Au-PLGA Hybrid Nanoparticles with Catalase-Mimicking and near

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Au-PLGA hybrid nanoparticles with catalase-mimicking and near-infrared photothermal activities for photoacoustic imaging-guided cancer therapy Juqun Xi, Wenjuan Wang, Lanyue Da, Jingjing Zhang, Lei Fan, and Lizeng Gao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00901 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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ACS Biomaterials Science & Engineering

Au-PLGA hybrid nanoparticles with catalasemimicking and near-infrared photothermal activities for photoacoustic imaging-guided cancer therapy †

†

Juqun Xi, a,b Wenjuan Wang,c Lanyue Da,a Jingjing Zhang,a Lei Fan, d* Lizeng Gaoa

a. Institute of Translational Medicine, Department of Pharmacology, School of Medicine, Yangzhou University, Yangzhou, Jiangsu 225002, China. b. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China. c. Department of Anesthesiology, The Second Affiliated Hospital of Soochow University, Suzhou, 215006, Jiangsu, China. d. School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, 225002, China.

*E-mail: [email protected] (L. Fan)

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ABSTRACT Imaging-guided diagnosis and therapy has been highlighted in the area of nanomedicines. However, integrating multiple functions with high performance in one theranostic (‘all-in-one’) still presents considerable challenges. Here, ‘all-in-one’ nanoparticles with drug-loading capacity, catalase-mimetic activity, photoacoustic (PA) imaging ability and photothermal properties were prepared by decorating Au nanoparticles on doxorubicin (DOX) encapsulated poly (lactic-co-glycolic acid) (PLGA) vehicle. The results revealed that the as-prepared Au-PLGA hybrid nanoparticles possessed high photothermal conversion efficiency of up to approximately 69.0%, meanwhile their strong acoustic generation endowed them with efficient PA signal sensing for cancer diagnosis. On an 808-nm laser irradiation, the O2 generation, DOX release profile and reactive oxygen species (ROS) level were all improved, which were beneficial to relieving tumor hypoxia and enhanced the cancer chemo/PTT combined therapy. Overall, the multifunctional Au-PLGA hybrid nanoparticles with these integrated advantages shows promise in PA imaging-guided diagnosis and synergistic tumor ablation.

Keywords: Au-PLGA hybrid nanoparticles, photoacoustic imaging, catalase-mimicking activity chemo/photothermal therapy


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INTRODUCTION Photothermal therapy (PTT) is a new strategy with minimal side effects in tumor treatment, which has drawn a great deal of attention in the last few years.1-4 PTT agents absorb near-infrared (NIR) light and transform it into heat, causing death to tumor cells. However, the tumor cells usually cannot be killed completely just by photothermal manner, finally leading to tumor recurrence. The reason lies in the limitations of radiant power and penetration depth of NIR light.5 Thus, the combination of PTT with gene therapy,6 immunotherapy,7 photodynamic therapy8 or chemotherapy9 has proposed as a more feasible strategy for cancer treatment. Moreover, to further enhance the therapeutic efficiency of PTT, imaging-guided strategies are also in development to realize real-time visualization of PTT to assist with cancer therapy. Gold nanoparticles (Au NPs) are utilized as attractive photothermal agents due to their efficient local heating upon excitation of surface plasmon oscillations.10 Previous literatures have presented that NIR-responsive Au NPs, such as nanostars,11 nanorods12 and nanoshells13, shows effective cancer photothermal therapy. In addition, duo to their tunable and strong light absorption in near-infrared region, Au NPs are also an excellent candidate as a photoacoustic (PA) contrast agent for PA imaging,14 which is an emerging diagnostic method with easy applicability in clinical treatments.15 Some other studies also revealed that Au NPs exhibit enzyme-like activities. For example, cysteaminemodified Au NPs show peroxidase-mimicking activity,16 and citrate-modified Au NPs have glucose oxidase-like activity.17 Moreover, on account of the reactivity of Au NPs with H2O2 to produce O2 as a catalase-mimicking enzyme,18 Au or Au-containing NPs can reconstruct the oxygen supply to the tumor. Hypoxia is now a well-recognized phenomenon in animal and human solid tumors, leading to chemotherapy resistance.19 The catalase-mimicking performance of Au NPs can be exploited to relieve tumor hypoxia, subsequently contributing to enhanced cancer chemotherapy.20 These above functions of Au NPs make it possible to construct Au-based theranostic platforms in the ‘all-in-one’ form. However, development of ‘all-in-one’ theranostic systems with excellent biocompatibility and versatility still remains a considerable challenge for specific application purposes.


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For spherical solid Au NPs, only those larger than 50 nm can exhibit strong NIR absorption, which is also essentially required for PTT as well as PA imaging.21 However, smaller particles are more preferable for tumor theranostic applications due to their longer blood residence time and shorter biological half-life.22 Besides, the enzyme-mimicking catalytic activity is also strongly affected by the particle size of Au NPs, and those with a smaller diameter are expected to display higher catalytic activity.23 Herein, we constructed Au NP-decorated poly (lactic-co-glycolic acid) (PLGA) nanoparticles wherein smaller Au nanoparticles are aggregated on the surfaces of polymer spheres to solve the above conundrum. This strategy could not only prevent the aggregation of Au NPs and keep these Au catalysts stable and active, but also maintain excellent PTT efficiency and PA properties, thus the compelling characteristics of Au nanoparticles could be utilized for effective tumor therapy. As schematically shown in Scheme 1, we synthesized Au-PLGA hybrid nanoparticles using polydopamine (PDA) as a bridge to link Au and PLGA, where PEG acted as a hydrophilic shell and Aumodified drug-loaded PLGA NPs served as a hydrophobic core (PLGA/DOX@PDA-Au-PEG NPs). Compared with graphene and its derivatives which present more excellent properties than gold,24,25 our obtained Au-PLGA hybrid nanoparticles showed several advantageous features: (1) intrinsic theranostic properties and high photothermal conversion efficiency of up to 69.0% could be obtained. (2) NIR-responsive Au-PLGA systems facilitated heat-triggered drug release in the tumor tissues and cells to enhance anticancer efficacy. (3) Au NPs could modulate tumor microenvironments through their catalase-mimicking activity toward H2O2 to improve the hypoxia environment in the tumor to inhibit tumor growth and metastasis. (4) NIR triggered the Au NPs to generate higher levels of ROS, thus further killing the tumor. We believe that the ‘allin-one’ Au-PLGA hybrid nanoparticles combining these advantages can be utilized for PA-imaging guided disease diagnosis and synergistic tumor therapy.

EXPERIMANTAL SECTION Materials. PLGA (lactic acid/glycolic acid=50/50, Mw=15-25 kDa) was obtained from Akina Inc. (Indiana, USA). Hydrogen peroxide (H2O2), tris (hydroxymethyl) aminomethane and Dopamine hydrochloride were purchased from Aladdin (Shanghai, China). Poly (vinyl alcohol) (PVA) was brought from Acros Organics (Beijing, China). Doxorubicin hydrochloride (DOX) was got from


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Adamas Reagent Co., Ltd (Shanghai, China). Gold (III) chloride trihydrate (HAuCl4⋅3H2O), dichloromethane (DCM) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM) was brought from Thermo Fisher Scientific (Waltham, USA). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). 4,6-diamidino-2-phenylindole (DAPI) was purchased from KeyGEN BioTech (Nanjing, China). Synthesis of PLGA/DOX@PDA-Au NPs. PLGA/DOX@PDA NPs were prepared according to our previous work,26 and then 16 µL HAuCl4⋅3H2O (0.24 M) was injected into the PLGA/DOX@PDA NP suspension (1.0 mg/mL, 20 mL). After stirring for 12 h at 25 °C in dark condition, the nanoparticles

were

centrifuged

(10

min,

10,000

rpm,)

and

washed,

and

the

obtained

PLGA/DOX@PDA-Au NPs were re-dispersed in 2 mL H2O. For comparison, PLGA@PDA-Au NPs were also synthesized, and the process was similar to the above except with no DOX addition. Synthesis of PEG-Modified PLGA/DOX@PDA-Au NPs. 2 mL PLGA/DOX@PDA-Au NP suspension (10 mg/mL) was mixed with 8 mL H2O containing 10 mg of PEG-NH2 (Mw = 5000). The o mixture was stirred at 25 °C for 6 h, centrifuged at 10,000 rpm for 10 min and washed with water. The obtained PEG-modified PLGA/DOX@PDA-Au NPs (PLGA/DOX@PDA-Au-PEG NPs) were redispersed in H2O for further use. For comparison, PLGA@PDA-Au-PEG NPs were also synthesized. Photothermal Property and PA Imaging of PLGA@PDA-Au-PEG NPs in Vitro. To evaluate the photothermal

efficiency of

PLGA@PDA-Au-PEG

NPs, 1.5 mL

H2O, PLGA@PDA,

PLGA@PDA-Au and PLGA@PDA-Au-PEG nanoparticles were dispersed in a cuvette at different concentrations and separately exposed to 808-nm laser (10 min and 1.0 W/cm2). A thermocouple thermometer (Fluke 52Ⅱ, USA) was employed to determine the temperature every 60 s. The PA imaging experiments were performed on a multi-spectral optoacoustic tomography system (inVision 128, iThera, Germany). PLGA@PDA and PLGA@PDA-Au-PEG nanoparticles with different concentrations were embedded in agar gel cylindrical tubes and excited by a photoacoustic illumination source.


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Oxygen Generation by PLGA@PDA-Au-PEG Catalysis. Oxygen generated by PLGA@PDAAu-PEG NPs was measured by a Dissolved Oxygen Meter (JPSJ-606L, Leici, China). All assays were carried out in 10 mL tubes with phosphate-buffered saline solution (pH 7.0). After addition of different concentrations of nanoparticles and H2O2 (30%, w/v) in the buffer system, the dissolved oxygen generated by H2O2, PLGA@PDA and PLGA@PDA-Au-PEG NPs were measured at given time intervals, respectively. To evaluate the effect of the NIR irradiation on O2 generated, the tubes were exposed to 808-nm laser (1.0 W/cm2, 5 min), and the changes in oxygen generation were recorded. Drug Loading and in Vitro Releasing Properties. Briefly, 5.0 mg of PLGA/DOX@PDA-AuPEG NPs were dissolved in 3.0 mL CH2Cl2, and then 10 mL water was added. After solvent extraction, the DOX was transferred from organic phase into aqueous phase. The content of DOX in the aqueous phase was determined by a UV-vis spectrophotometer at 480 nm. NIR-triggered release profile of DOX was also measured. Briefly, the PLGA/DOX@PDA-AuPEG NPs were dissolved in 1.5 mL PBS solution (pH=7.4). At different time intervals, the dispersions were exposed to the NIR laser for 10 min at 0.8 W/cm2 and 1.0 W/cm2, respectively. After irradiation, 200 µL of the dispersion was collected and centrifuged. The content of free DOX in the supernate was tested by a microplate reader (SPARK 10M, Tecan, Switzerland). Celluar Experiments. CCK-8 assays were used to evaluate the cell viability. In detail, CT26 cells (mouse colon cancer cells) or 4T1 cells (murine breast cancer cells) were seeded into 96-well plates at 1×104 per well and grew overnight. After that, culture media containing PLGA@PDA, PLGA@PDA-Au or PLGA@PDA-Au-PEG NPs at 25, 50, 100, 200 µg/mL were added to the cells and cultured for 48 h respectively. Then, CCK-8 solution was added to determine the relative cell viability using a microplate reader at 450 nm. To determine in vitro chemo-photothermal efficiency, 4T1 cells were incubated with PLGA@PDA, PLGA@PDA-Au-PEG and PLGA/DOX@PDA-Au-PEG at different concentrations for 24 h and then exposed to the 808-nm laser irradiation (10min, 1.0 W/cm2,). After NIR exposure, the cells were incubated for another 24 h. Finally, the cell viability was tested. Cellular uptake


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characteristic of PLGA/DOX@PDA-Au-PEG NPs in 4T1 cells was observed by laser scanning confocal microscopy. Cells were labled by DAPI to standing nuclei. Tumor Model. Balb/c mice (female, 18-22 g, 4-6 weeks) were obtained from Yangzhou University Comparative Medicine Centre and used under the agreement of Medical Ethics Committee of Yangzhou University Medical Academy. To develop the tumor model, 2×106 4T1 cells suspended in DMEM medium were subcutaneously injected into the back of each mouse. After 7 days of tumor xenografts, the tumor volumes reached approximately 50-100 mm3, and were ready for in vivo studies. In Vivo Photoacoustic Imaging. To evaluate the in vivo photoacoustic tumor imaging ability of the obtained particles, PLGA@PDA-Au-PEG NPs dispersion (5.0 mg/mL) was intratumor injected into a Balb/c mouse. Then photoacoustic images of the tumor section were acquired by the photoacoustic scanner and the PA signal intensity was recorded (LAZR, Visualsonics, Canada). In Vivo Chemo-Photothermal Therapy. Balb/c mice bearing subcutaneous 4T1 tumors (~ 70 mm3) were randomly divided into five groups (n=3 per group): (1) control; (2) PLGA/DOX@PDAAu-PEG; (3) PLGA@PDA + laser; (4) PLGA@PDA-Au-PEG + laser; (5) PLGA/DOX@PDA-AuPEG + laser. The administration of various nanoparticles was tail intravenous injection and the dose was 20 mg/kg in all cases. On day 1, 3, 5, 8, 11, 14 and 17, each group was administrated with different nanoparticles solutions. A thermal imaging camera was used to record the real-time temperature changes. The tumor size of each mouse was measured by a digital calliper and calculated according to the following formula: V=width2 × length/2. The relative tumor volumes were calculated as V/V0 (V0 was the initial tumor volume).

RESULTS AND DISCUSSION In this work, preparation of drug-loaded Au-PLGA hybrid nanoparticles (PLGA/DOX@PDA-Au-PEG NPs) comprised three critical steps (Scheme 1), including construction of DOX-loaded PLGA NPs, synthesis of Au NPs on the surface of PLGA spheres, and PEG modification. Although the preparation methods of drug-loaded PLGA nanoparticles are extensively reported,27 it is still hard to directly grow Au NPs on PLGA surface due to the lack of attachment sites. Herein, in order to solve this problem,


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we modified PLGA nanoparticles with PDA by in situ self-polymerization of dopamine as our previous work,28 and then the PDA-covered PLGA NPs were immersed in HAuCl4 solution, wherein AuCl4- ions absorbed on the PDA layer and were reduced to Au NPs by reducing groups on the PDA chains. Finally, the PLGA/DOX@PDA-Au NPs were stabilized by PEG-NH2 through the interactions between amino groups and Au NPs,29 result in the formation of PLGA/DOX@PDA-Au-PEG NPs. SEM (Figure 1a) and TEM (Figure 1b) images revealed that PLGA@PDA-Au NPs exhibited obvious mono-disperse character with large-scale domains of ~160 nm, and Au NPs immobilized on PLGA@PDA surface (inset of Figure 1b) were well dispersed with an average size of 8 nm. The TEM elemental mapping also displayed the distribution of Au element in an individual particle (Figure 1c). Furthermore, Figure 1d depicted the optical absorbance spectra of as-prepared nanoparticles. The obviously distinct color in the inset photos indicated the difference in composition. Compared with PLGA@PDA and PLGA NPs, PLGA@PDA-Au NPs exhibited a major absorption peak at 520 nm, belonging to Au NPs.30 Next, the PLGA@PDA-Au hybrid structure was further confirmed by X-ray powder diffraction, X-ray photoelectron spectroscopy and thermogravimetric analysis (TGA). As shown in Figure 2a, XPS measurement of PLGA@PDA-Au NPs showed the presence of C, N, O and Au elements. Au 4f peaks were clearly detected at 84.38 and 88.08 eV (Figure 2b), which were not obtained in PLGA@PDA NPs (Figure S1), verifying the success of Au NP-decoration on PLGA@PDA NPs. Furthermore, as presented in the XRD pattern (Figure 2c), the PLGA@PDA-Au NPs exhibited four peaks, which were indexed as (111), (200), (220) and (311) reflections of the facecentred-cubic structure of crystalline Au0. TGA was used to determine the content of Au in the AuPLGA hybrid NPs. The result shown in Figure 2d indicated that the Au content in PLGA@PDA-Au NPs was about 5.31%. Taken together, the above results proved the successful assembly of PLGA@PDA-Au hybrid nanoparticles. In order to prevent the aggregation of nanoparticles in biofluids and endow them with excellent water solubility, PEG-NH2 molecules were introduced onto the PLGA@PDA-Au NPs through Au–N bond.31 As shown in Table S1, the size distribution showed a trend of increasing size after coating PDA, subsequent formation of Au NPs and final PEG modification. Additionally, the zeta potentials


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were -11.6 ± 1.26, -2.87 ± 0.32, -16.7 ± 0.44 and -29.5 ± 0.58 mV for PLGA, PLGA@PDA, PLGA@PDA-Au

and

PLGA@PDA-Au-PEG

NPs

respectively,

which

also

indicated

the

compositional changes of the as-obtained nanoparticles. Importantly, the PEG layer could improve the water solubility and stability of PLGA@PDA-Au NPs.32 As shown in Figure S2, compared with PLGA@PDA-Au NPs, PEGylated Au-PDA@PLGA NPs showed much better chemical stability in PBS and cell culture medium (DMEM with 10% FBS) during long-term storage. Having successfully accomplished construction of the PLGA@PDA-Au-PEG NPs, we then tested their properties. As seen in Figure 1d, the present of Au NPs affected the optical absorption of PLGA@PDA NPs. After capping by PDA, the absorption of PLGA@PDA NPs in the NIR region was increased. When further introducing Au NPs, an increase in absorption was observed. The relatively high absorbance value in the NIR region ensured that our obtained nanoparticles could serve as an effective NIR agent for photothermal therapy. So, the photothermal properties of Au-PLGA hybrid nanoparticles were evaluated firstly. An 808-nm laser was chosen as a power source. As shown in Fig. 3a, PLGA@PDA NPs at 100 µg/mL presented a temperature increase of 6.2 °C after exposure of NIR laser for 10 min. Moreover, PLGA@PDA-Au NPs at the same dose exhibited a rapid temperature increase of 11.1 °C. With the PLGA@PDA-Au NP concentration increasing from 100 to 200 µg/mL, the solution temperature showed an increase of 15.9 °C. On the contrary, no obvious temperature rise (< 2 °C) was detected for the water sample. This result demonstrated that the small Au NPs assembled on the surface of nanoparticles exhibited excellent photothermal conversion efficiency and enhanced the photothermal capability of Au-containing nanocomposites significantly.33 Infrared thermal images were also acquired to monitor the photothermal effects of the PLGA@PDA-Au NPs, demonstrating irradiation time-dependent photothermal behavior (Figure 3b). We then measured the photothermal conversion efficiency (η) values of as-prepared nanoparticles using a method reported in the literature.34 On the basis of the data in Figure 3c, the η value of PLGA@PDA-Au was ~76.7%, which was much higher than that of PLGA@PDA (Table S2). This result also suggested that PLGA@PDAAu NPs could generate considerable heat upon 808-nm laser exposure, superior to the performance of PLGA@PDA NPs. Meanwhile, PLGA@PDA-Au NPs remained stable even after irradiation. After


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three cycles of on-off irradiation, PLGA@PDA-Au NPs still maintained their high photothermal conversion efficacy (Figure 3d). More importantly, PEG modification had almost no effect on the photothermal conversion efficiency of the PLGA@PDA-Au NPs (Figure S3). While improving the system stability, the photothermal conversion efficiency remained as high as 69.0%. Thus, PLGA@PDA-Au-PEG NPs had excellent stability and efficient photothermal conversion, showing their potential as a candidate for PTT. To evaluate the potential of our nanoparticles for in vitro PA imaging, PLGA@PDA-Au-PEG and PLGA@PDA NPs were embedded in agar gel cylinders to generate photoacoustic images on a multispectral optical tomography imaging system. As shown in Figure 4a, a concentration dependent PA signals could be obtained. A quantitative analysis also showed a linear relationship between the nanoparticle concentration and PA signal intensity (Figure 4b). It should be pointed out that that PA signal of PLGA@PDA-Au-PEG NPs was much stronger than that of PLGA@PDA NPs, coinciding with the results of the obtained by photothermal conversion studies. These results verified again that the decoration of Au NPs could improve the imaging ability of PLGA@PDA-Au-PEG NPs, acting as an ideal contrast agent for PA imaging. The introduction of Au NPs also endowed the PLGA@PDA-Au-PEG NPs with the catalasemimicking ability to catalyse the decomposition of H2O2. As shown in Figure 5a, PLGA@PDA-AuPEG NPs could trigger efficient O2 generation in H2O2 solution, while PLGA@PDA NPs showed almost no effect on the decomposition of H2O2. This catalase-mimicking activity of PLGA@PDA-AuPEG NPs was dependent on the concentrations of H2O2 and nanoparticles (Figure 5b and 5c). During the detection of O2, observable bubbles appeared in the PLGA@PDA-Au-PEG solution (Figure 5d). Interestingly, upon NIR irradiation, the O2 level was enhanced (Figure 5e). This observation was consistent with a previous report describing the enhanced catalytic activity promoted by light.35 These results demonstrated that Au-containing nanocomposites as a catalase-mimicking enzyme could react with H2O2 to generate O2. It has been proven that a deficit of oxygen may result in promoting the growth of the tumor, whose tissues and tumor cells expressed much higher levels of H2O2 than normal tissues and normal cells do.36 The generation of O2 through PLGA@PDA-Au-PEG NPs might help to


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regulate the oxygen level in the tumor, maintaining oxygen at a normal level inside the tumor to inhibit cancer growth.37 Thus, due to their H2O2 reduction ability, PLGA@PDA-Au-PEG NPs could act as an oxygen supplier in tumor cells, especially during PPT cancer therapy. Although the Au nanoparticles possess lower photothermal efficiency than that of Au-based nanorods,38 the enzyme-mimicking catalytic activity is strongly affected by the size of AuNPs, and those with a smaller diameter are expected to display higher catalytic activity. Therefore, our strategy balances the performance between photothermal efficiency and catalase-mimicking activity. To demonstrate the drug-loading and drug-release capabilities of the PLGA@PDA-Au-PEG NPs, DOX, a model anti-tumor drug, was loaded as a guest molecule. The drug loading process followed the O/W/O emulsion solvent extraction method, and the loading efficiency was 14.1%. The drug release profiles of DOX-loaded Au-PLGA hybrid nanoparticles were shown in Figure 6a. After 48 h later, 46.7% of DOX was released from Au-PLGA hybrid nanoparticles at 45 °C, while in contrast only 15.7% of DOX was released at 37 °C. Such a temperature-triggered release feature was due to the thermal instability of PLGA.39 Since the temperature in our system was a stimulus to trigger the drug release, we wondered whether the photothermal heating caused by NIR irradiation could also stimulate the drug release. In order to test this hypothesis, the PLGA/DOX@PDA-Au-PEG suspension in PBS (pH=7.4) was exposed to the 808-nm laser at different powder densities. As shown in Figure 6b, the DOX release was enhanced upon NIR irradiation. Compared with lower laser power (0.8 W/cm2), the DOX release with higher laser power (1.0 W/cm2) was more pronounced. These results confirmed that NIR irradiation was beneficial to triggering the drug release from the PLGA/DOX@PDA-Au-PEG NPs. Hence, PLGA/DOX@PDA-Au-PEG NPs, as an environmental temperature-responsive drugloading platform, could trigger the drug release in the assistant of NIR irradiation, which would promote the antitumor efficacy and minimize the drug side effects during combination therapy. Before conducting in vitro chemo/PTT therapy in cells, we tested the cellular uptake of PLGA/DOX@PDA-Au-PEG NPs. 4T1 cells were first cultured with PLGA/DOX@PDA-Au-PEG NPs, and then the cell nuclei were stained with DAPI. DOX is an inherently florescent molecule, which enables direct analysis of its uptake by tumor cells.40 The fluorescence imaging of both DOX


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and DAPI was performed using confocal microscopy.41 As displayed in Figure 7a, the green fluorescence of DOX was observed inside the cells, indicating the efficient cellular uptake of DOXloaded nanoparticles. Next, we performed a CCK-8 assay to evaluate the cytotoxicity of the Au-PLGA hybrid nanoparticles. Different amounts of as-prepared nanoparticles were added to wells containing 4T1 cells. After incubation for 48 h, we found that even at a high concentration (200 µg/mL), PLGA@PDA-Au-PEG NPs induced no appreciable negative effects on the viability of 4T1 cells (Figure 7b). Similar results were also obtained in CT26 cell lines (Figure S4). The results indicated that Au-PLGA hybrid nanoparticles possessed good biocompatibility and were feasible for further biorelated applications. As designed, the PLGA/DOX@PDA-Au-PEG NPs would possess chemotherapy and PTT ability together upon 808-nm illumination. As shown in Figure 7c, when the AuPDA@DOX/PLGA-PEG suspension (200 µg/mL) was cultured with 4T1 cells and subsequently irradiated using 808-nm laser (1.0 W/cm2, 10 min), about only 7.2% of the cells survived. However, about 22.3% or 65.7% of 4T1 cells survived when the cells were incubated with PLGA@PDA-AuPEG (200 µg/mL) or PLGA@PDA (200 µg/mL) after irradiation for 10 min. In the absence of laser irradiation, 70.6% of 4T1 cells were observed to survive in PLGA/DOX@PDA-Au-PEG group due to the DOX anticancer effect. We also examined the intracellular reactive oxygen species (ROS) level upon NIR irradiation. ROS are considered to cause the oxidation of intracellular DNA and protein, and here DCFH-DA was chosen as the ROS fluorescence probe. When cells were incubated with the Au-PLGA hybrid nanoparticles (Figure S5), the fluorescence signal level was nearly the same as that of the control group, demonstrating the low cytotoxicity of our system. In contrast, a higher fluorescence signal, indicating ROS existence, was observed when the wells containing PLGA@PDA-Au-PEG NPs were exposed to NIR irradiation. This suggested that Au NPs could act as excellent electron acceptors to inhibit the quick recombination of photo-excited electron-hole pairs, thus promoting the generation of ROS.42 On the basis of our findings, we concluded that PLGA/DOX@PDA-Au-PEG NPs could not only produce heat but also increase the intracellular redox state in response to NIR irradiation. It must also be mentioned that the ROS might improve the PLGA/DOX@PDA-Au-PEG NPs catalytic activity,


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which was consistent with the previous results on O2 level detection in vitro. Thus, PLGA/DOX@PDA-Au-PEG NPs had strong photothermal conversion ability, promoted the production of ROS under laser irradiation and delivered DOX into tumor cells. Use of hyperthermia in combination with ROS and chemotherapy was a more efficient therapeutic protocol against tumor cells than either PTT or chemotherapy alone. Next, we carefully studied the in vivo behavior of Au-PLGA hybrid nanoparticles. After establishing the tumor models, the biodistribution of PLGA@PDA-Au-PEG NPS in tumor tissues and major organs (heart, liver spleen, lung and kidney) was determined by ICP-AES at 24 hours postinjection. As shown in Figure 8a, PLGA@PDA-Au-PEG NPs exhibited the largest amounts of Au accumulated in the reticuloendothelial (liver) system, which was in accordance with previous reports.43, 44

In particular, the Au accumulation in the tumor was 8.3% of the injected dose, indicating a relatively

high tumor targeting efficacy for PLGA@PDA-Au-PEG NPs; thus, efficient PTT could be obtained. To test the potential of PLGA@PDA-Au-PEG NPs using as a PA imaging agent in vivo, each 4T1 tumor-bearing mouse was administered 50 µL of PLGA@PDA-Au-PEG NPs in PBS, and crosssectional PA images were acquired. As presented in Figure 8b, compared with the control group, the average PA intensity derived from the tumor site was increased. Quantitative analysis show in in Figure 8c showed that the PA intensity in vivo was enhanced by 1.4-fold, suggesting that PLGA@PDA-Au-PEG NPs could serve as a PA contrast agent. We further investigated the synergistic antitumor efficacy of Au-PLGA hybrid nanoparticles in vivo (Figure 9). When the tumor volume reached 70 mm3, the mice were divided into five groups (1) PBS, (2) PLGA/DOX@PDA-Au-PEG, (3) PLGA@PDA + Laser, (4) PLGA@PDA-Au-PEG + Laser and (5) PLGA/DOX@PDA-Au-PEG + Laser. During the chemo-photothermal therapy, the various dispersions were intravenous injection into each mouse through a tail vein at a dose of 20 mg/kg. After that, these mice were exposed to 808-nm laser (10 min, 1.0 W/cm2). As expected, the temperature of the tumor site was closely related to the irradiation time after administration. As illuminated in Figure 9a, the tumor temperature of group (5) increased from 34.1 to 57.3 °C rapidly when NIR laser irradiated for 10 min. In comparison, the tumor temperature of group (3) only increased slightly from


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33 to 42 °C under the same conditions (Figure S6). Thus, the PLGA/DOX@PDA-Au-PEG NPs were capable of inducing hyperthermia with high efficiency in the tumor area. The tumor volumes of different groups were recorded during the subsequent days. As shown in Figure 9b, the mice administrated with only PLGA/DOX@PDA-Au-PEG exhibited slight tumor inhibition. Although obvious inhibition of tumor growth was observed with administration of PLGA@PDA-Au-PEG plus NIR laser, the tumor was not destructed completely, showing lower efficiency in inhibition of tumor growth. In contrary, the tumor growth with administration of PLGA/DOX@PDA-Au-PEG plus NIR laser was effectively inhibited, demonstrate the chemo-photothermal synergistic therapeutic effect. At 17 days after PTT, the tumor weights, tumor photographs and 4T1 tumor-bearing mice in each group were shown in Figure 9c, Figure 9d and Figure S7. The tumor volume and weight in group (5) were the lowest values among all the groups, revealing that the tumors were completely eradicated without recurrence during the treatment. Toxicity is a critical factor in assessing the biocompatibility of photothermal agents. During the observational period (within 17 days), a series of observations was taken in vivo, including examining daily behavior (eating, drinking, activity) and body weight. There were no abnormalities in the daily behaviour and body weight (Figure 9e). We also carefully observed the behaviors of healthy mice after PLGA@PDA-Au-PEG NPs injection (as high as 50 mg/kg) over a long-term period (30 days). Meanwhile, the major organs of mice after administration of PLGA@PDA-Au-PEG NPs for 30 days were sliced, and then stained by H&E for histology analysis (Figure S8). The pictures showed that there were no recognizable changes in organ damage or inflammatory lesions in all major organs of the mice, including the hepatic and renal tissue. Altogether, PLGA@PDA-Au-PEG showed high biocompatibility and could act as a theranostic agent in PA imaging with a chemo/PTT synergetic effect.

CONCLUSIONS In summary, ‘all-in-one’ PEG coated Au-PLGA hybrid nanoparticles were synthesized in this work for PA imaging-guided synergistic cancer chemo/photothermal therapy. In this strategy, the structure of


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smaller Au NPs decorated on PLGA NPs not only keeps and protects the high catalytic activity of Au NPs, but also endows the hybrid nanoparticles with excellent photothermal conversion efficacy and PA imaging ability. Thus, the obtained PLGA/DOX@PDA-Au-PEG NPs show good catalytic activity to trigger H2O2 decomposition to overcome the intrinsic hypoxia-associated resistance to chemotherapy. After being concurrently exposed to NIR laser, PLGA/DOX@PDA-Au-PEG NPs make a contribution to more effective synergistic anti-tumor effect, compared to chemo- or photothermomono-therapy. It is worth emphasizing that NIR irradiation not only generates heat to kill the tumor cells, but also enhances the catalytic efficacy of Au-PLGA hybrid nanoparticles in the generation of oxygen from H2O2 and ROS generation to further destruct the tumor cells. This work presents the ‘allin-one’ PLGA-based nanoparticles for further clinical application in PA imaging-guided combined chemo/photothermal therapy of cancers.

ASSOCIATED CONTENT Supporting Information Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.xxxxxx. Characterization, ROS detection, biodistribution of nanoparticles, histology analysis; XPS spectrum, stability performance; Cell viability, ROS level; IR thermal images, photos of tumor-bearing mice, H&E staining; Sizes, zeta-potentials and photothermal conversion efficiency; Figures S1-S8 and Tables S1-S2 (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (L. Fan). ORCID Lei Fan: 0000-0003-0049-5819 Author Contributions


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†J. Xi and W. Wang contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEGGEMENTS This project was funded by the National Natural Science Foundation of China (No. 21703198), the Interdisciplinary Subject Construction Foundation of Yangzhou University (No. jcxk 2015-19), the University Natural Science Foundation of Jiangsu Province (16KJD150004) and the Social Development Project of Yangzhou City (YZ2016074). The authors also gratefully acknowledge financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions.

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TOC

Au-PLGA hybrid nanoparticles with catalase-mimicking and near-infrared photothermal activities for photoacoustic imaging-guided cancer therapy

†

†

Juqun Xi, a,b Wenjuan Wang,c Lanyue Da,a Jingjing Zhang,a Lei Fan, d* Lizeng Gaoa

Au-PLGA hybrid nanoparticles with drug-loading capacity, catalase-mimetic activity, photoacoustic (PA) imaging ability and photothermal properties were prepared, and the compelling characteristics could be utilized for effective tumor therapy.


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Figures

Scheme 1. Schematic diagram of PLGA/DOX@PDA-Au-PEG nanoparticles synthesis.

Figure 1. SEM (a) and TEM (b) images of PLGA@PDA-Au NPs. The inset of (b) showed the images of Au NPs on the surface of hybrid nanoparticles. (c) TEM elemental mapping of Au signal. (d) UVvis spectra of PLGA, PLGA@PDA, PLGA@PDA-Au NPs. Inset: the optical appearance of asobtained nanoparticles; 1-PLGA, 2-PLGA@PDA, 3-PLGA@PDA-Au.


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Figure 2. (a) XPS spectrum of PLGA@PDA-Au NPs. (b) Au 4f XPS spectrum of PLGA@PDA-Au NPs. (c) XRD patterns of PLGA@PDA, PLGA@PDA-Au NPs. (d) TGA curves of PLGA@PDA, PLGA@PDA-Au NPs.

Figure 3. (a) The curves of temperature increase curves of water, PLGA@PDA and PLGA@PDA-Au solutions with 808-nm laser irradiation. The total irradiation time was 10 min. (b) Temperature profiles of PLGA@PDA-Au solution (200 µg/mL) during the laser irradiation recorded by an IR camera. (c) Photothermal conversion of PLGA@PDA (200 µg/mL) and PLGA@PDA-Au (200 µg/mL) solutions under 808-nm laser irradiation. (d) Thermal curves of PLGA@PDA (200 µg/mL) and PLGA@PDAAu (200 g/mL) in aqueous solution over three on/off cycles of the 808-nm laser. NIR power: 1.0 W/cm2.


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Figure 4. (a) PA imaging phantoms of PLGA@PDA and PLGA@PDA-Au-PEG NPs. (b) Linear relationship between concentration of nanoparticles and PA signal intensity

Figure 5. (a) Oxygen generated by H2O2, PLGA@PDA (100 µg/mL) and PLGA@PDA-Au-PEG (100 µg/mL) for 10 min. Oxygen generated by PLGA@PDA-Au-PEG at different concentrations of H2O2 (b) and PLGA@PDA-Au-PEG nanoparticles (c). (d) Photos of oxygen generated by H2O2 and PLGA@PDA-Au-PEG for 10 min. 1-H2O2; 2-H2O2 + PLGA@PDA-Au-PEG. (e) Oxygen generated by H2O2, PLGA@PDA (100 µg/mL) and PLGA@PDA-Au-PEG (100 µg/mL) NPs with or without 808-nm laser irradiation (5 min, 1.0 W/cm2).


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Figure 6. (a) DOX release prolifes from PLGA/DOX@PDA-Au-PEG nanoparticles in PBS at different temperatures. (d) NIR-response release of DOX from PLGA/DOX@PDA-Au-PEG nanoparticle with an 808-nm NIR laser (1.0 and 0.8 W/cm2) for 10 min at different time points as indicated by the arrows.

Figure 7. (a) Confocal ﬂuorescence images of 4T1 cells incubated with PLGA/DOX@PDA-Au-PEG NPs. (b) Relative viabilities of 4T1 cells after being cultured with PLGA@PDA, PLGA@PDA-Au and PLGA@PDA-Au-PEG for 48 h. (c) Relative viabilities of 4T1 cells cultured with PLGA@PDA, PLGA@PDA-Au-PEG and PLGA/DOX@PDA-Au-PEG NPs with or without 808 nm laser irradiation (10 min, 1.0 W/cm2).


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Figure 8. (a) Biodistribution of the obtained nanoparticles determined at 24 h post intravenous of PLGA@PDA-Au-PEG NPs by measuring the content of Au using ICP-AES. (b) In vivo PA images of tumor tissues taken before (upper) and after (bottom) injection with PLGA@PDA-Au-PEG NPs. (c) PA signals in the tumor before and after administrationwith PLGA@PDA-Au-PEG NPs.


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Figure 9. In vivo photothermal therapy of 4T1 tumor-bearing mice. (a) IR thermal images of 4T1 tumor-bearing mice after intravenous injection of PLGA/DOX@PDA-Au-PEG NPs under 808-nm laser irradiation (1.0 W/cm2, 10 min). (b) Tumor growth curves of different groups after treatment. (c) Tumor weights of each group after excision. (d) Photos of tumors from (1) control group, (2) PLGA/DOX@PDA-Au-PEG group, (3) PLGA@PDA + Laser group, (4) PLGA@PDA-Au-PEG + Laser group, (5) PLGA/DOX@PDA-Au-PEG + Laser group. (e) Body weight after treatment indicated over 17 days.


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Au-PLGA Hybrid Nanoparticles with Catalase-Mimicking and near

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