Facile Approach to Synthesize Gold Nanorod@Polyacrylic Acid/Calcium Phosphate Yolk–Shell Nanoparticles for Dual-Mode Imaging and pH/NIR-Responsive Drug Delivery
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A facile strategy to fabricate gold nanorod@polyacrylic acid/calcium phosphate (AuNR@PAA/CaP) yolk–shell nanoparticles (NPs) composed with a PAA/CaP shell and an AuNR yolk is reported. The as-obtained AuNR@PAA/CaP yolk–shell NPs possess ultrahigh doxorubicin (DOX) loading capability (1 mg DOX/mg NPs), superior photothermal conversion property (26%) and pH/near-infrared (NIR) dual-responsive drug delivery performance. The released DOX continuously increased due to the damage of the CaP shell at low pH values. When the DOX-loaded AuNR@PAA/CaP yolk–shell NPs were exposed to NIR irradiation, a burst-like drug release occurs owing to the heat produced by the AuNRs. Furthermore, AuNR@PAA/CaP yolk–shell NPs are successfully employed for synergic dual-mode X-ray computed tomography/photoacoustic imaging and chemo-photothermal cancer therapy. Therefore, this work brings new insights for the synthesis of multifunctional nanomaterials and extends theranostic applications.
KeywordsYolk–shell structure Calcium phosphate Dual-mode imaging Photothermal therapy Drug delivery
We report a facile strategy to fabricate gold nanorod@polyacrylic acid/calcium phosphate (AuNR@PAA/CaP) yolk–shell nanoparticles.
The as-obtained AuNR@PAA/CaP yolk–shell nanoparticles (NPs) possess ultrahigh doxorubicin (DOX) loading capability (1 mg DOX/mg NPs), superior photothermal conversion property (26%) and pH/near-infrared (NIR) dual-responsive drug delivery performance, which were employed for synergic dual-mode X-ray computed tomography (CT)/photoacoustic (PA) imaging and chemo-photothermal cancer therapy.
This work brings new insights for the synthesis of multifunctional nanomaterials and extends theranostic applications.
Multifunctional nanoparticles (NPs) with complementary capacities of multimodal imaging and therapeutic functions have drawn extensive attention in biomedical areas [1, 2, 3, 4, 5, 6]. Nowadays, calcium phosphate (CaP) NPs have gained increasing attention in anticancer drug delivery because of their excellent biocompatibility and pH-sensitivity, originating from their chemical nature and mimics the inorganic component of biological hard tissues, such as bone and tooth [7, 8, 9, 10, 11]. However, owing to the lack of the theranostic capability, single CaP NPs are difficult to achieve simultaneous imaging and cancer theranostics. The main strategy turns to synthesize CaP-based multifunctional NPs with the capability of diagnosis and therapeutics. For example, Liu et al. developed a synthetic route to obtain amphiphilic gelatin–iron oxide core/CaP shell NPs, integrating magnetic resonance imaging and chemotherapy for killing cancer cells . It should be noted that sole modality imaging or therapy cannot enhance the anticancer efficiency in comparison with multimodal imaging and multiple therapeutic. Therefore, it is absolutely imperative to propose a simple synthetic method for fabricating CaP-based multifunctional NPs that possess the capacities of simultaneous dual-mode imaging diagnosis and chemo-photothermal therapy.
Among numerous photothermal nanomaterials, the optical property of gold nanorod (AuNRs) presented good photothermal therapy (PTT) effect owing to their tunable localized surface plasmon resonance (LSPR) across the NIR region [13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27]. Meanwhile, AuNRs possess photoacoustic (PA) and X-ray computed tomography (CT) imaging capacity, due to the strong near-infrared (NIR) absorption and X-ray opacity [28, 29]. Recently, Lu and co-workers prepared yolk–shell AuNR@hollow periodic mesoporous organosilica nanospheres only for chemo-photothermal therapy of breast cancer . However, their materials still have problems that need to be tackled including large particle size, poor biocompatibility and low drug loading capability. Moreover, up to now, there have been no reports on the synthesis of CaP-based yolk–shell NPs composed of a CaP shell and a removable AuNR yolk. It is worth mentioning that the yolk–shell architecture is a promising candidate for developing drug loading systems compared with the core–shell structure, owing to the unique cavity, large surface area and excellent loading capacity [31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41]. Hence, developing a facile synthetic strategy to fabricate well-dispersed AuNR core/CaP shell NPs with ultrahigh doxorubicin (DOX) loading capability and pH/NIR dual-responsive drug delivery performance for dual-mode CT/PA imaging and synergic chemo-photothermal therapy of cancer cells still remains a great challenge.
Herein, we have successfully developed for the first time a mild and facile route to fabricate AuNR@PAA/CaP yolk–shell NPs for synergistic dual-mode CT/PA imaging and chemo-photothermal therapy of cancer cells.
3 Experimental Details
Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), silver nitrate (AgNO3), hexadecyltrimethylammonium bromide (CTAB), L-ascorbic acid (LAA), sodium borohydride (NaBH4), polyacrylic acid (PAA, M W ≈ 1800) and doxorubicin hydrochloride (DOX) were purchased from Sigma-Aldrich (USA). Disodium hydrogen phosphate (Na2HPO4), hydrochloric acid (HCl, 37% weight in water), tetraethylorthosilicate (TEOS), methanol (HCHO, 37% weight in water), anhydrous ethanol (CH3CH2OH), sodium hydroxide (NaOH), calcium hydroxide (Ca(OH)2) and isopropyl alcohol (IPA) were purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. All chemicals were used without any further purification. All glassware was first cleaned with freshly prepared aqua regia and extensively rinsed with water before it was used. The deionized (DI) water was used in all experiments.
Transmission electron microscope (TEM) measurements were taken on a JEOL-2100F transmission electron microscope at 200 kV (Hitachi, Japan). The irradiation was performed using a NIR laser with a center wavelength of 808 nm (Beijing Kaipulin Optoelectronic Technology Co.). The scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) spectrum were carried out with a JEOL JSM-7610F scanning electron microscope. X-ray photoelectron spectra (XPS) were measured on an ECSALAB 250 using non-mono-chromatized Al-Kα radiation. Fourier transform infrared (FTIR) spectra were performed by a Magna 560 FTIR spectrometer (Nicolet, USA). The UV–Vis spectra were recorded at room temperature on a Japan JASCO V-570 spectrometer fluorescence spectrophotometer. The inductively coupled plasma atomic emission spectroscopy (ICP-AES) was determined by a Leeman ICP-AES Prodigy instrument.
3.3 Synthesis of AuNRs
AuNRs with an aspect ratio of ~4 was prepared by a seed-mediated growth method using CTAB surfactants as reported previously . Firstly, the synthesis of gold nanoseeds: HAuCl4 (10 mM, 0.25 mL) and CTAB (0.1 M, 10 mL) were added into a 10 mL glass bottle with gentle mixing before a freshly prepared and ice-bathed NaBH4 solution (10 mM, 0.6 mL) was injected and then the mixture was magnetically stirred until the color changed from golden yellow to brown indicating that 3.5 nm gold nanoseeds were obtained. The seed solution was then kept at 30 °C for at least 2 h before usage. Secondly, the growth of gold nanorods: AgNO3 (10 mM, 1 mL) and HAuCl4 (10 mM, 5 mL) were added into CTAB (0.1 M, 100 mL) in a 250 mL glass bottle, followed by the addition of HCl (0.1 M, 700 μL), LAA (0.1 M, 700 μL) and gold seeds (100 μL). The growth solution was left at 30 °C overnight. Finally, CTAB-stabilized AuNRs were prepared, then the sample solution was centrifuged several times to remove superfluous CTAB and redispersed into 100 mL of DI water for further use.
3.4 Synthesis of AuNR@mSiO2 Core–Shell NPs
When the pH value of 16 mL AuNRs solution was adjusted to about 10 with NaOH (0.1 M) solution under stirring, 30 μL of TEOS was subsequently injected slowly under vigorous stirring. The reaction mixture was allowed to proceed for 4 h to form an approximately 15-nm thick silica layer on the surface of AuNRs. Finally the AuNR@mSiO2 core–shell NPs were isolated by centrifugation and washed with DI water several times and then re-dissolved in 5 mL DI water for further use.
3.5 Synthesis of AuNR@PAA/CaP Yolk–Shell NPs
In a 50 mL of flask, 8 mg Ca(OH)2 and 100 μL of PAA aqueous solution (0.2 g mL−1) were firstly added to 10 mL DI water under magnetic stirring. In succession, 5 mL of AuNR@mSiO2 core–shell NPs solution was dispersed into the solution to form a suspension. Then, 20 mL of IPA was dripped into the suspension under magnetic stirring. Afterward, 24 mg Na2HPO4 was added to the above mixed suspension under magnetic stirring for 10 h. After being centrifuged and washed with DI water, the AuNR@mSiO2@PAA/CaP core–shell NPs were obtained. Finally, after etching the mSiO2 layer, the AuNR@PAA/CaP yolk–shell NPs were obtained.
3.6 DOX Loading and Release
Two portions of the prepared DOX-loaded AuNR@PAA/CaP yolk–shell NPs at equal amount were redispersed in pH 7.4 and pH 5.0 PBS (0.5 mL) and then transferred into pretreated semipermeable dialysis bags at 37 °C, respectively. After the two bags were immersed into 5 mL of PBS buffer (pH 7.4 and pH 5.0) at 37 °C, the amount of released DOX moving into the solution was determined by measuring the absorbance at 480 nm in a UV–Vis spectrophotometer at selected time intervals. To confirm that the laser irradiation can induce the drug release, another experiment was also carried out under the same procedures. The sample was immersed in PBS buffer at pH 5.0 with NIR irradiation (808 nm, 1.0 W cm−2) at selected time intervals. DOX concentration in the supernatant was determined by UV–Vis spectrophotometer as well. The samples (1 mg) were put into pH 5.0 PBS (3 mL). The supernatants were collected by centrifugation at selected time intervals and analyzed by ICP-AES to measure the Ca content.
3.7 Cell Culture
Human hepatocellular carcinoma (HeLa) cells were grown as a monolayer in a humidified incubator at 37 °C in a 95% air 5% CO2 in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum.
3.8 The Photothermal Therapy of AuNR@PAA/CaP Yolk–Shell NPs
The photothermal effect of AuNR@PAA/CaP yolk–shell NPs was measured in aqueous solution. Briefly, AuNR@PAA/CaP yolk–shell NPs (1 mL) with various concentrations were exposed to the 808-nm NIR laser (power density 1.0 W cm−2) for 5 min. The temperature was recorded every 30 s. Subsequently, the photothermal effect in the cell level was analyzed by using calcein AM staining method. Calcein AM can only penetrate in live cells and emit green fluorescence. The cells were seeded in a 24-well plate (2.5 × 104 cells per well) for 24 h. Then, the cells were divided into four groups: group 1 with PBS only; group 2 incubated with AuNR@PAA/CaP yolk–shell NPs (25 μg mL−1); group 3 incubated with NIR; group 4 incubated with both NPs and NIR laser. The NIR laser used in this experiment was 1.0 W cm−2 and the irradiation time was 5 min. After all the treatment, the cells were finally stained with calcein AM.
3.9 Calculation of the Photothermal Conversion Efficiency (η)
3.10 In Vitro Cytotoxicity Evaluation Against HeLa Cells
3.11 CT Imaging of AuNR@PAA/CaP Yolk–Shell NPs In Vitro
AuNR@PAA/CaP yolk–shell NPs with the Au concentrations in a range of 0–0.25 M were poured into tubes and placed in a self-made scanning holder. CT images were acquired under the following parameters: thickness, 1.0 mm; pitch, 120 kV, 280 mA; field of view, 300 mm; gantry rotation time, 4.95 s. CT data were analyzed by recording the Hounsfield units (HUs) for regions of interest. The raw data were reconstructed using 3D-Med software to acquire the CT images and calculate the CT values.
3.12 PA Signal Measurement In Vitro
To test the linearity of the PA signal as a function of AuNR@PAA/CaP yolk–shell NPs, 0.2 mL of the AuNR@PAA/CaP yolk–shell NPs aqueous suspension with different Au concentrations (0, 0.4, 0.5, 0.7 and 0.9 mM) was added to the agar-phantom container and placed in the MOST inVision 128 (iThera) system for signal detection. A complete PA image of the phantom was collected at 808 nm.
4 Results and Discussion
4.1 Synthesis and Characterization of AuNR@PAA/CaP Yolk–Shell NPs
To further confirm the details of the AuNR@PAA/CaP yolk–shell NPs, a series of characterization were performed. Figure S2 displays the FTIR spectrum of the AuNR@PAA/CaP yolk–shell NPs. The broad peak around 3500 cm−1 originates from moisture in the samples. The absorption bands at 1558 and 1419 cm−1 are attributed to the characteristic peaks of the carbonyl (C = O) of the carboxylic acid (–COO–) group, confirming the presence of PAA. Moreover, the characteristic peaks appeared at 565 and 1087 cm−1 can be assigned to the characteristic peaks of O–P–O bending and asymmetric stretching of PO4 3− ions. To further verify the crystallinity of obtained AuNR@PAA/CaP yolk–shell NPs, the XRD pattern of the sample was carried out. The diffraction peaks well match the planes of Au crystals, indicating the formation of crystalline Au. In addition, the characteristic peak at around 25°–35° is assigned to the amorphous CaP (Fig. S3, ESI). Surface information of the AuNR@PAA/CaP yolk–shell NPs was characterized by XPS in Fig. S4. The peaks at 190 and 132 eV are assigned to P 2s and P 2p, respectively. The Ca 2s and Ca 2p peaks appear at binding energies of 438 and 347 eV, and the binding energies of 531 and 87 eV are attributed to O 1s and Au 4f, respectively. EDX spectrum confirms that the AuNR@PAA/CaP yolk–shell NPs are composed of Au, Ca, C, P, O (Fig. S5, ESI). Meanwhile, an ICP-AES analysis quantified the weight percentage of Au and Ca is 13.5% and 19.4%, respectively.
4.2 Photothermal Effect
Next, to investigate the photothermal effect in vitro, the fluorescence microscopy images were obtained by staining the live cells with calcein AM, which can emit strong green fluorescence in live cells. As shown in Fig. 2e, when the HeLa cells were treated with both 808-nm laser irradiation and AuNR@PAA/CaP yolk–shell NPs, the dark region matches the laser irradiation area very well, suggesting the death of HeLa cells upon exposure to the laser irradiation. As a contrast, the cell viability and cell density are not reduced when the samples were treated by only the yolk–shell NPs or laser irradiation, compared with the control group. Obviously, the results illustrate that the yolk–shell NPs can transform laser energy into heat energy, which could kill HeLa cells and reduce adverse side effects to normal tissues as photothermal agents.
4.3 DOX Loading, pH/NIR-Responsive Controlled Release and Cytotoxicity Assays in vitro
All the results demonstrate that the AuNR@PAA/CaP yolk–shell NPs can achieve pH/NIR dual-responsive release, making it possible for minimizing the drug stimulation to normal cells and improving antitumor efficacy. As depicted in Fig. 3c, the cell viability was as high as 98.2% when the HeLa cells were treated with AuNR@PAA/CaP yolk–shell NPs at a high concentration of 50 μg mL−1, proving that the yolk–shell NPs exhibit excellent biocompatibility. DOX-loaded AuNR@PAA/CaP yolk–shell NPs reveal a similar cell toxicity against HeLa cells with the same concentration of DOX, confirming that the yolk–shell NPs are effective drug vehicles. It should be noted that single yolk–shell NPs and DOX-loaded yolk–shell NPs under 808-nm laser irradiation, respectively, exhibit different ability for photothermal ablation of cancer cells, and the latter shows a higher cell toxicity. The results indicate that the combination of the chemo- and photothermal therapy of DOX-loaded AuNR@PAA/CaP yolk–shell NPs could significantly enhance the efficiency of tumor ablation.
4.4 Confocal Laser Scanning Microscopy
4.5 CT and PA Imaging in vitro
In summary, we have successfully fabricated novel AuNR@PAA/CaP yolk–shell NPs which possess ultrahigh anticancer drug loading, superior photothermal conversion ability, good biocompatibility and pH/NIR dual-responsiveness. The yolk–shell NPs can serve as promising theranostic agents for simultaneous dual-mode CT/PA imaging and chemo-photothermal therapy. Furthermore, this work could encourage further study in the construction of CaP-based multifunctional yolk–shell NPs using NIR absorbing, fluorescent and magnetic nanomaterials for cancer theranostics.
We would like to thank the National Natural Science Foundation of China (Grant Nos. 21573040 and 21603029), the Natural Science Foundation and Science and Technology Development Planning of Jilin Province (20150204086GX and 20170520148JH), 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 Advanced Energy Materials (Northeast Normal University).
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