Gold nanoparticles coated with a polydopamine layer and dextran brush surface for diagnosis and highly efficient photothermal therapy of tumors

Abstract

In this study, multifunctional nanoparticles were fabricated for tumor diagnosis and therapy. The gold nanoparticles, which were prepared from HAuCl₄ and aspartate via in situ UV irradiation, were coated with polydopamine (PDA) by self-polymerization of dopamine. Subsequently, the Au@PDA nanoparticles were coated with the BSA–dextran conjugate (BD). The photothermal transduction efficiency of the Au@PDA@BD nanoparticles was 17.0%. During 808 nm laser irradiation, the temperature rise and photothermal cytotoxicity of the Au@PDA@BD nanoparticle solution depended on the polydopamine concentration. Because of the Au core, the Au@PDA@BD nanoparticles improved the brightness and resolution of tumor CT imaging after intratumoral injection of the nanoparticles into H22 tumor-bearing mice. Due to the dextran brush surface, Au@PDA@BD nanoparticles could prolong the circulation time in blood and accumulate at the tumor site after intravenous injection, and thus NIR laser irradiation could cause the tumor temperature to rise to ablate the tumor completely. Only once treatment by intravenous injection of Au@PDA@BD nanoparticles into the tumor-bearing mice followed by 808 nm laser irradiation for 10 min could completely cure the tumors even if the original tumor volume was about 250 mm³. At the same conditions, Au@PDA nanoparticles without the dextran brush surface had no treatment efficacy. Furthermore, Au@PDA@BD nanoparticles plus the laser irradiation had no histological toxicity on the main organs of the treated mice. The Au@PDA@BD nanoparticles show a great potential for highly efficient photothermal therapy of tumors.

---

Introduction

As an innovative therapy for tumors, photothermal therapy has received more and more attention.^1,2 Photothermal therapy has many advantages, such as a high effect for tumor therapy,³ non-toxicity for normal tissues, good tissue-penetrating ability,⁴ and minimal invasiveness.⁵ Many nanomaterials, such as gold nano-rods,⁶ magnetic nanoparticles,⁷ carbon nanotubes⁸ and graphene⁹ have been reported as photothermal therapy agents. Recently, Liu et al. discovered that polydopamine (PDA) was also a promising photothermal therapy agent which could efficiently kill cancer cells and suppress tumor growth without damaging healthy tissues.¹⁰ Furthermore, PDA possesses many merits, including paramagnetism,¹¹ adhesive property,¹² chemical reactivity,¹³ biocompatibility¹⁰ and biodegradation.¹⁴

X-ray CT (computer tomography) imaging, a non-invasiveness diagnostic technology with deep tissue penetration and high spatial resolution, has been widely used in clinical diagnostics.^15,16 Gold nanoparticles (Au NPs) have favourable X-ray attenuating property due to the high density and high atomic number of Au,^17,18 and have been investigated as new CT imaging agents.¹⁹ Besides, Au NPs have potential applications in therapy,²⁰ bio-sensing,²¹ and drug delivery²² due to their unique size- and shape-related electronic, magnetic, catalytic and optical properties.²³

Recently, multifunctional theranostic agents, which combine tumor imaging and therapeutic functions together, have received great attention.^24–26 PDA-coated Au NPs (Au@PDA NPs), which can be synthesized by self-polymerization of dopamine on the surfaces of Au NPs, have well controlled Au core size and PDA coating layer thickness as well as excellent biocompatibility as reported by Liu et al.²⁷ Au@PDA NPs are potential theranostic agent for CT imaging and photothermal therapy of tumors. However, most of Au@PDA NPs were quickly cleared out from blood after intravenous injection into mice, and the NPs were mainly accumulated in the liver and spleen because of the strong phagocytosis in reticuloendothelial system organs.²⁷

Alhareth et al. reported that the NPs with a dense brush dextran surface were less cleared out by liver and they could reach highly vascularized organs at higher concentrations.²⁸ Our previous study proved that the paclitaxel loaded emulsion with dextran brush surface had better tumor inhibition and survivability efficacies after intravenous injection into murine ascites hepatoma H22 tumor-bearing mice compared with commercial paclitaxel injection.²⁹ In order to prolong the circulation time of Au@PDA NPs in blood and increase the accumulation of Au@PDA NPs in tumor via enhanced permeability and retention (EPR) effect, herein, we fabricated Au@PDA@BD NPs with Au core, PDA layer, and dextran brush surface by coating Au@PDA NPs with BSA–dextran conjugate (BD). Intratumoral injection and intravenous injection of Au@PDA@BD NPs into H22 tumor-bearing mice with original tumor volumes of 100–250 mm³ and then NIR laser irradiation were performed to investigate the tumor photothermal therapy efficacy of the NPs. The tumor CT imaging of the NPs was also evaluated. In addition, the cell toxicity and histological toxicity were investigated to prove the biocompatibility of the NPs.

Experimental

Materials

L-Glycine (Gly, 99.0%) was purchased from Bio Basic Inc. L-Aspartate (Asp, 98.0%) was from J&K Scientific Ltd. Dopamine hydrochloride (DA, 98%) was from Sigma. Bovine serum albumin (BSA, fraction V, 99%) and dextran (10 kDa) were from Sangon Biotech Shanghai Co., Ltd. Chloroauric acid (HAuCl₄, AR) was from Sinopharm Chemical Reagent Co. Ltd. DMEM cell culture medium and fetal bovine serum were from GIBCO BRL Life Technologies Inc. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was from Promega Co. All other chemicals were from Sinopharm Chemical Reagent Co. Ltd.

Preparation and characterization of Au NPs, Au@PDA NPs and Au@PDA@BD NPs

Au NPs with Gly and Asp surfaces, named Au@Gly and Au@Asp, respectively, were synthesized from the mixed solutions of HAuCl₄ and the corresponding amino acid by in situ UV light irradiation at pH 10 with Au concentration of 0.05 mg mL⁻¹ as reported previously.³⁰ The Au@Gly solution was added into freshly prepared dopamine solution to reach an Au concentration of 0.025 mg mL⁻¹. The mixture was adjusted to pH 8.5 and then stirred at room temperature for 24 h to produce PDA coating layer on the surface of Au NPs. The Au@PDA NPs with desired PDA layer thicknesses were obtained at the weight ratios of dopamine to Au 2 : 1, 4 : 1 and 8 : 1; the corresponding NPs were named as Au@PDA-1, Au@PDA-2 and Au@PDA-3. Au@PDA-4 NPs were prepared by using Au@Asp and a weight ratio of dopamine to Au 8 : 1.

BD was synthesized by Maillard reaction as described previously with a molar ratio of BSA to dextran 1 : 4.³¹ BD solution was mixed with Au@PDA-4 solution at a weight ratio of BD to dopamine 3.2 : 1, that is, a weight ratio of BD to Au@PDA-4 2.8 : 1. The mixed solution was adjusted to pH 2.5 and then stirred at room temperature for 24 h followed by a heat treatment at 90 °C for 1 h to coat Au@PDA-4 NPs with BD. The produced NPs were named as Au@PDA-4@BD NPs.

All the NPs described above were used directly without purification. The NPs were freeze-dried and then re-dispersed in aqueous solution to obtain desired concentration.

Z-Average hydrodynamic diameter (D_h), polydispersity index (PDI) and ζ-potential values of the NPs were measured on a Zetasizer Nano instrument (ZS 90, Malvern Instruments). UV-vis absorption spectra of the NPs were measured on a UV-vis spectrometer (UV-2550, Shimadzu). FTIR spectra were obtained on a FTIR spectrometer (Nicolet 6700, Thermo Fisher). The NPs were purified before the FTIR measurement to remove the excess amino acid and BD in the solutions. Transmission electron microscopy (TEM) observations were conducted on an electron microscope (FEI Tecnai G2 TWIN, FEI company). TEM sample was prepared by depositing diluted solution onto a carbon-coated copper grid and drying at room temperature.

In vitro photothermal conversion of the NPs

Various Au@PDA and Au@PDA-4@BD solutions of 0.1 mL with different PDA concentrations were separately exposed to 808 nm laser (MDL-III-808-2.5W, Changchun New Industries Optoelectronics Technology Co., Ltd.) irradiation for 10 min at a power density of 5 W cm⁻² and a light spot size of 5 mm × 8 mm. The solution temperature was measured every 60 s using a thermoelectric couple. The photothermal conversion efficiency of Au@PDA-4@BD NPs was calculated as reported in the literature.³²

Cytotoxicity and photothermal-induced cell death of Au@PDA-4@BD NPs

In vitro cell viability against Au@PDA-4@BD NPs was investigated by MTS assay.²⁹ Human oral squamous carcinoma KB cells were seeded in 96 well plates and incubated in complete culture medium (DMEM medium supplemented with 10% fetal bovine serum, 100 IU mL⁻¹ penicillin G sodium and 100 μg mL⁻¹ streptomycin sulfate) at 37 °C. After 24 h incubation, the cells were treated with 0.1 mL per well fresh complete culture media containing Au@PDA-4@BD NPs with 1, 10, 100 and 200 μg mL⁻¹ PDA concentrations in triplicate. After incubation for another 24 h, the cells were assayed as described previously.²⁹ For the photothermal cytotoxicity, the cells were exposed to the NIR laser irradiation for 10 min after the cells were incubated in the media containing Au@PDA-4@BD NPs for 24 h; after another 4 h of incubation, the cell viability was assayed.

In vivo tumor photothermal therapy using Au@PDA-4@BD NPs

The animal experiments of this study were carried out at Experimental Animal Center of School of Pharmacy of Fudan University in full compliance with the guidelines approved by Shanghai Administration of Experimental Animals. Male ICR mice (SPF, 18–22 g) from Sino-British SIPPR/BK Lab were inoculated in right hindquarter with 3 × 10⁶ H22 cells. When the tumors grew to about 100 mm³, the mice were randomly divided into different treatment groups with five in each group. A 0.1 mL of physiological saline or Au@PDA-4@BD solution with 2 mg mL⁻¹ PDA concentration was injected into the tumor. For the groups with NIR irradiation, each tumor was exposed to 808 nm laser at a power density of 5 W cm⁻² for 10 min immediately after the injection. The tumor sizes were measured every other day using a caliper and the tumor volumes were calculated according to the literature.³³

The tumor photothermal therapy after intravenous injection of Au@PDA-4 and Au@PDA-4@BD NPs was also carried out. When H22 tumors grew to about 100 or 250 mm³, the mice were separately injected via the tail veins with 0.2 mL physiological saline, Au@PDA-4 and Au@PDA-4@BD solutions containing 2 mg mL⁻¹ PDA. For the groups with NIR irradiation, each tumor was exposed to the laser for 10 min after 24 h of the injection. The tumor sizes, body weights and death of the mice were monitored.

In vivo biocompatibility of Au@PDA-4@BD NPs

The histological toxicity of Au@PDA-4@BD NPs plus the laser irradiation was evaluated by examining hematoxylin–eosin (H&E) stained histological sections. The 250 mm³ tumor-bearing mice were treated with the NPs via tail vein injection and the laser irradiation as described above. The mice were sacrificed at 18 day post-treatment; the organs of heart, liver, spleen, lung and kidney were surgically taken out, fixed, dehydrated and embedded in paraffin in succession. The specimens were cut into 4 μm thick sections, and the sections were stained with H&E and then observed on a microscope (Olympus BX53).

In vitro and in vivo CT imaging

Au@PDA-4@BD solution of 0.2 mL with 0, 1, 4, 8, 16 or 32 mg mL⁻¹ Au concentration was added into a 2 mL Eppendorf tube. The CT images were acquired on an In Vivo Xtreme imaging system (Bruker). For in vivo CT imaging, when H22 tumors grew to about 200 mm³, the mice were injected with 0.2 mL Au@PDA-4@BD solution containing 32 mg mL⁻¹ Au concentration via intratumoral injection, and then the mice were immediately anesthetized with isoflurane; the CT images were acquired at 10 min post-anesthesia. The resulting ROI (region of interest) data were acquired to obtain the average signal intensity of the tumor.

Blood concentrations of Au@PDA-4 and Au@PDA-4@BD NPs

Male ICR mice (20 g) were randomly assigned to two treatment groups with 35 mice in each group. Au@PDA-4 and Au@PDA-4@BD solutions of 0.2 mL containing 50 μg Au were separately injected into the mice via the tail veins. At each predetermined interval (10 min, 0.5, 1, 2, 4, 6 and 8 h post-injection), five mice in each group were sampled. About 500 μL of the blood was collected from eye ground vein for each mouse. The Au NPs in the blood samples were digested and/or dissolved according to the method described in the literature.³⁴ The Au concentration in the digested solution was measured on an inductively coupled plasma atomic emission spectrometer (ICP-AES, Hitachi P-4010).

Results and discussion

Preparation and characterization of Au NPs, Au@PDA NPs and Au@PDA@BD NPs

In this study, we fabricated NPs with gold core, PDA layer and dextran brush surface for tumor CT imaging and photothermal therapy. The fabrication process of the NPs is illustrated in Scheme 1. Firstly, Au NPs were prepared from the mixed solution of HAuCl₄ and glycine or aspartate by reduction via in situ UV irradiation at pH 10.0 and room temperature. Secondly, Au@Gly and Au@Asp NPs were coated with PDA by self-polymerization of dopamine at pH 8.5 and room temperature. Thirdly, Au@PDA was coated with BD by electrostatic attraction between PDA and BSA as well as gelation of BSA at pH 2.5 and 90 °C.

Scheme 1 Illustration of the fabrication process of Au@PDA@BD NPs.

As mentioned above, Au@Gly and Au@Asp NPs were produced via in situ UV irradiation. After the irradiation, almost all the Au³⁺ ions changed to Au NPs, therefore, no purification was performed for the Au NPs. The surface plasmon resonance (SPR) absorption of Au@Gly NPs (data not shown) and Au@Asp NPs shown in Fig. 1 are similar to the previous results.³⁰ The D_h values of Au@Gly and Au@Asp NPs are 63 and 27 nm, respectively (Table 1), which are also consistent with the previous results.³⁰ The Au NPs solution was mixed with freshly prepared dopamine solution and the mixture was adjusted to pH 8.5 as reported in the literature.²⁷ After 24 h stirring under air, Au@PDA NPs were obtained. The thickness of PDA coating layer can be adjusted by changing the weight ratio of dopamine to Au as shown in Table 1. For Au@PDA-1, Au@PDA-2 and Au@PDA-3 NPs, which used Au@Gly as the core, the average thicknesses of the PDA layers are 3, 11 and 24 nm, respectively. For Au@PDA-4 NPs, which used Au@Asp as the core, the average thickness of the PDA layer is 31 nm. The SPR absorption of Au@PDA-4 NPs is a bit of red shift compared with the absorption of Au@Asp NPs as shown in Fig. 1. The FTIR peaks of 1600 and 1510 cm⁻¹ of Au@PDA-4 NPs shown in Fig. S2 of ESI† are consistent with the PDA peaks reported in the literature,²⁷ which confirm the formation of PDA layer on Au@Asp surface. The TEM images in Fig. 2A–D confirm that the NPs have a structure of Au core and PDA coating layer. The PDA layer thicknesses shown in the TEM images are close to the thicknesses obtained from the D_h values shown in Table 1.

Fig. 1 UV-vis spectra of Au@Asp, Au@PDA-4 and Au@PDA-4@BD NPs.

Table 1 Preparation conditions as well as D_h and PDI results of the NPs

Sample	Au core	WDA/WAu	WBD/WDA	Dh (nm)	PDI	Dh of Au core (nm)	PDA thickness (nm)	BD thickness (nm)
Au@Gly	Au@Gly	—	—	63 ± 1	0.21 ± 0.01	63	—	—
Au@PDA-1	Au@Gly	2	—	68 ± 1	0.23 ± 0.01	63	3	—
Au@PDA-2	Au@Gly	4	—	85 ± 1	0.21 ± 0.01	63	11	—
Au@PDA-3	Au@Gly	8	—	110 ± 1	0.14 ± 0.02	63	24	—
Au@Asp	Au@Asp	—	—	27 ± 1	0.21 ± 0.01	27	—	—
Au@PDA-4	Au@Asp	8	—	89 ± 1	0.07 ± 0.03	27	31	—
Au@PDA-4@BD	Au@Asp	8	3.2	105 ± 3	0.19 ± 0.01	27	31	8

---

Fig. 2 TEM images of (A) Au@PDA-1, (B) Au@PDA-2, (C) Au@PDA-3, (D) Au@PDA-4 and (E) Au@PDA-4@BD NPs.

Au@PDA-4 NPs were coated with BD to produce the NPs with dextran brush surface. BD was produced by Maillard reaction, in which the reducing-end carbonyl group of dextran was conjugated to the amine group in N-terminal or lysine residue of BSA without chemicals. After the Maillard reaction, each BSA molecule was conjugated with about 2.7 dextran chains on average.³⁵ Coating Au@PDA-4 with BD was performed at pH 2.5, at which Au@PDA-4 NPs were negatively charged with ζ-potential of −11.5 mV, BD was positively charged with ζ-potential of 16.1 mV, and BD was adsorbed on the PDA surface via electrostatic attraction between PDA and BSA. After the adsorption, the solution was heated at 90 °C for 1 h to induce the BSA gelation and also to eliminate the anaphylaxis of BSA.²⁹ After the heat treatment, irreversible BD shell layer was produced on Au@PDA-4 surface. The FTIR peaks of 1654 and 1540 cm⁻¹ shown in Fig. S2 of ESI† confirm the formation of BD layer on Au@PDA-4 surface. The D_h of Au@PDA-4@BD NPs is 105 nm (Table 1), indicating that the thickness of BD shell is about 8 nm. The TEM images show that Au@PDA-4@BD NPs have similar morphology as Au@PDA-4 NPs (Fig. 2D and E). The data in Table 2 show that Au@PDA-4@BD NPs are negatively charged in pH 7.4 PBS solution (10 mM phosphate buffer containing 0.15 M NaCl). Au@PDA-4@BD NPs did not aggregate after one month of storage in PBS, suggesting that the NPs are dispersible at physiological condition. The D_h of Au@PDA-4@BD NPs is 122 nm after freeze-dry and re-dispersion in aqueous solution, indicating that Au@PDA-4@BD NPs can be freeze-dried for storage and re-dispersed for application.

Table 2 D_h, PDI and ζ-potential results of the NPs before and after one month of storage in pH 7.4 PBS

Sample	Freshly in PBS			After one month in PBS
	Dh (nm)	PDI	ζ-Potential (mV)	Dh (nm)	PDI
Au@Asp	31 ± 2	0.15 ± 0.01	−31 ± 3	33 ± 1	0.22 ± 0.03
Au@PDA-4	90 ± 1	0.09 ± 0.02	−37 ± 2	95 ± 3	0.13 ± 0.02
Au@PDA-4@BD	107 ± 2	0.21 ± 0.01	−27 ± 1	112 ± 3	0.24 ± 0.03

---

In vitro photothermal conversion of the NPs

The NPs solutions were separately exposed to 808 nm laser irradiation at a power density of 5 W cm⁻² to investigate their photothermal conversions. The results in Fig. 3 show that the temperature changes of the solutions during the irradiation depend on the PDA concentrations. For Au@Gly, Au@PDA-1, Au@PDA-2 and Au@PDA-3 NPs, which contained the same Au concentration and 0, 50, 100 and 200 μg mL⁻¹ PDA, respectively, the temperature changes are in the order of Au@Gly < Au@PDA-1 < Au@PDA-2 < Au@PDA-3 (Fig. 3A). For Au@PDA-4 and Au@PDA-4@BD solutions with 200 μg mL⁻¹ PDA concentration, the temperatures rose from 25.2 to 55.8 °C and from 26.5 to 52.3 °C, respectively, after 10 min of the irradiation (Fig. 3B and C). Dilution of the Au@PDA-4 and Au@PDA-4@BD solutions to PDA concentrations of 100 and 50 μg mL⁻¹ would reduce the temperature changes in sequence. At the same PDA concentration, the temperatures of Au@PDA-4 solution rose faster than those of Au@PDA-3 solution (Fig. 3A and B). Therefore, we used Au@PDA-4 to prepare Au@PDA-4@BD NPs. Using the method reported in the literature,³² we measured that the photothermal transduction efficiency of Au@PDA-4@BD NPs was 17.0%.

Fig. 3 Temperature changes during the NIR laser irradiation; (A) Au@Gly, Au@PDA-1, Au@PDA-2, Au@PDA-3 solutions containing the same Au concentration and different PDA concentrations, as well as the solution of PDA NPs without Au core; (B) Au@PDA-4 and (C) Au@PDA-4@BD solutions with different PDA concentrations.

In vitro cytotoxicity and photothermal-induced cell death of Au@PDA-4@BD NPs

KB cell viability against Au@PDA-4@BD NPs was investigated. When the PDA concentration in the culture was 200 μg mL⁻¹ or less, the cell viability was higher than 80% after 24 h incubation (Fig. 4), implying that Au@PDA-4@BD NPs have very low cytotoxicity. It was reported that dopamine is highly cytotoxic,³⁶ but PDA has good biocompatibility both in vitro³⁷ and in vivo.²⁷ In this study, we did not purify Au@PDA-4 NPs after the self-polymerization of dopamine. The low cytotoxicity of Au@PDA-4@BD NPs indicates that the dopamine in the solution had changed to PDA after 24 h self-polymerization reaction. Fig. 4 also demonstrates the photothermal cytotoxicity of the NPs. After the incubation with the NPs and exposure to the irradiation for 10 min, more than 95% of the cells were killed when the PDA concentration in the culture was 100 μg mL⁻¹ or higher. This result confirms that the hyperthermia caused by the NPs and NIR irradiation can effectively kill the tumor cells.

Fig. 4 KB cell viability after 24 h incubation with Au@PDA-4@BD NPs at different PDA concentrations followed by the NIR laser irradiation for 10 min or not.

In vitro and in vivo CT imaging

Fig. 5A shows in vitro CT images of Au@PDA-4@BD solutions containing 0, 1, 4, 8, 16 and 32 mg mL⁻¹ Au; the brightness of the image increases with the Au concentration. Fig. 5B shows the CT images of H22 tumor-bearing mice before and after intratumoral injection of 0.2 mL Au@PDA-4@BD solution containing 32 mg mL⁻¹ Au concentration. The average signal intensities of the tumor tissue were 726 and 931 before and after the injection, respectively. The images prove that both the brightness and resolution of the tumor site are enhanced after the injection of the NPs. This result suggests that Au@PDA-4@BD NPs can serve as a contrast agent for tumor CT imaging.

Fig. 5 (A) In vitro CT images of Au@PDA-4@BD solutions containing different Au concentrations; (B) CT image of H22 tumor-bearing mouse; (C) CT image of H22 tumor-bearing mouse after intratumoral injection of Au@PDA-4@BD NPs. The insets are the enlarged images of the tumor sites. The average signal intensities of the tumor tissue were 726 and 931 before and after the injection, respectively.

In vivo tumor photothermal therapy by intratumoral injection of Au@PDA-4@BD NPs

Tumor photothermal therapy of Au@PDA-4@BD NPs was evaluated by H22 tumor-bearing mice. In order to keep the Au@PDA-4@BD concentration in tumor site for effective photothermal therapy, we firstly carried out intratumoral injection. When the tumors grew to about 100 mm³, the treatment was performed by a single intratumoral injection of 0.1 mL physiological saline or Au@PDA-4@BD solution with 2 mg mL⁻¹ PDA concentration followed by 808 nm laser irradiation or not. The tumor volume changes and body weight changes after the treatment are shown in Fig. 6A and B, respectively. After the treatment, all the tumors in Au@PDA-4@BD + laser group disappeared within two days and did not recrudesce, whereas the tumors in other groups grew faster and faster. For saline + laser group and Au@PDA-4@BD group, the tumors were smaller than those of saline group without irradiation. These results imply that only NIR laser irradiation or Au@PDA-4@BD injection has some effect on tumor inhibition. Fig. 6C presents the photo of the mice after 19 days of the treatment for visualization. Except for Au@PDA-4@BD + laser group, death occurred after 16 days of the treatment. In Au@PDA-4@BD + laser group, no tumor recrudesced and no mouse died even after 100 days of the treatment, demonstrating that the tumors can be completely cured by a single treatment of Au@PDA-4@BD injection plus NIR irradiation.

Fig. 6 (A) Relative tumor volume changes and (B) relative body weight changes of four treatment groups (n = 5) after single intratumoral injection followed by NIR laser irradiation or not; (C) photo of the mice from the four groups taken at 19 day post-treatment, and tumors are indicated by circles. The treatments were performed when the tumors grew to about 100 mm³.

In vivo biocompatibility, blood concentration, and tumor photothermal therapy after intravenous injection of Au@PDA-4@BD NPs

We further evaluated the tumor photothermal therapy efficacy of Au@PDA-4@BD NPs by intravenous injection. When the tumors grew to about 100 mm³, the treatment was performed by a single injection of 0.2 mL physiological saline or Au@PDA-4@BD solution with PDA concentration of 2 mg mL⁻¹ via tail veins followed by the laser irradiation or not. The results in Fig. 7 prove that intravenous injection of Au@PDA-4@BD NPs plus NIR laser irradiation can also cure H22 tumors completely. For Au@PDA-4@BD + laser group, after the single treatment, all the tumors disappeared completely within two days and did not appear anymore, the mice increased their body weights gradually, and no mouse died during our observation period of 50 days. On the contrary, all the mice in the other groups increased their tumor volumes gradually and died after 33 days of the treatment. Fig. 3C reveals that the temperature rising during the irradiation depends on Au@PDA-4@BD concentration. Fig. 4 demonstrates that the photothermal cytotoxicity also relies on Au@PDA-4@BD concentration. The results in Fig. 7 can be explained by that Au@PDA-4@BD NPs could effectively accumulate at the tumor site after the intravenous injection and the laser irradiation could cause the tumor temperature to rise to ablate the tumor completely.

Fig. 7 (A) Tumor volume changes and (B) relative body weight changes of four treatment groups (n = 5) after single intravenous injection followed by NIR laser irradiation or not; (C) survival curves of the mice after the treatments. The treatments were performed when the tumors grew to about 100 mm³.

Au@PDA-4@BD NPs possess dextran brush surface because the hydrophilic dextran chains extend in aqueous solution. In order to prove that it is the dextran brush surface that prolongs the circulation time of the NPs in blood and enhances the accumulation of the NPs in tumor tissue via EPR effect, we analyzed the Au concentrations in blood after intravenous injection of Au@PDA-4 and Au@PDA-4@BD. Fig. 8A shows that Au@PDA-4@BD group had higher Au concentration in the blood than Au@PDA-4 group, confirming that the NPs with the dextran brush surface can prolong the circulation time in blood.

Fig. 8 (A) Au concentrations in blood after intravenous injection of Au@PDA-4 and Au@PDA-4@BD (n = 5); (B) tumor volume changes, (C) relative body weight changes, and (D) survival curves of six treatment groups (n = 5) after a single intravenous injection followed by NIR laser irradiation or not; (E) representative H&E strained histological images of the major organs excised from H22 tumor-bearing mice at 18 day post-treatment and the images of the organs from normal mice without treatment. The treatments were performed when the tumors grew to 250 mm³.

Furthermore, we synchronously evaluated the tumor photothermal therapy efficacy of Au@PDA-4 and Au@PDA-4@BD NPs by intravenous injection. When the tumors grew to about 250 mm³, 0.2 mL Au@PDA-4 or Au@PDA-4@BD solution containing 2 mg mL⁻¹ PDA concentration was injected via tail veins. For Au@PDA-4 and Au@PDA-4@BD groups without the irradiation, due to the large tumor volume before the treatment, all the mice died after 23 days of the treatment as shown in Fig. 8. Similarly, Au@PDA-4 + laser group did not present significant enhancement in tumor inhibition or survival compared with the saline group. For Au@PDA-4@BD + laser group, however, after the single treatment, the tumor volume decreased gradually, and most importantly, all the tumors with original volume of about 250 mm³ disappeared completely at 13 day post-treatment. After that, no tumor appeared and all mice were normal. The monitoring was ended at 90 day post-treatment because the tumors had been cured completely. These results clearly demonstrate the function of the dextran brush surface of Au@PDA-4@BD NPs. Without the dextran brush surface, Au@PDA-4 NPs cannot accumulate in the tumor sites after intravenous injection and therefore the NIR irradiation cannot ablate the tumors.

Similarly, we performed CT imaging by intravenous injection via tail vein with 0.2 mL Au@PDA-4@BD solution containing 32 mg mL⁻¹ Au concentration. The signal intensities of the tumor tissue before and after the injection were not significant difference (data not shown). Fig. 5A shows that the CT signal was not significantly improved when the Au concentration was 8 mg mL⁻¹ in vitro. These results suggest that Au@PDA-4@BD NPs are not a very sensitive CT contrast agent. The tumor CT imaging can only be improved by intratumoral injection of the NPs. However, intravenous injection of 0.2 mL Au@PDA-4@BD solution containing 2 mg mL⁻¹ PDA concentration can improve photothermal therapy and cure tumor completely, demonstrating that Au@PDA-4@BD NPs are an excellent photothermal therapy agent.

To examine in vivo biocompatibility of Au@PDA-4@BD NPs, we observed the H&E stained histological sections of the organs from the mice with original tumor of 250 mm³ and treated by the intravenous injection of Au@PDA-4@BD plus the laser irradiation as described above. The organs were taken out at 18 day post-treatment, and their histological sections have no significant difference compared with the sections of normal mice without the treatment (Fig. 8E). These results verify that the intravenous injection of Au@PDA-4@BD plus the laser irradiation have no notable histological toxicity on the main organs.

Conclusions

In this study, multifunctional Au@PDA-4@BD NPs with gold core, PDA layer and dextran brush surface were produced for tumor diagnosis and photothermal therapy. The thickness of PDA layer can be adjusted by changing the weight ratio of dopamine to Au. The temperature rise and photothermal cytotoxicity of the NPs during 808 nm laser irradiation depended on the PDA concentration. Both in vitro and in vivo investigations demonstrate that Au@PDA-4@BD NPs are biocompatible. Au@PDA-4@BD NPs improved the brightness and resolution of tumor CT imaging after intratumoral injection of the NPs into H22 tumor-bearing mice. Au@PDA-4@BD NPs could prolong the circulation time in blood and accumulate at tumor site after intravenous injection due to the dextran brush surface, and the NIR laser irradiation could cause the tumor temperature to rise to ablate the tumor completely. By only once treatment, Au@PDA-4@BD NPs plus NIR laser irradiation could cure the tumors completely even the original tumor volume was 250 mm³. This study demonstrates that Au@PDA@BD NPs are a great potential nanomaterial for highly efficient photothermal therapy of tumor.

Acknowledgements

Financial supports of National Natural Science Foundation of China (NSFC Project 21274026) and Ministry of Science and Technology of China (973 Program 2011CB932503) are gratefully acknowledged.

Notes and references

1. V. Shanmugam, S. Selvakumar and C. S. Yeh, Chem. Soc. Rev., 2014, 43, 6254–6287.
2. D. Jaque, L. M. Maestro, B. del Rosal, P. Haro-Gonzalez, A. Benayas, J. L. Plaza, E. M. Rodriguez and J. G. Sole, Nanoscale, 2014, 6, 9494–9530.
3. W. Choi, J.-Y. Kim, C. Kang, C. C. Byeon, Y. H. Kim and G. Tae, ACS Nano, 2011, 5, 1995–2003.
4. X. Song, Q. Chen and Z. Liu, Nano Res., 2015, 8, 340–354.
5. F. Helmchen and W. Denk, Nat. Methods, 2005, 2, 932–940.
6. B. Feng, Z. Xu, F. Zhou, H. Yu, Q. Sun, D. Wang, Z. Tang, H. Yu, Q. Yin, Z. Zhang and Y. Li, Nanoscale, 2015, 7, 14854–14864.
7. O. Penon, M. J. Marin, D. B. Amabilino, D. A. Russell and L. Perez-Garcia, J. Colloid Interface Sci., 2016, 462, 154–165.
8. L. Zhang, Y. Li, Z. Jin, K. M. Chan and J. C. Yu, RSC Adv., 2015, 5, 93226–93233.
9. G. Darabdhara, M. R. Das, V. Turcheniuk, K. Turcheniuk, V. Zaitsev, R. Boukherroub and S. Szunerits, J. Mater. Chem. B, 2015, 3, 8366–8374.
10. Y. Liu, K. Ai, J. Liu, M. Deng, Y. He and L. Lu, Adv. Mater., 2013, 25, 1353–1359.
11. K. Y. Ju, Y. Lee, S. Lee, S. B. Park and J. K. Lee, Biomacromolecules, 2011, 12, 625–632.
12. H. Lee, S. M. Dellatore, W. M. Miller and P. B. Messersmith, Science, 2007, 318, 426–430.
13. L. Wang, D. Wang, Z. Dong, F. Zhang and J. Jin, Nano Lett., 2013, 13, 1711–1716.
14. C. J. Bettinger, J. P. Bruggeman, A. Misra, J. T. Borenstein and R. Langer, Biomaterials, 2009, 30, 3050–3057.
15. X. Li, N. Anton, G. Zuber and T. Vandamme, Adv. Drug Delivery Rev., 2014, 76, 116–133.
16. D. P. Clark and C. T. Badea, Phys. Med., 2014, 30, 619–634.
17. I. C. Sun, J. H. Na, S. Y. Jeong, D. E. Kim, I. C. Kwon, K. Choi, C. H. Ahn and K. Kim, Pharm. Res., 2014, 31, 1418–1425.
18. K. Ye, J. Qin, Z. Peng, X. Yang, L. Huang, F. Yuan, C. Peng, M. Jiang and X. Lu, Nanoscale Res. Lett., 2014, 9, 529–541.
19. L. E. Cole, R. D. Ross, J. M. Tilley, T. Vargo-Gogola and R. K. Roeder, Nanomedicine, 2015, 10, 321–341.
20. K. Kobayashi, J. Wei, R. Iida, K. Ijiro and K. Niikura, Polym. J., 2014, 46, 460–468.
21. C. C. Mayorga-Martinez, A. Chamorro-Garcia and A. Merkoci, Biosens. Bioelectron., 2015, 67, 53–58.
22. R. Cao-Milan and L. M. Liz-Marzan, Expert Opin. Drug Delivery, 2014, 11, 741–752.
23. X. Yang, M. Yang, B. Pang, M. Vara and Y. Xia, Chem. Rev., 2015, 19, 10410–10488.
24. P. Sanpui, A. Paul and A. Chattopadhyay, Nanoscale, 2015, 7, 18411–18423.
25. L. L. Ren, S. Z. Chen, H. D. Li, Z. Y. Zhang, C. H. Ye, M. L. Liu and X. Zhou, Nanoscale, 2015, 7, 12843–12850.
26. T. Dreifuss, O. Betzer, M. Shilo, A. Popovtzer, M. Motiei and R. Popovtzer, Nanoscale, 2015, 7, 15175–15184.
27. X. S. Liu, J. M. Cao, H. Li, J. Y. Li, Q. Jin, K. F. Ren and J. Ji, ACS Nano, 2013, 7, 9384–9395.
28. K. Alhareth, C. Vauthier, F. Bourasset, C. Gueutin, G. Ponchel and F. Moussa, Eur. J. Pharm. Biopharm., 2012, 81, 453–457.
29. J. Qi, C. Huang, F. He and P. Yao, J. Pharm. Sci., 2013, 102, 1307–1317.
30. H. Cai and P. Yao, Colloids Surf., B, 2014, 123, 900–906.
31. H. Hao, Q. Ma, C. Huang, F. He and P. Yao, Int. J. Pharm., 2013, 444, 77–84.
32. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell and B. A. Korgel, Nano Lett., 2011, 11, 2560–2566.
33. T. J. Li, C. C. Huang, P. W. Ruan, K. Y. Chuang, K. J. Huang, D. B. Shieh and C. S. Yeh, Biomaterials, 2013, 34, 7873–7883.
34. L. M. Wang, Y. Liu, W. Li, X. M. Jiang, Y. L. Ji, X. C. Wu, L. G. Xu, Y. Qiu, K. Zhao, T. T. Wei, Y. F. Li, Y. L. Zhao and C. Y. Chen, Nano Lett., 2011, 11, 772–780.
35. H. Hao and P. Yao, Chem. J. Chin. Univ., 2014, 35, 652–659.
36. A. H. Stokes, T. G. Hastings and K. E. Vrana, J. Neurosci. Res., 1999, 55, 659–665.
37. X. Zhang, S. Wang, L. Xu, L. Feng, Y. Ji, L. Tao, S. Li and Y. Wei, Nanoscale, 2012, 4, 5581–5584.

---

Footnote

† Electronic supplementary information (ESI) available: FTIR spectra and size distributions of Au, Au@PDA-4 and Au@PDA-4@BD NPs. See DOI: 10.1039/c6ra02684b

---