Small Gold Nanorod

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Rationally designed calcium phosphate/small gold nanorod assemblies using poly(acrylic acid calcium salt) nanospheres as templates for chemo-photothermal combined cancer therapy Shengnan Li, Lingyu Zhang, Haipeng Zhang, Zhongcheng Mu, Lu Li, and Chungang Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00612 • Publication Date (Web): 14 Nov 2017 Downloaded from http://pubs.acs.org on November 15, 2017

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ACS Biomaterials Science & Engineering

Rationally designed calcium phosphate/small gold nanorod assemblies using poly(acrylic acid calcium salt)

nanospheres

as

templates

for

chemo-

photothermal combined cancer therapy Shengnan Li,a Lingyu Zhang,a Haipeng Zhang,b Zhongcheng Mu,a Lu Li,*a and Chungang Wang*a a

College of Chemistry, Northeast Normal University, Renmin Street 5268, Changchun, 130024, P. R. China b

The First Hospital of Ji Lin University, Xinmin Street 71, Changchun, 130021, P. R. China E-mail: [email protected]; [email protected]

KEYWORDS: calcium phosphate, multifunctional nanoparticles, gold nanorod, drug delivery, chemo-photothermal therapy

ABSTRACT: Elaborately designed novel multifunctional therapeutic agents are highly desired for efficient cancer therapy. In this work, a new therapeutic nanoplatform based on calcium phosphate/small gold nanorod assemblies modified with methoxy-poly(ethylene glycol)-thiol (designated as PEGylated CaP/Au NR assemblies) is created via a mild, reproducible and simple

ACS Paragon Plus Environment

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route for the first time. The obtained PEGylated CaP/Au NR assemblies possess a lot of virtues including outstanding drug loading capacity, excellent photothermal conversion efficiency (η, ~38.5%), pH/near infrared (NIR) dual-responsive release property and good biocompatibility. After loading doxorubicin (DOX) in PEGylated CaP/Au NR assemblies, the DOX-loaded PEGylated CaP/Au NR assemblies can simultaneously supply intense heating effect and increased DOX release under 808 nm NIR laser, achieving excellent antitumor therapeutic effect in vitro and in vivo. Furthermore, the combination of DOX-loading and photothermal treatment upon PEGylated CaP/Au NR assemblies displays better therapeutic effect than single chemotherapy or photothermal therapy. Furthermore, the comprehensive methyl thiazolyl tetrazolium (MTT), hemolysis and histological assays manifest no obvious toxicity of PEGylated CaP/Au NR assemblies. Our work elucidates the great prospect of PEGylated CaP/Au NR assemblies as a therapeutic agent for synergistic chemo-photothermal cancer therapy.

INTRODUCTION In recent years, multifunctional nanoparticles (NPs) with multiple therapeutic functions are highly demanded to improve therapeutic efficacy.1-7 Such multifunctional therapeutic platforms can be constructed with well-designed configurations and components that synchronously possess targeting, stimuli response, and synergistic treatment.8, 9 These nanocarriers often use environmental stimuli to achieve desired release of therapeutic drugs to targeted sites, enhancing therapeutic efficacy as well as attenuating side effects to normal cells.10 The pH value is a prominent internal stimulus to accelerate the release of drug in tumor tissue, which is due to the lower extracellular pH (6.5-6.8), especially intracellular pH (4.5-6.5) in tumor tissues than normal tissues and the blood stream (pH 7.2-7.4).11-14 Nowadays, calcium phosphate (CaP) have gained a growing number of interest due to their potential in therapeutic delivery applications,


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because they are excellent biocompatible, biodegradable, nontoxic, pH-responsive and native to the major inorganic mineral components of bone and teeth.15-21 Recently, Zhao et al. fabricated CaP hybrid NPs by using poly(diallyldimethylammonium chloride) and poly (acrylate sodium) as dual templates for anticancer drug delivery.22 Gregor et al. synthesized the CaP/poly(D,Llactide-co-glycolide acid) NPs as delivery system for hydrophilic anionic drugs.23 It should be noted that single chemotherapy is not sufficient to achieve the desirable therapeutic efficacy in general.24-26 Currently, the main strategy is the combination of chemotherapy and photothermal therapy (PTT). Nowadays, numerous materials have been extensively studied as promising photothermal agents, such as gold-based NPs,27 carbon nanomaterials,28 CuS NPs,29 polymer NPs.30 Especially, the assembly of gold NPs have drawn much attention by virtue of their strong plasmonic coupling effect between adjacent gold NPs.31 Therefore, the integration of the advantages of CaP, gold assemblies and pH/near infrared (NIR) light dual-responsive property into a single NP can guarantee a prospective nanotheranostic agent for advanced therapeutic purposes. Nevertheless, there are no reports about combining eminent photothermal conversion capability, outstanding drug loading capacity and pH/NIR dual-responsive property into the composite NPs of CaP and gold assemblies for simultaneous therapeutic applications in vitro and in vivo. Herein, we report a mild, reproducible and simple route to generate multifunctional CaP/small gold nanorod assemblies (designated as CaP/Au NR assemblies). The obtained CaP/Au NR assemblies simultaneously combine good biocompatibility, wonderful photothermal conversion capability, superior drug loading capacity and pH/NIR dual-stimuli responsive drug delivery property into one single CaP/Au NR assembly, which can serve as a efficient therapeutic agent of cancer cells via synergistic chemo-photothermal therapy in vitro and in vivo.


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EXPERIMENTAL SECTION Materials. Poly(acrylic acid) (PAA, Mw ≈ 1800), hydroge tetrachloroaurate trihydrate (HAuCl4·3H2O), doxorubicin hydrochloride (DOX) and methoxy-poly(ethylene glycol)-thiol (PEG, Mw ≈ 2000) were obtained from Sigma-Aldrich (USA). L-ascorbic acid (AA) was purchased from Energy Chemical. Calcium hydroxide (Ca(OH)2) was purchased from Aladdin. Diammonium hydrogen phosphate ((NH4)2HPO4) was purchased from Tianjin Guangfu Technoligy Development Co., Ltd. Isopropyl alcohol (IPA) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd. All the chemicals were analytically pure and used without further purification. Deionized water was used throughout all experiments. Characterization. The morphology of the samples was measured on a TECNAI G2 F20 highresolution transmission electron microscope (TEM). UV-vis absorption spectra were determined by a U-3010 spectrophotometer (Hitachi, Japan). Fourier transform infrared (FTIR) spectra were performed with a Magna 560 FTIR spectrometer (Nicolet, USA). Confocal laser scanning microscopy (CLSM) was monitored by Olympus Fluoview FV1000. The X-ray photoelectron spectrum (XPS) was determined by an ECSALAB 250 using nonmonochromatized Al Kα radiation. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) was recorded using a Leeman ICP-AES Prodigy instrument. N2 adsorption/desorption measurement was taken with an intelligent gravimetric analyzer Autosorb-iQ (Quantachrome). Thermal imaging was taken by a T460SC, infrared camera (FLIR, Sweden). A continuous-wave diode laser (LSR808H) was applied for the laser irradiation experiment (1 W cm-1). Synthesis of poly(acrylic acid calcium salt) nanospheres (PAA-Ca NSs). Briefly, 5 mg of Ca(OH)2 and 100 µL of PAA aqueous solution (0.2 g mL-1) were added to 10 mL of deionized


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water. After agitating for 30 min, 20 mL of IPA was dripped into the above solution at ambient temperature to form the PAA-Ca NSs. Synthesis of PEGylated CaP/Au NR assemblies. To obtain the PEGylated CaP/Au NR assemblies, 100 µL HAuCl4 (30 mM) was added into the above solution under moderate stirring for 1 h. Subsequently, 50 µL AA (0.1M) was added and kept reacting for 4 h. Afterwards, 6 mg of (NH4)2HPO4 was added into the obtained mixture, which left to react for 12 h. Finally, the CaP/Au NR assemblies obtained from the above process were collected by centrifugation (5000 rpm, 7 min) and washed twice with water. Then, 1 mL of PEG (10 mg mL-1 in H2O) was added drop by drop into 1mL aqueous solution of CaP/Au NR assemblies (2 mg mL-1). The above mixture was dispersed with ultrasonic for 30 s and kept reacting over night. After that, the PEGylated CaP/Au NR assemblies was separated centrifugally and washed by water to remove excess PEG. DOX loading and release study in vitro. 50 µL of 10 mg mL-1 free DOX was dispersed in aqueous solution of PEGylated CaP/Au NR assemblies (1 mg/mL) for 24 h. The DOX-loaded PEGylated CaP/Au NR assemblies were centrifuged and washed twice with deionized water to wipe off the unloaded DOX. The DOX-loading efficiency (LE%) can be calculated by eq 1: LE%=

minitial DOX-m(remanent DOX) ×100% minitial DOX

(1)

Release profile was examined by using semipermeable dialysis bag at 37 oC. Three equal portions of the as-prepared PEGylated CaP/Au NR assemblies were redispersed in 500 µL of PBS (pH = 5.3, 7.4 and 5.3 with NIR laser) after centrifugation. The release media were moved into preprocessed dialysis bags and then immerged in corresponding PBS (3 mL). To prove that the laser irradiation can trigger drug release, one of the sample in PBS (pH = 5.3) was exposed to


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laser illumination (1 W cm-2). At selected time points, dialysis solution was quantified by UV-vis spectrophotometer at 490 nm. Measurement of photothermal performance. Briefly, NP aqueous suspensions (500 µL) with diverse concentrations (0-0.3 mg/mL) were subjected to 1 W cm-2 of NIR laser for 5 min. The temperature was recorded every 30 s. Then, the thermal images of above solutions at 0, 1, 2, 3, 4, 5 min were obtained via an infrared camera. Subsequently, the photothermal effect in the cell level was analyzed. The cells treated with PEGylated CaP/Au NR assemblies (25 µg mL-1) and NIR laser (5 min) were stained with DMEM medium containing 20 µM of calcein AM for 15 min. In addition, the groups treated with only laser radiation or PEGylated CaP/Au NR assemblies and without any treatment were investigated as comparison, respectively. Calculation of the photothermal conversion efficiency (η). The photothermal conversion efficiency (η) of PEGylated CaP/Au NR assemblies was calculated according to previous reports.32, 33 The temperature change of the sample (0.2 mg mL-1, 1 mL) was recorded under continuous irradiation (1 W cm-1). Subsequently, the irradiation by the 808 nm laser was discontinued when a short equilibration period was reached. The cooling period was monitored to determine the linearized time data versus lnθ to obtain the system time constant. In vitro cytotoxicity of PEGylated CaP/Au NR assemblies. The cytotoxicity of PEGylated CaP/Au NR assemblies on HepG-2 cells was conducted by the MTT assay. 100 µL of 1 × 105/mL cells were cultured for 12 h in 96-well plates to obtain a monolayer. Afterwards, the original medium was substituted by fresh medium containing diverse concentrations of PEGylated CaP/Au NR assemblies and DOX-loaded PEGylated CaP/Au NR assemblies. After cultivated 24h, the cells which need irradiation were treated by NIR laser (1 W cm2, 5 min). The standard MTT assay was subsequently utilized to evaluate the cell viabilities.


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Hemolysis Assay of PEGylated CaP/Au NR assemblies. Human red blood cells were first washed with physiological saline for several times. After that, 1ml of blood cells was diluted with 10 mL of PBS. 300 µL of the diluted cell suspension was separately added into 1.2 mL of PBS (negative control), deionized water (positive control) and product solutions with varying concentrations. After shaking and holding for 2 h, the supernatants were acquired by centrifugation (2000rpm, 10 min) and measured by a UV-vis spectrophotometer. The hemolysis percentage of each sample was calculated as follow: hemolysis %=

Abssample -Absnegative control ×100% Abspositive control -Absnegative control

(2)

In vivo antitumor efficacy. The chemo-photothermal combination therapeutic efficacy of our samples was investigated with Balb/c mice bearing H-22 tumors on the right shoulders. Thirtyfive tumor-bearing mice were allocated randomly to seven groups (n=5): (1) PBS (control, 200 µL), (2) PBS with NIR laser, (3) free DOX (2.5 mg kg-1), (4) free DOX with NIR laser, (5) PEGylated CaP/Au NR assemblies with NIR laser, (6) DOX-loaded PEGylated CaP/Au NR assemblies (containing 2.5 mg kg-1 DOX), (7) DOX-loaded PEGylated CaP/Au NR assemblies with NIR laser. The tumors were irradiated with laser (808 nm, 1 W cm-2, 5 min) after injection for 24 h. Meanwhile, the mice in group 2 and group 5 were photographed to acquire real-time thermal images using an infrared camera. After treatment, each mouse was weighted with a balance and the dimension of each tumor was monitored by a caliper every other day. The tumor volume (V) was computed as follow: 1 V= ×(tumor length)×(tumor width)2 2

(3)

At 11 days, all tumors were retrieved and weighted to calculate the tumor growth inhibition rate using the equation:


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inhibition %=

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(MControl group -MTreated group ) ×100% (4) MControl group

Here, MControl group and MTreated group are the average tumor mass. Histology examination. Five H-22 tumor-bearing Balb/c mice in each group were administered via tail vein of PEGylated CaP/Au NR assemblies and PBS (200 µL) as control. After treatment for 11 days, the organs (heart, liver, lung, kidney and spleen) were routinely sectioned and stained with hematoxylin and eosin (H&E). Finally, an optical microscope was used to detect the histological sections. RESULTS AND DISCUSSIONS

Scheme 1. Schematic illustration of the synthetic route of PEGylated CaP/Au NR assemblies for chemo-photothermal cancer therapy in vitro and in vivo. As shown in Scheme 1, Ca(OH)2 firstly dissolved in PAA aqueous solution to obtain PAA-Ca aqueous solution. Then, IPA was dropwise added into the mixture under constantly stirring to form PAA-Ca NSs. When the chloroauric acid (HAuCl4) and L-ascorbic acid (AA) were added, PAA-Ca/Au NR assemblies were obtained. Then, diammonium hydrogen phosphate ((NH4)2HPO4) as phosphate source was added to form the final CaP/Au NR assemblies


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Subsequently, the CaP/Au NR assemblies were functionalized with PEG to improve the physiological stability. The PEG modified CaP/Au NR assemblies were employed as pH/NIR dual-responsive drug delivery carriers for synergistic chemo-photothermal cancer therapy. Figure 1A shows the mean diameter of PAA-Ca NSs is 100 nm. Notably, the as-prepared PAA-Ca NSs can absorb water molecules inside their structure because PAA is a high waterabsorbent polymer. Owing to the good water solubility of HAuCl4 and AA, they are easily enriched in the PAA-Ca network, where the nucleation of Au0 firstly form via the redox reaction between them. Then, the final CaP/Au NR assemblies were obtained via anisotropic growth. The well-dispersed CaP/Au NR assemblies with the size of 100 nm are observed in Figure 1B and 1C clearly. High-resolution TEM image of the square-marked area in Figure 1C presents spacing of lattice fringes of 0.235 nm, which matches well with Au (Figure 1D). Elemental mapping images

Figure 1. TEM images of (A) PAA-Ca NSs. (B) PEGylated CaP/Au NR assemblies. (C) HRTEM image of a single CaP/Au NR assembly. (D) The magnified HRTEM image of squaremarked area. (E-H) The elemental mapping images of a single CaP/Au NR assembly.


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confirm that the CaP/Au NR assemblies are composed of Au (purple), Ca (blue), P (red), and O (green) elements (Figure 1E-H). ICP-AES further verifies the existence of Ca, P and Au with a percentage of 12.84 wt%, 4.75% and 24.4%, respectively. Furthermore, the FTIR spectra were measured to validate the presence of CaP, PAA and the successful graft of PEG (Figure S1). The characteristic bands at 570 and 1079 cm-1 are assigned to O–P–O stretching vibration of PO43ions.34 In the meantime, the peaks at 1411 and 1557 cm-1 are attributed to the carbonyl asymmetric stretching band (C=O) of –COO− groups, demonstrating the presence of PAA. The typical absorption band of -CH3 groups locates at 2895 cm-1, indicating the successful modification of PEG on the surface of CaP/Au NR assemblies. Notably, compared with unmodified CaP/Au NR assemblies, no precipitation of PEGylated CaP/Au NR assemblies was observed in water, PBS, or culture medium after 24 h (Figure S2), suggesting that the PEG was successfully modified and effectively improved their stability for biomedical applications. Moreover, the XPS was also utilized to investigate the surface information of CaP/Au NR assemblies (Figure S3). The binding energies at 190 and 133 eV can be allocated to P 2s and P 2p. The peaks of Ca 2s and Ca 2p at 438 and 347 eV are attributed to Ca2+. The binding energy at 531 eV is the characteristics of O 1s and the appearance of Au 4f peaks at 87.2 and 87.5 eV indicates the presence of Au.35,

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Additionally, N2 adsorption/desorption measurement was

performed to study the porous nature of PEGylated CaP/Au NR assemblies. Figure S4 exhibits that the as-fabricated PEGylated CaP/Au NR assemblies have a uniform pore width of 3.5 nm, which permit the drug to enter and be released. All the above results testify the successful synthesis of PEGylated CaP/Au NR assemblies and their potential for drug delivery. The optical property of the as-prepared PEGylated CaP/Au NR assemblies was then characterized with UV-vis absorption spectroscopy, as displayed in Figure 2A. A pronounced


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absorbance peak is displayed at 790 nm, which motivates us to study the potential of PEGylated CaP/Au NR assemblies in PTT. Therefore, the PEGylated CaP/Au NR assemblies solutions with diverse concentrations were irradiated by laser for 5 min (1 W cm-2, 808 nm). Figure 2B and 2C clearly demonstrate that photothermal conversion effect can increase monotonically with PEGylated CaP/Au NR assemblies concentration and the solution temperature can be easily controlled to above 42 oC, which is effective for causing cell damage and death.37, 38 Furthermore, the photothermal conversion efficiency (η) is calculated to be 38.5% (Figure S5), which is markedly higher than the corresponding values for gold nanoshells (13%), gold vesicles (18%) and gold nanorods (22%) (Table S1 of the Supporting Information). These results prove that the PEGylated CaP/Au NR assemblies can act as superb photothermal conversion agents for cancer therapy.

Figure 2. (A) UV-vis absorption spectrum of PEGylated CaP/Au NR assemblies. (B) Photothermal curves and (C) infrared thermal images of PEGylated CaP/Au NR assemblies at different concentrations in aqueous solution (1 W cm-2, 5 min). (D) The release profiles of DOX from DOX-loaded PEGylated CaP/Au NR assemblies in PBS buffer.


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To test the drug storage ability and pH/NIR dual-responsive release property of PEGylated CaP/Au NR assemblies, we selected DOX as a modal anticancer drug. The loading efficiency (LE) and the drug loading content is 84.5% and 0.422 mg DOX per milligrame PEGylated CaP/Au NR assemblies (Figure S6), which may be due to the mesoporous structure and electrostatic attraction between positively charged DOX and negatively charged carboxylic acid groups on PAA. Figure 2D shows the curves of cumulative DOX release from DOX-loaded PEGylated CaP/Au NR assemblies at different conditions. It can be found that the release amount of DOX is only 13.5% after releasing for 7 h at neutral condition, whereas approximately 49.8% of DOX is released from PEGylated CaP/Au NR assemblies at pH 5.3, which simulates the pH of endosomes and lysosomes in tumors. Hence, the PEGylated CaP/Au NR assemblies with the pH-responsiveness are advantageous for targeting tumor sites as drug vehicles and minimizing the side effects.39, 40 Additionally, we measured the DOX release profile in pH 5.3 PBS with and without NIR laser. After the suspension of drug-loaded PEGylated CaP/Au NR assemblies is exposed to laser irradiation for 10 min, a higher rate release of drug is observed. When the laser irradiates the suspension again, the amount of DOX release increases sharply once more. The phenomenon can be ascribed to the extraordinary photothermal conversion effect of PEGylated CaP/Au NR assemblies, which can generate heat and effectively accelerate the release of DOX. Our results indicate that the PEGylated CaP/Au NR assemblies can achieve pH/NIR dual-sensitive release and are invaluable therapeutic agents for minimizing side effects and improving therapeutic efficacy. The remarkable pH/NIR dual-responsive property and photothermal conversion performance of PEGylated CaP/Au NR assemblies suggest that they can serve as eminent therapeutic agents for chemo-photothemal therapy of cancer if they have good biocompatibility. Therefore, a


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Figure 3. In vitro cell viabilities of HepG-2 cells with (A) various concentrations of PEGylated CaP/Au NR assemblies and (B) different treatments. (C) Fluorescence images of HepG-2 cells stained with calcein AM after in vitro photothermal ablation under NIR laser irradiation with and without the addition of PEGylated CaP/Au NR assemblies (scale bars: 100 µm). (D) The hemolysis property in the human blood red cells. standard MTT assay was used to assess the potential cytotoxicity of PEGylated CaP/Au NR assemblies on HepG-2 cancer cells. The cell viability reaches 96% even at a high dose (100 µg mL-1) when HepG-2 cells were treated with PEGylated CaP/Au NR assemblies (Figure 3A). Comparatively, the viability is extremely low of the cells treated with both DOX-loaded PEGylated CaP/Au NR assemblies and NIR irradiation due to the combined chemophotothermal therapy effect, as shown in Figure 3B. These results strongly verify that the PEGylated CaP/Au NR assemblies have good biocompatibility and show a great potency for synergetic chemo-photothermal therapy. The live staining images with calcein AM was investigated to confirm the photothermal ablation of cancer cells in vitro (Figure 3C). The cells show no apparent apoptosis when they are exposed to PEGylated CaP/Au NR assemblies or laser alone. In contrast, the region, where the cells treated by PEGylated CaP/Au NR assemblies with


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NIR laser, exhibits black, suggesting conspicuous cell death. The results confirm that the PEGylated CaP/Au NR assemblies can effectively ablate cancer cells assisted by NIR laser irradiation. The hemolysis assay was applied to clarify the biocompatibility in vivo of PEGylated CaP/Au NR assemblies (Figure 3D). No visible red color is found in the centrifuge tubes containing PEGylated CaP/Au NR assemblies with varied concentrations. The hemolytic value of PEGylated CaP/Au NR assemblies with maximum concentration (1000 µg mL-1) is only 2.84%, indicating that the PEGylated CaP/Au NR assemblies are hemocompatible and can be potentially used in vivo. The in vivo antitumor performance was further examined. Thirty-five Balb/c mice bearing H-22 tumors were assigned to seven groups (n = 5): PBS (control), PBS with NIR laser, free DOX,

Figure 4. (A) IR thermal images of tumor-bearing mice after injection with PBS or PEGylated CaP/Au NR assemblies exposed to the 808 nm laser at 1 W cm-2. (B) Body weight recorded for mice after treatment of each group. (C) Relative tumor volume recorded for mice after treatment of each group. Statistical significance: **p < 0.01, ***p < 0.001.


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free DOX with NIR laser, PEGylated CaP/Au NR assemblies with NIR laser, DOX-loaded PEGylated CaP/Au NR assemblies, DOX-loaded PEGylated CaP/Au NR assemblies with NIR laser. In order to clarify the photothermal effect of PEGylated CaP/Au NR assemblies in vivo, the thermal images of the mouse injected with PEGylated CaP/Au NR assemblies or PBS were captured at different time points in tumor regions (Figure 4A). The tumor temperature of the mouse injected with PEGylated CaP/Au NR assemblies is significantly higher than the PBS group. The outcomes reveal the superb photothermal performance of PEGylated CaP/Au NR assemblies in vivo. The body weight of the mice in each group was shown in Figure 4B. Obviously, no significant weight change is observed, displaying no noticeable side effects during therapy period. The tumor volume of each mouse is plotted in Figure 4C. The average tumor

Figure 5. (A) Representative photograph of excised tumors from each group at the 11th day post treatment. (B) The mean tumor weights for each group on the last day of experiment and (C) the tumor inhibition rate of each group. Statistical significance: **p < 0.01, ***p < 0.001. (D) Hematoxylin and eosin (H & E) stained histological sections of major organs (heart, liver, lung, kidney and spleen).


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volume of the control group is the largest among all the groups. Under the treatment with PEGylated CaP/Au NR assemblies with NIR laser and DOX-loaded PEGylated CaP/Au NR assemblies, the efficient tumor restraint is observed. Unfortunately, these two methods can not eliminate the tumors completely. However, the DOX-loaded PEGylated CaP/Au NR assemblies with NIR laser group has the smallest tumor volume and almost achieves the complete elimination of tumors. After 10 days of injection, the tumors of all mice were excised (Figure 5A) and weighed to obtain the tumor inhibition rates (Figure 5B and 5C). The mice treated with DOX-loaded PEGylated CaP/Au NR assemblies upon NIR laser show the highest tumor inhibition rate of about 97%, compared to PEGylated CaP/Au NR assemblies with NIR laser (73%), DOX-loaded PEGylated CaP/Au NR assemblies (56%), free DOX with NIR laser (34%) and free DOX treated group (29%). All the results imply that the synergistic chemophotothermal therapy of our system can effectively restrain tumor growth. Such a potent antitumor effect can be attributed to the massive accumulation of DOX-loaded PEGylated CaP/Au NR assemblies due to the enhanced permeability and retention (EPR) effect in the cancerous tumor space.41, 42 To test the biocompatibility and potential toxicity of PEGylated CaP/Au NR assemblies in vivo, we performed histological analysis of major organs (heart, liver, lung, kidney and spleen), respectively. As displayed in Figure 5D, there is no obvious histological lesion or inflammation appearing in staining histological sections. The results clearly confirm that the PEGylated CaP/Au NR assemblies have no detectable toxicity in vivo and can be promising candidates as synergistic therapeutic agent in biological medicine applications. CONCLUSIONS In summary, the PEGylated CaP/Au NR assemblies have been designed and synthesized for the first time to serve as novel combinational therapeutic nanoplatforms for the cancer treatment.


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The obtained ultimate products possess excellent photothermal conversion efficiency owing to structure of small gold nanorod assemblies. Simultaneously, the PEGylated CaP/Au NR assemblies can be employed as nanocarriers with pH/NIR dual-responsive release property. Furthermore, the comprehensive MTT, hemolysis and a series of animal experiments reveal the good physiological stability and biocompatibility of PEGylated CaP/Au NR assemblies. Importantly, the outstanding synergistic chemo-photothermal antitumor effect is achieved both in vitro and in vivo after treatment with DOX-loaded PEGylated CaP/Au NR assemblies and NIR irradiation. Taken together, the PEGylated CaP/Au NR assemblies hold enormous potential for the combined chemo-photothermal therapy of cancer and provide technical guidance for the rational design of theranostic agents.

ACKNOWLEDGMENT We would like to thank the National Natural Science Foundation of China (Grant No. 21573040 and 21603029), the Natural Science Foundation and Science and Technology Development Planning of Jilin Province (20150204086GX and 20170520148JH), the Program for New Century Excellent Talents in University (NCET-13-0720), the Fundamental Research Funds for the Central Universities (2412016KJ007 and 2412016KJ020), the China Postdoctoral Science Foundation (2016M600224), the Jilin Provincial Research Foundation for Basic Research (20160519012JH) and Jilin Provincial Key Laboratory of Micro-Nano Functional Materials (Northeast Normal University). Supporting Information. Calculation method and comparison of photothermal conversion efficiency; FTIR spectra of PEGylated CaP/Au NR assemblies and CaP/Au NR assemblies; photographs of CaP/Au NR assemblies modified with and without PEG in water, PBS buffer and


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Table of Contents Rationally designed calcium phosphate/small gold nanorod assemblies using poly(acrylic acid calcium salt) nanospheres as templates for chemo-photothermal combined cancer therapy Shengnan Li,a Lingyu Zhang,a Haipeng Zhang,b Zhongcheng Mu,a Lu Li,*a and Chungang Wang*a

The PEGylated calcium phosphate/small gold nanorod assemblies have been first explored for synergistic chemo-photothermal therapy.


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Small Gold Nanorod

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