal effect tests. NIR light source was an optical-fiber-coupled powertunable diode laser centred at 808 nm with maximal power of 5 W (Hi-Tech Optoelectronics, China). Upon the exposure of NIR laser with different power intensity for 20 min, the temperature elevation of DI water as a control, PSiNPs solution, PSiNPs@Au solution, CS hydrogels, and CS/PSiNPs@Au hydrogels with the volume


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of 400 μL were recorded, and their corresponding thermal imaging were also taken by a IR thermal camera (Fluke, FLK-Ti32s, USA), respectively. 2.5. Rheology measurements. Temperature sweep experiments of CS-based hydrogels were monitored by HAAKE MARS60 rheometer（Thermo Scientific, USA）equipped with a Rheonaut and a IS10 with temperature ranging from 20 to 70 ºC, at a constant temperature rate of 1 ºC/min and a fixed frequency of 1 Hz. A flat geometric rotor (20 mm) was utilized to establish their storage modulus (G') and loss modulus (G'') curves. 2.6. Degradation, drug loading and release studies in vitro. At 37 °C, a serial of PSiNPs, PSiNPs@Au nanocomposites, CS/PSiNPs@Au hydrogels containing equivalent PSiNPs concentration (50 μg/mL) was incubated in PBS buffer (3 mL, pH = 7.4). At different time points, 450 μL of as-preapred solutions was taken, and then ultra-centrifugated (1 × 106 rpm, 15 min) to obtain supernatants. Subsequently, a molybdenum blue colorimetric method was used to analyze silicon elemental amount of these resultant supernatants.22 During 28-day incubation under the same conditions, the weight loss of CS and CS/PSiNPs@Au hydrogels was also measured. 100 µL β-GP aqueous solution (4%, w/w) was added to 900 µL CS solution containing DOX (1 mg/mL) at 37 ºC for 20 min to prepare DOX/CS and DOX/CS/PSiNPs@Au hydrogels, respectively. As-prepared hydrogels were incubated in 1 mL PBS buffer (pH = 5.4 or 7.4) at 37 ºC, respectively. At different time points, the supernatant via the centrifugation (1.3 × 104 rpm, 10 min) was measured by UV-vis spectra at 480 nm to monitor DOX release. Besides, hydrogels were also exposed under NIR irradiation with the power intensity of 1.6 W/cm2 for 10 min at day 3, 6 and 9. The



kinetics of DOX release could be plotted, according to the standard curve method in our previous study.44 2.7. In situ gelling, biodegradation and toxicity analysis in vivo. All animal experiments were carried out accroding to the protocols provided by Laboratory Animal Center of Simcere Pharmaceutical Group in Nanjing. 100 μL of PBS as acontrol, PSiNPs@Au or CS/PSiNPs@Au solution with the dose of 20 mg/kg was subcutaneously injected into BALB/c mice, respectively. After 20 min, mice were sacrificed to observe hydrogels formation in body. And then at day 7, 14, and 28 post injections, the mice were sacrificed to observe samples degradation in body. Moreover, the subcutaneous tissues surrounding the injected sites of mice were taken and examined by a pathologist using hematoxylin-eosin (H&E) staining. During 28 days, the weight of each mouse was also measured to study the toxicity of these samples. And at day 28, heart, kidney, liver, lung, and spleen tissues were collected from the mice, treated with H&E staining, and then examined by a pathologist. 2.8. Localized chemo-photothermal therapy in vivo. First, 1 × 106 4T1 cells were subcutaneously injected into the region of right upper extremity of every Balb/C mouse to develop the tumor model. And then 30 mice bearing 4T1 tumor with the size of ~ 100 mm3 were randomly divided into 6 groups with 5 mice per group in our experiment: (1) PBS + NIR laser (as a control), (2) DOX/CS + NIR laser, (3) PSiNPs@Au + NIR laser, (4) CS/PSiNPs@Au + NIR laser, (5) DOX/CS/PSiNPs@Au, and (6) DOX/CS/PSiNPs@Au + NIR laser. Subsequently, 100 μL all agents including PBS, PSiNPs@Au, CS/PSiNPs@Au, DOX/CS, and DOX/CS/PSiNPs@Au solution with the


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same concentration of 1 mg/mL were intratumorally injected into mice, respectively. At day 0, 7, 14, and 28, the tumor regions were repeatedly irradiated by NIR laser (1.6 W/cm2, 10 min), and their corresponding thermal imaging and temperature elevation was also observed by an IR thermal camera. Eevery other day, the tumor size of every mouse was measured by a calliper in our experiments. At day 28, the tumor tissues of these mice were removed, photographed and weighed. Moreover, their sections were also observed by H&E staining and terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) assay. The tumor size and the inhibition efficiency of tumor growth was calculated as the reported method in our previosu study.22 2.9. Statistical analysis. SPSS statistics software was used for statistical analysis. Significant differences (***p < 0.001, **p < 0.01, or NS (non-significant difference, p > 0.05)) were statistically calculated by ANOVA with Tukey’s post-test, and the data in our experiment were reported as mean ± standard deviation.

3. RESULTS AND DISCUSSION 3.1. Characterization of PSiNPs@Au nanocomposites. As shown in Figure 1a, silicon wafers were electrochemical etched into porous silicon samples, and then sonicated in toluene under nitrogen protection to prepare hydrogen-terminated PSiNPs. Next, as-prepared PSiNPs were incubated in HAuCl4 solution to fabricate PSiNPs@Au nanocomposites, via in situ reductive synthesis of AuNPs. In Figure 1b, compared with bare PSiNPs solutions, the color of PSiNPs@Au solutions changed from turbid yellow to deep purple. UV-vis spectra also showed that a new absorbance peak at 548 nm



appeared, indicating the reductive of HAuCl4 into AuNPs by hydrogen-terminated PSiNPs.36,45 Besides, TEM, XPS and XRD analysis were also adopted to monitor the in situ growth of AuNPs onto PSiNPs. According to Figure 1c, in contrast to XRD patterns of bare PSiNPs (Si (111), (220), (311) and (400)), both AuNPs (Au (111), (200), (220)) and PSiNPs (Si (111), (220)) were found in PSiNPs@Au nanocomposites. Moreover, Si (111) and (220) peaks weakened and Si (311) and (400) peaks almost disappeared, because AuNPs layer coated on PSiNPs could hinder the transmission of X-ray and decreased their detection depth for silicon crystals. Compared with Figure 1d, HRTEM results clearly showed that AuNPs with the size range of 10 – 20 nm were randomly distributed into PSiNPs matrices in Figure 1e, and Au (111) and (220) lattice planes were also easily observed except for Si (220) lattice plane on PSiNPs@Au samples. From XPS results in Figure 1g, Au elemental signal of PSiNPs@Au nanocomposites was also detected at 84.2 eV, compared with PSiNPs alone. And Figure 1h also presented the high-resolution spectra of Au elemental signal with Au (4f5/2) and Au (2f7/2) centred at 87.9 and 84.2 eV, and typical binding energy difference (Δ = 3.7 eV). According to the results of atomic concentrations in Figure 1f, Au 4f intensity increased from 0.0% (PSiNPs) to 5.8% (PSiNPs@Au), and Si 2p intensity decreased from 22.9% to 18.2%. Overall, these above-mentioned results provided the sufficient proof for in situ reductive growth of AuNPs on PSiNPs’ surfaces. To improve the water-dispersibility of these resultant PSiNPs@Au nanocomposites, oxidized PSiNPs@Au nanocomposites were obtained via simple sonication in DI water. According to SEM images in Figure 2a, oxidized PSiNPs@Au nanocomposites with


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the average size of ~200 nm were observed. An energy-dispersive X-ray spectroscopy (EDS) was also used to analyze their elemental compostions. Especially, the detection of Au content pointed that AuNPs were still bound with PSiNPs, even after long-time sonication in water. In Figure 2b, DLS results showed that their mean hydrodynamic size was 224.8 (± 11.8) nm in water, similar with SEM observations. Compared with oxidized PSiNPs alone with negative surface charges reported in our previous studies,32 the zeta potential of oxidized PSiNPs@Au nanocomposites was 8.2 (± 4.6) mV, resulted from the residue of Au3+ ions on AuNPs’ surfaces. These above results indicated that without any extra hydrophilic coatings, oxidized PSiNPs@Au nanocomposites could be well dispersed in aqueous solution, which was benifit to next biological experiments. Finally, to test the photothermal effect of oxidized PSiNPs@Au nanocomposites in aqueous solution, an IR camera with thermal imaging was used to measure their temperature changes under 1.6 W/cm2 808-nm laser irradiation. From Figure 2c (left 1), with the exposure time increased from 0 to 20 min, the temperature of 1mg/mL PSiNPs@Au solution significantly increased (ΔT = 20.8 ºC, deep red). However, DI water (ΔT = 2.5 ºC, deep blue) or PSiNPs solution (ΔT = 4.9 ºC, light blue) revealed little change under the same condition. Compared with PSiNPs alone, the attachment of AuNPs with excellent conversion capability of NIR light to thermal energy could efficiently improve the photothermal effect of PSiNPs. Besides, as seen in Figure 1c (left 2, 3), the photothermal effect of PSiNPs@Au solution was dependent on NIR laser power and their concentration. To investigate the stability of their photothermal effect,



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1 mg/mL PSiNPs@Au solution was also irradiated by 1.6 W/cm2 NIR laser for 20 min (Laser ON), followed by tunring off laser irradiation (Laser OFF) and naturally cooling to room temperature. In Figure 1c (left 4), the temperature elevation of 21.2, 20.6, 21.8, and 20.7 °C was recorded after sequential cycles, respectively, revealing their stable photothermal effect upon the repeated exposure of NIR laser. 3.2.

Characterization

of

multifunctional

CS/PSiNPs@Au

nanocomposite

hydrogels. First, oxidized PSiNPs@Au nanocomposites were homogenously dispersed in acidic CS solution added with β-GP as precursor at room temperature, and then gradually changed into hydrogels when ambient temperature reached 37 oC, which was recorded in Figure 3a. Compared with simple CS hydrogels, the color of CS/PSiNPs@Au nanocomposite hydrogels changed from milk white to brown, because of the incorporation of PSiNPs@Au nanocomposites. FTIR spectra were used to monitor their gelation. According to Figure 3c, the peak at 961 cm-1 was caused by phosphate groups stretching of β-GP. The saccharide ether peaks of CS skeletal vibrations involving C–O stretching appeared at 1060 cm-1. The peaks at 1354, 1441 cm-1 were assigned to –CH3 symmetrical deformation mode. The peaks at 1640, 1560 cm-1 was attributed to amide I (C=O) and amide II (–NH) bonds of CS. Peaks at 2909, 2840 cm-1 were due to C–H stretch vibrations of CS. Another broad peak at ~3400 cm-1 was ascribed to O–H and N–H stretching vibrations of CS. By analyzing the intensity of these above-mentioned peaks, obvious changes could be found between precursor and hydrogel samples. With the formation of hydrogels, the peak intensity of phosphate groups stretching at 961 cm-1 decreased, due to their protonation. In addition, an


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obvious decreasing of these peaks intensities at 1354, 1441, 2840 and 2909 cm-1 happened, due to inter-chains hydrophobic interactions of CS. The ratio between amide I and II also decreased, indicating inter-chains hydrogen bonding between C=O, –NH, and –OH groups of CS. According to these results, we suggested that the gelation of CS/β-GP precursors was mainly caused by synergistic interactions of molecular forces including hydrogen bonding, electrostatic and hydrophobic interactions. When ambient temperature increasing, heat could help the electrons transfer from β-GP to CS, neutralize the ammonium groups of CS, and then improve their hydrogen bonding and hydrophobic interaction, which resulted in the physical crosslinking of CS molecules to form hydrogels. Moreover, rheological measurements were also used to analyze the gelation temperature of hydrogels. With ambient temperature increasing from 20 to 70 oC, G' and G'' of CS and CS/PSiNPs@Au hydrogels were determined, respectively. In Figure 3b, whether CS or CS/PSiNPs@Au precursors, G'' was higher than G' at room temperature, revealing they behaved like a free-flowing solution. With temperature increasing, G' increased more quickly than G'', and finally G'' was much lower than G', due to the formation of semisolid hydrogels. Therefore, gelation temperature could be defined as the temperature at which G' was equal to G'', as seen in Figure S1a, b. Comparing CS with CS/PSiNPs@Au hydrogels, the gelation temperature increased from 29.4 and 35.7 oC. The results indicated that more heat was need for the gelation of CS/PSiNPs@Au hydrogels than CS hydrogels, which was need to break down the hydrogen bonding between oxidized PSiNPs@Au nanocomposites and CS molecules.



Additionally, their gelation time was also measured in Figure S1c, d. Their gelation time was about 6 min at 37.0 oC, whether CS/PSiNPs@Au or CS hydrogels, which demonstrated that these precursor solutions could be fast gelled under physiological conditions. After dry-freezing process, SEM was used to observe the morphology of hydrogels samples, as seen in Figure 3d and Figure S2. CS hydrogels presented integrated sponge-like scaffolds with interconnected macroporous structures (20 − 200 μm). After further magnification, microporous structures (1 − 10 μm) were also found in one single chamber, and their surfaces were relatively homogeneous and flat. In contrast, macroporous structures of CS/PSiNPs@Au hydrogels became fragmentary with rougher and more textured surfaces. According to FTIR and rheological results, the incorporation of PSiNPs@Au nanocomposites slightly prevented the formation CS hydrogels and deteriorated the integrity of their crosslinking networks. To analyze their hyperthermia potential, IR camera was also utilized to monitor temperature elevation of CS-based hydrogels upon the exposure of 808-nm laser (1.6 W/cm2, 20 min). As seen in Figure 3e, compared with CS hydrogels (ΔT = 2.4 oC) and PSiNPs@Au nanocomposites (ΔT = 21.9

oC),

the temperature elevation of

CS/PSiNPs@Au nanocomposite hydrogels (ΔT = 32.2 oC) was the highest under the same NIR irradiation. Moreover, in Figure 3f, the temperature elevation of 33.2, 32.8, 32.6, and 33.1 °C was achieved after sequential cycles, respectively. The result showed that PSiNPs@Au nanocomposites encapsulated in CS hydrogels still had significant and stable photothermal effect, on the other hand, low thermal diffusion of CS hydrogels could help PSiNPs@Au nanocomposites generate higher local temperature


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under the same NIR irradiation. 3.3. Degradation, anticancer drugs loading and release in vitro. CS-based hydrogels rehydrated in physiological media could swell and ultimately dissolve, leading to a short life time ranging from a few days to a month.41,42 As shown in Figure 4a (top), similar degradation phenomena were also observed after 28-day incubation in PBS solution at 37 oC, whether CS or CS/PSiNPs@Au hydrogels. Meanwhile, their corresponding weight loss was also recorded in Figure 4c. On the first 2 days, CS/PSiNPs@Au or CS hydrogels fast degraded to 56% or 49%, and then gradually degraded to 72% or 71% until day 28, respectively. Besides, PSiNPs could be also thoroughly degraded into orthosilicic acid in physiological media via self-oxidation.25 Here, a molybdenum blue colorimetric method was used to analyze PSiNPs degradation of CS/PSiNPs@Au hydrogels or PSiNPs@Au nanocomposites under the same physiological conditions, respectively. In Figure 4d, after 2-day incubation, the degradation ratio of PSiNPs@Au nanocomposites or bare PSiNPs sharply increased to 71.3% or 94.1%, respectively. And then they slowly incresed to 85.2% or 100% after 28 days. In contrast to the fast degradation of bare PSiNPs or PSiNPs@Au nanocomposites, the degradition of PSiNPs contained in CS/PSiNPs@Au hydrogels could be significantly alleviated, due to the protection of outer CS hydrogels. After 6 days, they only reached at 60.4%, and then slowly increased to 78.1% at day 28. With the degradation of these samples, their photothermal effect was also monitored under the same conditions, recorded in Figure 4e. For PSiNPs@Au nanocomposites, ΔT sharply decreased from 21.8 to 9.3 oC only after 1 day, and then gradually decreased to



3.6 oC until day 28. However, for CS/PSiNPs@Au nanocomposite hydrogels, on the first 3 days, ΔT slightly decreased from 28.4 to 27.3 oC, and then slowly reached 9.7 oC until day 28. The result demonstrated that the heavy detachment of AuNPs from PSiNPs@Au nanocomposites without the protection of CS hydrogels could lead to a remarkable decay of their photothermal effect, with their fast degradation in physiological environment. However, if adding the protection of CS hydrogels, the stability of their photothermal effect could be remarkably improved, which had much favor for repeated photothermal therpay without repeated injection of hyperthermia agents during long-term cancer treatments. Furthermore, anticancer drugs (DOX) were loaded into CS or CS/PSiNPs@Au hydrogels to prepare DOX/CS or DOX/CS/PSiNPs@Au hydrogels, respectively, which was shown in Figure 4a (bottom). To evaluate DOX release behavior from hydrogels, two typical pH values of 5.4 (acidic tumor environments) and 7.4 (physiological environments) were chosen. In Fig. 4b, the cumulative release profiles of DOX were investigated by UV-vis spectra at different time points up to 11 days, which presented two release stages. Stage I: an early release happened, as a result of free DOX released from swelling hydrogels. Stage II: with the decomposition of CS hydrogels, a late release appeared. After 11-day incubation of DOX/CS hydrogels in PBS solution, DOX release reached 40.4% at pH 5.4, while only 20.1% released at pH 7.4. For DOX/CS/PSiNPs@Au hydrogels, similar phenomena (21.5% at pH 7.4, and 46.9% at pH 5.4)) could be also observed under the same conditions. This pH-responsive behavior was ascribed to higher solubility of DOX molecules and easier decomposition


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of CS hydrogels in weak acidic environments. Besides, we also found that NIR laser could promote DOX release from CS/PSiNPs@Au hydrogels. For example, at day 3 under NIR irradiation (1.6 W/cm2, 10 min), DOX release increased by 14.7% at pH 5.4, and increased by 8.4% at pH 7.4, respectively. After 11-day incubation plus repeated NIR irradiation at day 3, 6 and 9, the cumulative release amount significantly increased and reached 68.7% at pH 5.4 (or 34.5% at pH 7.4), compared with 47.4% at pH 5.4 (or 22.6% at pH 7.4) without NIR irradiation. In contrast, no obvious increasing of DOX release from CS hydrogels under the same NIR irradiation could be observed, such as, 43.8% at pH 5.4 (or 24.1% at pH 7.4), compared with 40.6% at pH 5.4 (or 20.6% at pH 7.4) without NIR irradiation. Accordingly, under the help of tumor acidic environments and NIR laser irradiation, high-efficiency and long-term sustained DOX release from CS/PSiNPs@Au hydrogels could be achieved. 3.4. In situ gelation, biodegradability and biocompatibility in vivo. To study in situ gelation, biodegradability and biocompatibility of CS/PSiNPs@Au hydrogels in vivo, PBS (group 1) as a control, PSiNPs@Au (group 2) or CS/PSiNPs@Au solutions (group 3) was subcutaneously injection into Balb/C mice, respectively. After 20 min, mice were sacrificed to observe hydrogels formation in body. From Figure 5a, compared with group 1, CS/PSiNPs@Au precursors gelled and adhered to subcutaneous tissues by visual observation of mice in group 3. In contrast, PSiNPs@Au nanocomposites simply accumulated on the muscles of injected sites in mice of group 2. After 7 days, CS/PSiNPs@Au hydrogels clearly swelled. After 14 days, their degradation and cutaneous absorption appeared, and trace residues still existed until day 28. For group



2, the degradation of PSiNPs@Au nanocomposites could be obviously found after 7 days, and then they completely disappeared after 28 days. These results demonstrated that in situ gelation and biodegradation of CS/PSiNPs@Au hydrogels happened in vivo, and the protection of outer CS hydrogels could improve the persistence of PSiNPs@Au nanocomposites at the injected sites in body. In our experiments, the induction of inflammation by CS/PSiNPs@Au hydrogels was further investigated via histological observation of the subcutaneous tissues surrounding the injected sites, recorded in Figure 5b. For group 1 and 2, only some inflammatory cells were found, implying negligible inflammation. For group 3, some lymphocyte infiltration was observed after 14 days, but these inflammatory responses were mostly alleviated after 28 days. Besides, the body weight of these mice was also recorded in Figure 5c. During 28 days, the increasing profile of body weight of the mice in group 2 and 3 were similar with that of the control mice in group 1, revealing that PSiNPs@Au or CS/PSiNPs@Au samples had no obvious toxic side effects on mice maturing. The degradation and metabolism of PSiNPs@Au nanocomposites or CS/PSiNPs@Au hydrogels in body might induce some damages to the some tissues. So we also used H&E staining assay to examine their toxicity in heart, liver, spleen, lung, and kidney tissues of mice at day 28, recorded in Figure 5d. Compared with the mice of group 1 as a control, there was the slight degeneration in hepatocytes of liver samples, but no inflammatory infiltrates and sinusoids was observed in group 2 or 3, respectively. For others organs including spleen, lung, kidney and heart samples showed no obvious changes in morphology. Overall, CS/PSiNPs@Au hydrogels with


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excellent biodegradability and biocompatibility were safe enough for their future clinical translation. 3.5. Long-term and repeatedly localized combinatorial therapy in vivo. To investigate in vivo anticancer effect of localized combinatorial therapy based on DOX/CS/PSiNPs@Au nanocomposite hydrogels, animal experiments were also carried out. Six groups of mice bearing 4T1 tumor were randomly chosen for different treatments in our experiments: (1) PBS + NIR laser as a control, (2) DOX/CS + NIR laser, (3) PSiNPs@Au + NIR laser, (4) CS/PSiNPs@Au + NIR laser, (5) DOX/CS/PSiNPs@Au, and (6) DOX/CS/PSiNPs@Au + NIR laser. All agents including PBS, PSiNPs@Au, DOX/CS, CS/PSiNPs@Au, or DOX/CS/PSiNPs@Au solutions were intratumorally injected into mice, respectively. When the tumor regions were repeatedly irradiated by 808-nm laser (1.6 W/cm2) at day 0, 7, 14, and 28, the temperature changes and thermal imaging of these sites were recorded in Figure 6a. After 10-min exposure of NIR laser, group 3 (42 – 45 ºC), group 4 (46 – 51 ºC), or group 6 (45 – 50 ºC) exhibited remarkable heating localized in the tumor region, respectively, while the temperature of the surrounding region near the tumor only reached ~30 ºC. In contrast, the tumor temperature of group 1 and 2 showed no obvious increasing (< 35 ºC) under the same condition. After 28 days, their corresponding thermal imaging were also taken, which showed that temperature changes of group 4 and 6 could still reach ~40 ºC, however, group 3 was similar with the control group (< 35 ºC). These results demonstrated that PSiNPs@Au nanocomposites with stable photothermal effect still generated an effective conversion of NIR light-to-heat even



after long-term incubation in body, due to the protection of CS hydrogels. As shown in Figure 6c, the tumor size of mice was also measured by a calliper every other day to monitor their tumor growth. Compared with group 1, the tumor growth rate of the mice in group 2 ― 5 became slower, but their tumor volume still increased. However, an inhibition effect with significant differences (***p < 0.001) on tumor growth could be found in group 6. Furthermore, to accurately analyze the inhibition efficiency of tumors growth, the tumors were harvested and weighed, shown in Figure 6d. Comparing the inhibition efficiency of tumor growth in group 3 (27.9%) with group 4 (50.8%), significant differences (**p < 0.01) were calculated by statistical analysis. The result demonstrated that CS/PSiNPs@Au hydrogels as hyperthermia agents had better therapeutic efficacy than PSiNPs@Au nanocomposites, attributed to their stronger and longer-term photothermal effect at target tumor site, which could avoid repeated injection of therapeutic agents during cancer treatments. Especially, the mice of group 6 had the smallest tumor with significant inhibition efficiency (85.2%), in contrast to group 2 (54.9%), group 4 (50.8%), and group 5 (54.5%). Statistical analysis implied that the synergistic anticancer effect of chemo-photothermal therapy with significant differences (***p < 0.001) based on DOX/CS/PSiNPs@Au hydrogels under repeated NIR irradiation had optimal tumor ablating activity. In Figure 6b, the body weight of all mice in group 1 – 6 for in vivo toxicity tests. In contrast to the control group, there was no obvious change in others experimental groups, similar with the above-mentioned results in Figure 5c. In addition, to analyze the antitumor activity at the cellular level, H&E staining and TUNEL assays of the


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tumor sections was further carried out. In Figure 6e, H&E staining results displayed no obvious destruction of tumor tissues in group 1 and 3. However, in group 2, 4 or 5, tumor cells nuclei partially shrunk, and their correponding TUNEL images also presented that a part of cells exsiting in apoptotic state had brown signals. Clearly, in group 6, highest level of tumor cell necrosis and apotosis and obvious tumor ablation could be found, because of the severe destroy of most tumor cells with massive nuclei absence. Accordingly, these above results also supported that localized combination therapy based on DOX/CS/PSiNPs@Au + NIR laser had excellent biosaftey and the optimal tumor ablating activity for clinical applications.

4. CONCLUSION In summary, the constrcution of injectable biodegradable CS/PSiNPs@Au nanocomposite hydrogels with significant and long-term stable photothermal effect was reported. These resultant nanocomposite hydrogels showed in situ gelation, biodegradability and biocompatibility, in vitro or in vivo. Apart from an efficient loading of DOX, a long-term sustained DOX release from CS/PSiNPs@Au nanocomposite hydrogels was achieved with the help of NIR irradiation and acidic tumor environments. Furthermore, based on CS/PSiNPs@Au nanocomposite hydrogels plus repeated NIR irradiation, localized chemo-photothermal therapy of cacner demonstrated a optimal synergistic antitumor efficacy in vivo. Overall, these multifunctional CS/PSiNPs@Au nanocomposite hydrogels would have wide potential applications on cancer treatments in future clinic.



ACKNOWLEDGEMENTS This work is funded by the National Natural Science Foundation of China (No. 30930077 and No. 31000164), Natural Science Foundation of Jiangsu Province (No. BK20130964), and bilateral Chinese-Croatian scientific project (No. 6-5).

Supporting Information SEM images and rheological measurements with higher solution were recorded. This information is available free of charge via the Internet at http://pubs.acs.org/.

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Figures

Figure 1. (a) Scheme of the fabrication of PSiNPs@Au nanocomposites, (b) UV-vis spectra and their corresponding photographs recorded in the inset, (c) XRD, (d,e) HRTEM and (f-h) full and high-resolution XPS spectra, and the table of different atomic concentrations of bare PSiNPs and PSiNPs@Au nanocomposties.


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Figure 2. (a) SEM image and EDS spectra (scale bar = 200 nm), (b) the hydrodynamic size and zeta potential of oxidized PSiNPs@Au nanocomposites, and (c) T – t curves of DI water, PSiNPs (1 mg/mL), and PSiNPs@Au nanocomposites (1 mg/mL) in aqueous solution under NIR laser irradiation (1.6 W/cm2, 20 min, left 1). PSiNPs@Au solution with different concentration under NIR laser irradiation (1.6 W/cm2, 20 min, left 2). 1 mg/mL PSiNPs@Au solution under NIR laser irradiation with different power intensity for 20 min (left 3). And photothermal stability of 1 mg/mL PSiNPs@Au solution (400 μL) under repeated NIR laser irradiation (1.6 W/cm2, 20 min, left 4). Their corresponding thermal imaging was recorded in the inset, and error bar were based on standard errors of the mean (n = 3).



Figure 3. (a) Photographs of CS and CS/PSiNPs@Au precursors before (20 oC) and after gelation (37 oC), (b) G' and G'' of CS and CS/PSiNPs@Au hydrogels against temperature ranging from 20 to 70 oC, (c) FTIR spectra of CS precursors, CS hydrogels and CS/PSiNPs@Au hydrogels, (d) SEM images of CS and CS/PSiNPs@Au hydrogels with different magnification, (e) T – t curves of CS hydrogels, PSiNPs@Au solutions, and PSiNPs@Au hydrogels with the equivalent concentration of 1 mg/mL under NIR laser irradiation with the power intensity of 1.6 W/cm2 for 20 min, and (f) photothermal stability of 400 μL PSiNPs@Au hydrogels under repeated NIR laser irradiation (1.6 W/cm2, 20 min). Their corresponding thermal imaging was recorded in the inset, and error bar were based on standard errors of the mean (n = 3).


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Figure 4. (a) Photographs of CS/PSiNPs@Au hydrogels with degradation (top) and DOX loading (bottom), (b) DOX release profiles at different pH value from hydrogels with or without adding NIR irradiation, (c) weight loss of 400 μL CS and CS/PSiNPs@Au hydrogels during 28-day incubation in PBS solution at 37 oC, (d) silicon elemantal amount exsited in supernatants monitored by UVvis-NIR spectra, and (e) photothermal effect of PSiNPs@Au and CS/PSiNPs@Au during 28-day incubation under the same physiological conditions. Error bar are based on standard errors of the mean (n = 3).



Figure 5. Three random groups of BALB/c mice with five mice per group with different treatments: (1) PBS + NIR laser, (2) PSiNPs@Au + NIR laser, and (3) CS/PSiNPs@Au + NIR. (a) In situ gelation and persistence in vivo of group 1― 3 after subcutaneous injection, (b) the corresponding histology sections at different time points (scale bar = 100 μm), (c) the changes in body weight of mice in group 1 – 3 during 28 days, (d) histology sections of heart, spleen, liver, kidney, and lung tissues harvested from mice of group 1 – 3 at day 28 after injection (scale bar = 40 μm). Error bar are based on standard errors of the mean (n = 5).


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Figure 6. Six random groups of mice bearing 4T1 tumor with different treatments: (1) PBS + NIR laser, (2) DOX/CS + NIR laser, (3) PSiNPs@Au + NIR laser, (4) CS/PSiNPs@Au + NIR laser, (5) DOX/CS/PSiNPs@Au, and (6) DOX/CS/PSiNPs@Au + NIR laser. (a) Thermal imaging of different groups mice under NIR laser irradiation (1.6 W/cm2, 10 min) at day 0 and 28, respectively, (b) the changes in body weight of mice in different groups druing 28 days, (c) the changes in tumor sizes of mice in different groups during 28 days, (d) the weight of tumors removed from all groups of mice after 28 day, and (e) histology analysis including H&E staining and TUNEL of tumor slices collected from mice at day 28 (scale bar = 40 μm). Error bar are based on standard errors of the mean (n = 5, ***p < 0.001, or **p < 0.01).



For Table of Content Use Only

Multifunctional chitosan/porous silicon@Au nanocomposite hydrogels with in situ gelation, excellent biodegradability, long-term sustanied drug release, and significant photothermal effect were fabricated, which showed a remarkable localized chemophotothermal therapeutic efficacy under repeated NIR laser irradiation in vivo.

Title: Multifunctional Chitosan/Porous Silicon@Au Nanocomposite Hydrogels for Long-Term and Repeatedly Localized Combinatorial Therapy of Cancer via a Single Injection

Authors: Bing Xia*, Weiwei Zhang, Haibei Tong, Jiachen Li, Zhenyu Chen, and Jisen Shi*


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Porous Silicon@Au Nanocomposite

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