Double Drug-Loaded and pH/Thermal Sensitive Multifunctional Drug Delivery System for Tumor Photoacoustic Imaging-Guided Enhanced Chemo/Photothermal Therapy

Background : Owing to the tunability of longitudinal surface plasmon resonance (LSPR), ease of synthesizing small size and excellent stability, AuNRs have been developed as photothermal agents for cancer therapy. However, PTT alone could not kill cancer cells completely due to the local heterogeneous distribution of heat in tumors, penetration depth of light, light scattering and absorption. In addition, the treatment systems based on AuNRs hold disadvantages of loading one antitumor drug or a low therapeutic efficiency. Therefore, the construction of the AuNRs theranostic system to achieve imaging-guided dual drug delivery and enhanced photothermal therapy for tumor still remains a great challenge. Methods: The AuNRs were prepared using a seedless method. A mesoporous silica shell layer was coated on the surface of the AuNRs by sol-gel method. Double anticancer drugs, DOX and Btz, were loaded into the AuNRs@MSN nanoparticles through physical absorption and covalent conjugation, respectively. Results: The release of DOX and Btz is found pH/thermal dual responsive in vitro. Compared with AuNRs@MSN, PDA-AuNRs@MSN exhibits an increased near-infrared (NIR) absorption at 808 nm and an enhanced photothermal effect. In contrast to chemotherapy or photothermal therapy alone, the integrated D/B-PDA-AuNRs@MSN nanoparticles show higher cell apoptosis and enhanced tumor treatment efficacy in vitro and in vivo . Conclusions: In this study, we designed a double-drug loading, enhanced chemo/photothermal therapy and pH/thermal responsive drug delivery system for photoacoustic (PA) imaging-guided tumor therapy. We believe that the multifunctional D/B-PDA-AuNRs@MSN theranostic probe could serve as an effective

4 probe for the treatment of cancers.

Background
Cancer is one of the greatest threats to human health [1]. At present, the most common ways for cancer treatment are chemotherapy, surgery and radiotherapy in clinical. However, these approaches usually result in adverse side effects. Recently, the near-infrared (NIR) light-driven nanomaterials-mediated photothermal therapy (PTT) has attracted extensive attention due to its advantages of being non-invasive and few side effects. The mechanism of the PTT is based on the principle that the photothermal agents absorb the NIR light converting into heat, leading to cancer cell ablation and death [2]. In this regard, gold nanomaterials, such as gold nanostructure (gold nanoparticles (AuNPs) [3][4][5], gold nanorods (AuNRs) [6-9], gold nanocages (AuNCs) [10-12], gold nanostars [13][14][15], gold nanoshell [16][17][18] and gold nanoflowers [19]) have been developed as photothermal agents for cancer therapy.
Among these gold nanomaterials, AuNRs have been widely used in the field of biomedicine due to the tunability of longitudinal surface plasmon resonance (LSPR), ease of synthesizing small size and excellent stability. However, PTT alone could not kill cancer cells completely due to the local heterogeneous distribution of heat in tumors, light scattering and absorption [20]. In addition, the energy of the light was gradually decreased with the depth of penetration into the tissues [21]. Compared with single therapy, combination therapy, such as chemo-photothermal therapy, is considered as an effective strategy to enhance therapeutic efficiency because this strategy has the advantages of reducing the negative effects and overcoming the drug resistance of tumor cells. However, the treatment systems based on AuNRs hold disadvantages of loading one antitumor drug or a low therapeutic efficiency 5 [22,23]. Therefore, the construction of the AuNRs theranostic system to achieve imaging-guided control over dual drug delivery and enhanced photothermal therapy for tumor still remains a great challenge.
In this study, we developed a double-drug loading and pH/thermal dual sensitive drug delivery system for imaging-guided multi-modal cancer therapy based on polydopamine-coated mesoporous silica-AuNRs. In this drug delivery system, nanoscale mesoporous silica-coated AuNRs encapsulating doxorubicin hydrochloric acid (DOX) was designed as a core. Polydopamine (PDA) was deposited on the surface of the mesoporous silica@AuNRs by oxidative self-polymerization for enhanced PTT and controlled drug release as gatekeepers. Another anti-tumor drug bortezomib (Btz) was combined to PDA through boronic acid of Btz and catechol of PDA conjugation. The resulting multifunctional nanocarriers possess the properties of enhanced chemo-photothermal therapy and pH/thermal-responsive controlled drug release.

Synthesis of AuNRs
The AuNRs were prepared using a seedless method with slight modifications [24,25]. Briefly, CTAB solution (30 mL, 0.2 M) was added to 30 mL of 1.0 mM HAuCl 4 , followed by the addition of 1.8 mL of 4 mM AgNO 3 and 72 μL of HCl (37%). Next, 450 μL of 85.8 mM AA was added and gently swirled as the solution became colorless.
Finally, 45 μL of 10 mM NaBH 4 was rapidly injected. The resulting solution was kept for 6 h at 30 °C.

Preparation of AuNRs with mesoporous silica shell (AuNRs@MSN)
AuNRs@MSN were synthesized according to the reported previously [26]. To remove excess CTAB from AuNRs, 30 mL of the as-synthesized AuNRs was centrifuged at 16 000 rpm for 30 minutes. The precipitate was redispersed in 30 mL of Milli-Q water and 300 μL of 0.1 M NaOH solution was added upon stirring. Then, three injections of 90 μL of 20% TEOS in methanol solution was added into the above solution at 30 minutes intervals. The mixture was stirred for 24 h at 30 °C. The AuNRs@MSN were separated by centrifugation. The precipitate was refluxed with 20 mL of 10 mg/mL NH 4 NO 3 -ethanol solution under 60 °C for 12 h to extract the surfactant template CTAB. The final product was collected by centrifugation at 16 000 rpm for 30 min and washed with ethanol three times. The as-synthesized solid was dried in the lyophilizer.
AuNRs@MSN (50 mg) was added to the DOX solution ( 1 mg/mL, 5 mL) and stirred in 7 dark at the room temperature for 24 h. The product was acquired by centrifugation respectively. The dispersion solution was then transferred into a dialysis bag (molecular weight cut off =8 000-14 000 kDa) and placed in 100 mL of PBS buffer 8 solution at 37 °C with or without 808 nm light irradiation and shaked at 150 rpm. At timed intervals, 3 mL of solution was withdrawn from the solution. The released DOX and Btz were analyzed by UV-vis spectrum. The volume of the release medium was kept constant by adding 3 mL fresh medium after each sampling.

In vitro cytotoxicity
The cell viability was determined by CCK-8 assay. 4T1 cells were seeded into a 96well plate at a density of 1 × 10 4 cells and cultured at 5% CO 2 and 37 °C for 24 h.

Temperature measurement in vitro
The aqueous of AuNRs@MSN and PDA-AuNRs@MSN containing the same Au concentration (20 mg/mL) were added into 0.5 mL centrifuge tube and irradiated by 808 nm laser at a power density of 1 W/cm 2 for 600 s. For the control group, 0.5 mL of deionized water was also irradiated under the same condition. To investigate different concentration of PDA-AuNRs@MSN photothermal effect, the as-prepared PDA-AuNRs@MSN was diluted to different concentrations (Au = 10, 20, 40 and 60 mg/mL) and 0.5 mL sample solution was added into the centrifuge tube and was irradiated by 808 nm laser (1 W/cm 2 ) for 600 s. For the control group, 0.5 mL of deionized water was also irradiated under the same condition. A thermal imager was used to measure the temperature changes and obtain the infrared thermal images.

In vivo biosafety analysis
Female BALB/c mice (4 weeks) were purchased from SLAC laboratory animal Co, Ltd.

Synthesis and characterizations of D/B-PDA-AuNRs@MSN
The synthesis of D/B-PDA-AuNRs@MSN was shown in Scheme 1. Firstly, a one-pot seedless-mediated growth method was adopted to synthesize CTAB-stabilized AuNRs. Transmission electron microscopy (TEM) images show that the AuNRs are about 25 nm in length and 8 nm in width (Fig. 1A) and longitudinal surface plasmon resonance (LSPR) peak of 730 nm (Fig. 1D). Then, a mesoporous silica shell layer coating on the surface of the AuNRs by sol-gel method is observed and the thickness of the silica shell is about 20 nm (Fig. 1B). After coating, the LSPR peak was found to shift to 757 nm due to the surface refractive index changes (Fig. 1D).  (Fig. 2B). Figure 2C and 2D show the nitrogen adsorption-desorption isotherms, and the pore size distribution diagram of the prepared AuNRs@MSN and PDA-AuNRs@MSN nanoparticles. For the AuNRs@MSN, the BET surface area is 564.76 m 2 g -1 , the pore volume is 0.82 cm 3 g -1 , and the pore size is about 2.33 nm. After depositing the PDA shell, the BET specific surface area, pore size and the pore volume of the PDA-AuNRs@MSN nanoparticles are smaller than the AuNRs@MSN nanoparticles, and are 62.35 m 2 g -1 , 1.89 nm and 0.2 cm 3 g -1 , respectively. This further suggests that the PDA shell has been successfully modified on the surface of the AuNRs@MSN nanoparticles.

In vitro drug release
Double anticancer drugs, DOX and Btz, were loaded into the nanoparticles through two different mechanisms. DOX loading was achieved by physical absorption, while Btz was realized by covalent conjugation. The UV-vis spectrum confirms both two anticancer drugs were successfully loaded in the PDA-AuNRs@MSN (Fig. 3). The loading contents of DOX and Btz in PDA-AuNRs@MSN were measured to be 115 mg/g and 6.1 mg/g by calculating the absorbance of the supernatant according to the DOX and Btz standard curve (Fig. 4A-4D). Fig. 4E and 4F show the drug release profiles of DOX and Btz in different pH solutions with or without the laser 13 irradiation. Both anticancer drugs release are pH-dependent. For DOX, the drug release rate is much less at pH 7.4 than that at pH 5.0 because of the PDA layer might be partially peeled from the surface of the nanoparticles at pH 5.0 [27,28].
For Btz, about 81.7 % of the Btz is released at pH 5.0, while only 18.8 % of the Btz is released at pH 7.4. This difference is attributed to the pH dependence of cleavage of the boronic ester bond.
The drug release rates of DOX and Btz are obviously faster under NIR irradiation at different pH values. The release of Btz from the nanoparticles can be increased to 88.2 % under laser irradiation and acidic condition. This condition can facilitate the cleavage of the boronic ester for drug release [30]. While for the release rate of DOX, it is a slightly increase, which can be ascribed to the heat that accelerates the DOX molecules movement at pH 5.0. These results indicate the release of DOX and Btz are sensitive to pH and heat-dependent.

Photothermal effect of PDA-AuNRs@MSN
In order to prove the deposition of the PDA layer could enhance the photothermal effect, the laser-induced heat generation of PDA-AuNRs@MSN and AuNRs@MSN by 808 nm laser irradiation at a power intensity of 1.0 W cm -2 for 10 min were measured under the same Au concentration, respectively. As seen from

In vitro cytotoxicity and cell uptake
The cytotoxicity of AuNRs@MSN and PDA-AuNRs@MSN were evaluated by CCK-8 assay. The cell viabilities of the 4T1 cells are over 90% after incubated with AuNRs@MSN and PDA-AuNRs@MSN even at 60 mg/mL of Au concentration, suggesting that the nanoparticles are biocompatibility (Fig. 7A). In order to evaluate the combination therapeutic effect, 4T1 cancer cells were treated with several groups with or without NIR laser irradiation. As shown in Fig (Fig. 7C).

In vivo photoacoustic imaging and photothermal-chemo therapy
Prior to in vivo application, we first studied the potential in vivo toxicity of the PDA-AuNRs@MSN. HE staining results demonstrate the main organs (heart, liver, spleen, lung, kidney and intestines) have no inflammation or abnormality after 15 days compared with the control group, showing the PDA-AuNRs@MSN could be applied to in vivo biomedical application (Fig. 7D).
Due to high NIR absorption, we further investigated the PDA-AuNRs@MSN for in vivo PA imaging, which is non-invasive biomedical imaging with high imaging depth and spatial resolution [33]. The optimal photoacoustic excitation wavelength in vitro was found to be 875 nm (Fig. 8A) and the PA intensity of the PDA-AuNRs@MSN becomes stronger with the increase of the concentration gradually, and exhibits a certain linear correlation (Fig. 8B). Furthermore, we conducted in vivo PA imaging of the PDA-AuNRs@MSN on 4T1 tumor-bearing mice. As shown in Fig. 8C   The infrared thermal images of 4T1-tumor-bearing mice with intravenous injection of PBS (20

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to 33 download. scheme1.JPG