hydrophobic drugs by multifunctional yolk-shell nanoparticles for hepatocellular carcinoma theranostics

Chemical Engineering Journal 389 (2020) 124416

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Co-delivery of hydrophilic/hydrophobic drugs by multifunctional yolk-shell nanoparticles for hepatocellular carcinoma theranostics ⁎

T

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Xiangjun Chena, Lixue Songa, Xiliang Lia, Lingyu Zhanga, Lu Lia, , Xiuping Zhangb, , ⁎ Chungang Wanga, a b

Faculty of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin 130024, China Department of Hepatobiliary and Pancreatic Surgical Oncology, The First Medical Center of Chinese People’s Liberation Army (PLA) General Hospital, Beijing, China

H I GH L IG H T S

of hydrophilic/hydrophobic drugs in separate rooms. • Co-delivery triggered drugs release from independent channels without mutual effect. • Bimodal • The multi-mode upconversion luminescence/computed tomography/magnetic resonance imaging are obtained.

A R T I C LE I N FO

A B S T R A C T

Keywords: Co-delivery of hydrophilic/hydrophobic drugs Multi-mode imaging Chemo-photothermal therapy Hepatocellular carcinoma theranostics

Co-delivery of hydrophilic/hydrophobic drugs in separate rooms and bimodal triggered drugs release from independent channels without mutual effect, coupled with multi-mode imaging are vitally significative for overcoming drug resistance and enhancing the therapeutic effects. Herein, we report the polydopamine@upconversion nanoparticle@mesoporous silica yolk-shell nanoparticles (PDA@UCNP@mSiO2 NPs) to simultaneously load the hydrophilic doxorubicin (DOX) and hydrophobic hydroxycamptothecin (HCPT) in their distinct domains for combinational chemotherapy. The oleic acid-coated UCNPs attaching onto the surface of polydopamine (PDA) exhibited the multi-mode upconversion luminescence (UCL)/computed tomography (CT)/ magnetic resonance (MR) imaging and the hydrophobic environment held great ability for storage of the hydrophobic HCPT. While the mSiO2 shell was used for loading the hydrophilic DOX and ensuring the good water dispersibility of the NPs. Additionally, the NPs with near-infrared (NIR) excitation possessed an efficient photothermal efficiency of 31.1% achieving through the PDA. In a word, the resulted NPs were successfully employed for multi-mode imaging-guided synergistic dual drug chemo-photothermal therapy of hepatocellular carcinoma.

1. Introduction Cancer is one of the most dreadful diseases in the world, killing almost 10 million people every year [1–5]. Chemotherapy is one of the main treatment used in cancer therapy [6]. However, traditional cancer chemotherapy is often subjected to the inherent drawbacks of small molecular drugs, such as poor water solubility, intrinsic or acquired drug resistance and severe side effects [7,8]. In recent years, it is inefficient to use a single drug in the field of cancer treatment due to the toxicity of the drug at high dosage, the heterogeneity of cancer cells and its drug resistance [9–11]. Therefore, the co-delivery of multiple drugs has attracted increasing attention in cancer therapy because of its unique advantages, such as promote synergistic actions, decrease side ⁎

effects and lack of drug resistance [12,13]. Multifunctional nanoparticles (NPs) such as synthetic polymere drug conjugates, liposomes, micelles, nanogels, microspheres and nanospheres have been developed for the drugs delivery of cancer therapeutics, to achieve the controlled drug release and co-delivery [14–21]. Kutty et al. fabricated micelles for the co-delivery of suberoylanilide hydroxamic acid and paclitaxel [22]. However, it is technically challenging to simultaneously load hydrophobic and hydrophilic drugs for synergistic efficacy. Hu et al. have successfully demonstrated a new kind of polyprodrug-gated crosslinked vesicles (GCVs) to deliver hydrophobic and hydrophilic therapeutic drugs for chemotherapy [23]. Dai and co-workers synthesized a liposome-like nanocapsule from an amphiphilic drug-drug conjugate of Janus

Corresponding authors. E-mail addresses: [email protected] (L. Li), [email protected] (X. Zhang), [email protected] (C. Wang).

https://doi.org/10.1016/j.cej.2020.124416 Received 31 October 2019; Received in revised form 20 January 2020; Accepted 10 February 2020 Available online 11 February 2020 1385-8947/ © 2020 Elsevier B.V. All rights reserved.

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Scheme 1. Schematic illustration of the synthetic strategy of PDA@UCNP@mSiO2 NPs for multi-mode imaging-guided dual-drug chemo-photothermal HCC therapy.

Fig. 1. TEM images of A) PDA NPs; B) PDA@PAA NPs; C) PDA@RE(OH)3/PAA NPs; D) PDA@RE(OH)3/PAA@SiO2 NPs; E) PDA@UCNP@mSiO2 NPs, inset: SEM image of an individual PDA@UCNP@mSiO2 NP; F) HR-TEM image of a single NP, inset: The corresponding high-resolution TEM image; G) The elemental mapping images of a PDA@UCNP@mSiO2 NP.

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Fig. 2. A) FTIR spectra of (a) PDA NPs; (b) PDA@PAA NPs; (c) PDA@RE(OH)3/PAA@SiO2 NPs; (d) PDA@UCNP@mSiO2 NPs. B) XRD analysis of PDA@UCNP@ mSiO2 NPs. C) N2 adsorption-desorption isotherm and pore size distribution curve (inset) of PDA@UCNP@mSiO2 NPs. D) PDA@UCNP@mSiO2 NPs under 980 nm laser excitation, insets: (a) Corresponding digital photographs under daylight and (b) under the excitation of a 980 nm laser, respectively. E) Inverted fluorescence microscope images of HepG2 cells (a) Bright-field image, (b) DAPI, (c) Upconversion luminescence (UCL) image, (d) Overlay. Scale bar: 50 μm.

demonstrated by the effectively against hepatocellular carcinoma (HCC) therapy.

camptothecin-floxuridine conjugate [24]. Nevertheless, the work mentioned is mainly focused on therapeutics but cannot fulfill the requirements of precision therapy without using imaging contrast agents [25,26]. Obviously, the integration of multi-mode imaging, co-delivery of various hydrophilic and hydrophobic anticancer drugs and effective response to stimuli capabilities into a single NP will ensure a promising chemo-photothermal therapeutic agent for simultaneous diagnostic and therapeutic applications [27–29]. Nevertheless, few studies have been performed to create a dual drug delivery system, especially independent rooms for storage and release hydrophilic/hydrophobic drugs without mutual effects by an individual NP, which will effectively realize multimodal imaging-guided multidrug chemo-photothermal therapy. In the present work, we firstly develop a facile and novel method to prepare the multifunctional polydopamine@upconversion nanoparticle@mesoporous silica (designed as PDA@UCNP@mSiO2) yolkshell NPs. The obtained NPs achieved the co-delivery of hydrophilic doxorubicin (DOX)/hydrophobic hydroxycamptothecin (HCPT) in separate rooms and pH/NIR triggered drugs release from independent channels without mutual effect, coupled with multi-mode imaging for synergistic chemo-photothermal therapy of cancer. In addition, the enhanced therapeutic effect of drug delivery systems (DDSs) was

2. Experimental section 2.1. Synthesis of PDA NPs Typically, 20 mL of ethanol and 45 mL of deionized water were added to a 100 mL flask to synthesize the PDA NPs under stirring at room temperature. Then, NH3·H2O (2 M, 3 mL) was added to mixed solution, where the pH value was 8.8–9.0. After stirring for 30 min, dopamine hydrochloride (0.25 g) was dispersed into deionized water (5 mL) and then was added into the solution drop by drop, and the reaction solution was stirred for 24 h at 30 °C. The PDA NPs were separated from mixture by centrifuging and washing with water for thrice. 2.2. Synthesis of PDA@Polyacrylic acid (PDA@PAA) NPs 150 μL of NH3·H2O solution was injected to 10 mL of as-prepared PDA NPs solution. Briefly, 200 μL of PAA (0.2 g mL−1) was added into 3

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Fig. 3. Temperature elevation of the PDA@UCNP@mSiO2 NPs with different concentrations A) and the corresponding IRT images B). C) Laser power-dependent temperature elevation of PDA@UCNP@mSiO2 NPs. D) Cyclic photothermal stability tests of PDA@UCNP@mSiO2 NPs. E) Heating and cooling curves of the PDA@ UCNP@mSiO2 NPs. F) A plot of ln θ versus time that was obtained from the cooling period. G) In vitro HCPT and DOX release profiles at pH 5.3 or pH 7.4, respectively. H, I) In vitro DOX and HCPT release profiles recorded for PDA@UCNP@mSiO2 NPs with periodic 808 nm laser illumination.

2.5. Synthesis of PDA@UCNP@mSiO2 yolk-shell NPs

the solution under ultrasonic at room temperature for 30 min. Then, isopropyl alcohol (IPA) (100 mL) was added to the solution drop by drop to obtain the PDA@PAA NPs.

The as-prepared PDA@RE(OH)3/PAA@SiO2 NPs were dispersed in 3 mL deionized water, subsequently, 4.5 mL of ethanol, and 1.5 mL of oleic acid (OA) were mixed together under stirring, to which NaF was added. The mixture was stirred for 15 min, transferred into autoclave, sealed, and then solvothermally treated at 180 °C for 24 h. The products were purified by centrifugation, washed with ethanol and deionized water several times. Finally, PDA@UCNP@mSiO2 yolk-shell NPs were thus formed.

2.3. Synthesis of PDA@Rare earth hydroxide/poly(acrylic acid) (designed as PDA@RE(OH)3/PAA NPs) To synthesize the PDA@RE(OH)3/PAA NPs, Y(NO3)3·6H2O, Yb (NO3)3·6H2O, Er(NO3)3·6H2O (Y:Yb:Er = 78:20:2) were added in asprepared PDA@PAA followed by stirring. After stirred for 5 h, the PDA@RE(OH)3/PAA NPs were collected by centrifugal separation and washed three times with water.

3. Results and discussion 3.1. Synthesis of PDA@UCNP@mSiO2 yolk-shell NPs

2.4. Synthesis of PDA@RE(OH)3/PAA@SiO2 NPs The controlled synthetic strategy for preparation of the multifunctional PDA@UCNP@mSiO2 NPs was presented in Scheme 1. The PDA NPs were synthesized by using an oxidation and self-polymerization reaction under ammonia condition [30]. Then, the PAA on the PDA NPs was achieved upon the addition of PAA to obtain well-dispersed PAA/PDA NPs. The resulted PAA/PDA could absorb and retain water molecules inside its net structure [31]. Moreover, it was found that Y3+ ,

The PDA@RE(OH)3/PAA NPs were redispersed in 40 mL distilled water, then IPA (160 mL) was introduced into the above solution. A suspension was adjusted to pH ≈ 8.8 with NH3·H2O (2 M, 3 mL) solution. TEOS (200 μL) was then injected to the suspension, left stirring at 30 °C for 15 h, the products were washed with deionized water and collected by centrifugation. 4

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Fig. 4. A) Cytotoxicity assays of HepG2 cells incubated with various concentrations of NPs. B) blank PDA@UCNP@mSiO2 Fluorescence images of HepG2 cells after costaining using calcein AM. (a) Cells only, (b) Cells with NIR laser, (c) PDA@UCNP@ mSiO2 NPs, (d) PDA@UCNP@mSiO2 NPs with 808 nm laser (scale bars: 200 μm). C) Cell viabilities of PDA@UCNP@mSiO2 NPs and PDA@UCNP@mSiO2 NPs with NIR laser. Power density: 0.7 W cm−2. D) Cell viabilities of HepG2 cells after treatment with DOX-NPs, HCPT-NPs, cocktail, HCPT/ DOX-NPs, and HCPT/DOX-NPs + NIR in various concentrations. Power density: 0.7 W cm−2. E) Apoptosis-related caspase-3 and Bcl-2 protein expressions. GAPDH was used as an internal control.

Yb3+ and Er3+ can easily convert into RE(OH)3 in the presence of water and oxygen. Subsequently, RE(OH)3 was produced on the PAA. The well-dispersed PDA@RE(OH)3/PAA NPs were further used as precursor to obtain PDA@RE(OH)3/PAA@SiO2 NPs upon the addition of TEOS. Then, we used a solvothermal method to synthesize PDA@UCNP@ mSiO2 NPs under 180 °C. Finally, the synthesized yolk-shell NPs were utilized as pH/NIR-responsive drug carriers for multi-mode imagingguided dual-drug chemo-photothermal therapy of HCC. As seen in Fig. 1A, TEM image showed the PDA NPs with size about 120 nm. Additionally, PAA aqueous solution, ammonia solution, and IPA were added to PDA aqueous solution, where the final ratio of water to IPA was about 1:10. Then, PAA was successfully assembled on each PDA NP to obtain about 150 ± 20 nm PAA/PDA NPs (Fig. 1B). As PAA can absorb and retain water molecules within its net structure, the rare earth salt was hydrolyzed in the PAA network to obtain the PDA@RE (OH)3/PAA NPs with about 150 ± 20 nm (Fig. 1C). Subsequently, SiO2 shell was coated on the surface of the PDA@RE(OH)3/PAA NPs, which were synthesized by the addition of the TEOS under the aqueous ammonia conditions. The size of the synthesized PDA@RE(OH)3/PAA@

SiO2 NPs were approximately 165 ± 20 nm (Fig. 1D, S1). Finally, under a solvothermal method at 180 °C, PDA@UCNP@mSiO2 NPs with a well-defined yolk-shell structure at the size of 165 ± 20 nm were prepared (Fig. 1E and Fig. S2). The SEM image of a broken NP clearly revealed that the PDA@UCNPs were inside of the mSiO2 shell. The core was composed of small particles of UCNPs with about 6 nm for average size around PDA (Fig. 1F). The appeared large cavity between core and shell was due to the dissolution of PAA. When the RE(OH)3 NPs were completely transformed into UCNPs, the PAA molecules were fully replaced with OA, forming the OA-coated UCNPs. The lattice distance (0.31 nm) was observed from the inset in HR-TEM image, which was consistent with the (1 1 1) planes of cubic NaYF4 (0.3158 nm). As shown in Fig. 1G, the elemental mapping further confirmed the yolkshell structure and the uniform distribution of Na Si, F, N, Yb, Y and Er elements.

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Fig. 5. A) The cell cycle distribution of HepG2 cells induced by culture medium, NPs, DOX-NPs, HCPT-NPs and HCPT/DOX-NPs with same drug concentrations, respectively. B) Flow cytometry analysis for apoptosis of HepG2 cells treated with culture medium, NPs, DOX-NPs, HCPT-NPs and HCPT/DOX-NPs with same drug concentrations, respectively. Q1: necrotic cells, Q2: later apoptotic cells, Q3: living cells, Q4: early apoptotic cells.

3.2. Fourier transform infrared (FTIR) spectra/X-ray diffraction/N2 adsorption/desorption analysis

PDA@UCNP@mSiO2 NPs reveals their high potential for bioapplication.

FTIR spectra were used for demonstrating the surface functional groups (Fig. 2A). Successful coating of PAA on the surface of PDA was suggested by the presence of C = O stretch at 1733 cm−1, which suggested the successful coating of PAA. Furthermore, in the curve of the PDA@RE(OH)3/PAA@SiO2 NPs, the bands at 1089 cm−1 and 800 cm−1 revealed the characteristic peaks of the symmetrical and asymmetrical vibrations of Si-O-Si. The 1623 cm−1 peak is assigned to the C = C stretching vibration of the OA molecules coated on the surfaces of the UCNPs. The crystalline structure of the PDA@UCNP@ mSiO2 NPs was also characterized using X-ray diffraction (XRD) (Fig. 2B). All the diffraction peaks can be ascribed to a structure known from NaYF4 (JCPDS no. 77-2042) [32]. To study the specific surface area and porous of PDA@UCNP@mSiO2 NPs, the N2 adsorption/desorption isotherm and pore-size distribution of PDA@UCNP@mSiO2 NPs were shown in Fig. 2C, depicting that the pore size distributions of the NPs mainly focused at 2 and 4.8 nm, and also accompanied with the wide pore size distributions ranging from 2 to 30 nm. The mesoporous pore size was immensely favorable for drug-delivery applications.

3.5. Photothermal effect Owing to the NIR absorption, the synthesized PDA@UCNP@mSiO2 NPs can be utilized as promising photothermal therapeutic (PTT) agents (Fig. S4A). PDA@UCNP@mSiO2 NPs were dispersed in water (0, 100, 200, 400, and 800 μg mL−1) were irradiated with an 808 nm laser (1.0 W cm−2) for 300 s. The photothermal effect of the PDA@UCNP@ mSiO2 NPs was pronounced, the temperature of the NPs was increased and showed a concentration-dependent relationship with the irradiation (Fig. 3A). In addition, the infrared thermal (IRT) photographs were carried out to evaluate the photothermal properties of the PDA@ UCNP@mSiO2 NPs suspensions (Fig. 3B). Such as, at 800 μg mL−1, the temperature of the NPs dispersion could be over 55.9 °C, whereas the temperature of the deionized water was barely changed. As depicted in Fig. 3C, the temperature increase was also shown to be laser-powerdependent. To evaluate their photostability, PDA@UCNP@mSiO2 NPs solutions were irradiated by an 808 nm laser at 1 W cm−2 for four cycles, there was no evident temperature change after four laser on/off cycles (Fig. 3D). Meanwhile, the photothermal conversion efficiency (η) of PDA@UCNP@mSiO2 NPs was determined to be ~ 31.1% (Fig. 3E and F) according to the previous reports [33–37], which was high compared with the reported PTT agents (Table S1). The high photothermal conversion efficiency has made the PDA@UCNP@mSiO2 NP highly superior as a promising PTT agent.

3.3. UCL properties The resultant PDA@UCNP@mSiO2 NPs could emit strong green fluorescence under 980 nm irradiation (Fig. 2D). The insets were digital photographs of NPs under daylight and under the excitation of 980 nm laser, respectively. After PDA@UCNP@mSiO2 NPs were incubated with HepG2 cells at 37 °C for 3 h, a significant UCL signal was observed by excitation of NPs at a 980 nm laser. The corresponding bright field image of HepG2 cells, the fluorescent microscopy images and the overlay of the fluorescent microscopy images and bright field images were shown in Fig. 2G. These results clearly demonstrate that the NPs could widely applications for UCL imaging.

3.6. Drug loading and release For cancer therapy, the combination of two drugs exhibits more effective for cancer cells suppression [38,39]. The beneficial prospect of PDA@UCNP@mSiO2 NPs is drug delivery, and they could carry ultrahigh payloads of hydrophobic and hydrophilic drugs within separate rooms. DOX and HCPT were selected as model drugs, as they have been demonstrated to inhibit cancer cells synergistically by the different action mechanism. DOX is adsorbed on the mSiO2 shell of PDA@ UCNP@mSiO2 NPs through electrostatic attraction, while HCPT is integrated in the PDA@UCNP@mSiO2 NPs via hydrophobic interactions [40,41]. Free DOX and HCPT exhibited the typical absorption peaks at 480 nm and 376 nm. Meanwhile, the amounts of DOX and HCPT were

3.4. The stability of PDA@UCNP@mSiO2 NPs The PDA@UCNP@mSiO2 NPs exhibited high stability in water, phosphate-buffered saline (PBS), culture medium (DMEM), and fetal bovine serum (FBS), and remained stable for 24 h without any detectable agglomeration. (Fig. S3). This favorable colloidal stability of 6

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Fig. 6. Images from confocal microscope showing the delivery of DOX and HCPT to HepG2 cells. DOX is shown in red, and HCPT is shown in blue. The scale bars are 50 μm.

under NIR laser irradiation (Fig. 3H and 3I), which was due to the NPsinduced thermal effect to quicken the release of drugs. Therefore, the results indicate that the NPs can be used simultaneously as agents for pH/NIR stimulus responsive release of the hydrophilic/hydrophobic drugs.

measured by UV–vis spectra (Fig. S4B, S4C). Their profiles clearly elucidated that the DOX and HCPT were loaded the NPs. The DOX and HCPT loading content into the PDA@UCNP@mSiO2 NPs were determined 0.17 mg DOX and 0.65 mg HCPT per mg PDA@UCNP@mSiO2 NPs, respectively. The ultrahigh loading capability of HCPT is mainly caused by the hydrophobic interaction between the OA and HCPT. The drug release profiles of two drugs from HCPT/DOX-NPs in PBS pH 7.4 and pH 5.3 were shown in Fig. 3G. Quantitatively, less than 20% drug leakage for both DOX and HCPT was observed at pH 7.4 in the duration of 3 day, whereas upon incubating at pH 5.3, a continuous release of DOX and HCPT, and the accumulative release reached ~80% and ~60%. Nearly 90% and 70% of DOX and HCPT were released at pH 5.3

3.7. Cytotoxicity Biocompatibility is vital for biomedical applications of nanomaterials. The viabilities of HepG2 cells treated with various formulations were determined by both MTT and live-cell staining assays. As shown in Fig. 4A, cell viabilities could reach 93% even at 400 μg mL−1 after 24 h 7

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Fig. 7. A) CT images and attenuation (HU) plot of Yb3+ with different concentrations in vitro. B) MR images and plot of 1/T2 over Yb3+ concentration of PDA@ UCNP@mSiO2 NPs. The slope means the specific relaxivity (r2). C) T2-weighted MR and CT images of rats after tail veins injection of the PDA@UCNP@mSiO2 NPs.

loaded NPs with a fixed molar ratio of HCPT:DOX (1:1) under NIR laser irradiation revealed superior therapeutic effect demonstrating the outstanding synergetic anticancer activity. It is mentioned that HCPT/ DOX-NPs under NIR treatment resulted in higher level of cytotoxicity compared with control, the enhancement in therapeutic behavior is due to the two different drug combinations as well as the NPs-induced photothermal effect to accelerate release of drugs, which could effectively conquer the limitations of drug resistance and enhance the therapy efficiency. In order to evaluate the apoptosis efficiency, the expressions of apoptosis-related proteins were detected. As shown in Fig. 4E, the HCPT/DOX-NPs + laser group depicted the highest protein level of caspase-3 among all the groups. The protein level of Bcl-2 in the HCPT/DOX-NPs + laser group was lower than those of any other group. These results display that the combination of photothermalchemotherapy achieved much preferable cancer cell killing.

of treatment with PDA@UCNP@mSiO2 NPs, demonstrating the excellent biocompatibility of the NPs. To further verify the photothermal effect, the cells were incubated with PDA@UCNP@mSiO2 NPs for 24 h and then irradiated with 808 nm laser for 5 min (Fig. 4B). After treatment, Calcein AM (which could mark living cells with green color) were employed to dye the HepG2 cells under various conditions. Cells treated with either PDA@UCNP@mSiO2 NPs or only NIR laser alone shown no detectable cytotoxicity. In contrast, the cell viability significantly decreased in the presence of the PDA@UCNP@mSiO2 NPs under laser irradiation. Then, the photothermal effects of PDA@ UCNP@mSiO2 NPs were further investigated on HepG2 cells with laser (808 nm, 0.7 W cm−2) and no laser at different concentrations. PDA@ UCNP@mSiO2 NPs exhibited low cytotoxicity to cancer cells, the NPs upon laser irradiation showed higher cancer cell killing efficiency than NPs without NIR laser group (Fig. 4C), confirming the laser irradiationinduced cancer cell death. The cell viability can be decreased to be 40% under the 2 W cm−2 laser density (Fig. S5). The results above prove that the PDA@UCNP@mSiO2 NPs can play as the excellent PTT agents. Due the higher drugs loading ability of the yolk-shell NPs, the controllable proportion of different types of drugs and the synchronized release of drugs in an individual NP unit were realized. Combination index (CI) value was used to evaluate the synergistic effect of two drugs. CI < 1, = 1 or > 1 represent synergism, addition and antagonism respectively [24,42].The relevant explanation and equation about how to calculate CI was provided in supporting information. The HCPT/DOX-loaded NPs with a ratio of HCPT:DOX (1:1) depicted the lowest CI value (0.3068) and achieved the optimal synergistic effect. The MTT assays were utilized to access the combined therapeutic effects, the viabilities of HepG2 cells incubated with DOX-NPs, HCPT-NPs, cocktail, HCPT/DOXNPs, and HCPT/DOX-NPs + NIR was assessed for 24 h at various concentrations. As demonstrated in Fig. 4D, the ability to kill HepG2 cells was described as follows: HCPT/DOX-NPs + NIR > HCPT/DOXNPs > cocktail > DOX-NPs > HCPT-NPs, the obtained dual-drug

3.8. Cell cycle and apoptosis To obtain a crucial understanding of how single drug loaded NPs and dual-drug loaded NPs might result in HepG2 cell death, the cell cycle and apoptosis were tested. The cells treated with HCPT-loaded NPs generated a slight increase in the percentage of cells in the S phase, proving that the HCPT killed cancer cells by interfering with the function of DNA topoisomerase I (TopI) during DNA replication in the S phase. Meanwhile, the DOX-loaded NPs treated cells presented significantly G2/M phase arrest, and the mechanism of DOX was act to against DNA. Notably, the treatment with HCPT/DOX-NPs caused the most significant decrease in the percentage of cells in G0/G1 phase while presented increased G2/M and S phase than the control group, the NPs group, DOX-loaded NPs and HCPT-loaded NPs (Fig. 5A), indicating that dual drug-loaded NPs exhibited the synergistic therapeutic effects. As seen in Fig. 5B, the HepG2 cells cultured with NPs, DOX-NPs, 8

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Fig. 8. A) In vivo IRT images of mice I.V. injected with PBS or the PDA@UCNP@mSiO2 NPs (5 mg/kg) upon laser irradiation. B) Average weights of the mice in different groups. C) Tumor growth curves in different groups. D) Representative mice photos after different treatments and the corresponding H&E staining of tumor sections isolated from mice on day 10. E, F) Mean tumor weight and tumor inhibition rate of each group. G) Western blot analyses of expressed protein levels in tumor tissues.

3.10. CT/MR imaging

HCPT-NPs and HCPT/DOX-NPs, the percentage of apoptotic HepG-2 cells treated with HCPT/DOX-NPs (51.1%), which are higher than NPs, DOX-NPs and HCPT-NPs groups. The above results display the superior therapeutic effects of the HCPT/DOX-NPs, demonstrating the enhanced cytotoxicity by dual-drug loaded NPs compared with sole drug-loaded NPs.

Owing to the high X-ray attenuation of Yb3+, the PDA@UCNP@ mSiO2 NPs with various concentrations were implemented to acquire the CT/MR images [43]. The CT/MR signals were gradually strengthened with the increased concentration of PDA@UCNP@mSiO2 NPs in aqueous solution (Fig. 7A), which proved the preeminent diagnosis abilities of the PDA@UCNP@mSiO2 NPs. Benefiting from the excellent MR and CT imaging abilities of Yb3+, tumor-bearing mice were supervised by injecting the PDA@UCNP@mSiO2 NPs with the tail vein. Based on Fig. 7C, the high contrast imaging of the tumors were detected in the T2-weighted MR and CT investigations after 24 h injection, which stated the highly promising of the NPs for multiplex theranostic nanoplatforms to regulate the treatment schedule.

3.9. Confocal laser scanning microscope (CLSM) CLSM photographs of HepG2 cells were incubated with HCPT/DOXNPs for 1, 3, 6, 12 and 6 h + NIR in order to observe the cell uptake process. The blue fluorescence of the HCPT and red fluorescence of the DOX inside the HepG2 cells were shown in Fig. 6. After 6 h incubation, the cells were exposed upon 5 min NIR irradiation (808 nm, 0.7 W cm−2), then the enhanced fluorescence compared with no irradiation suggested that PDA@UCNP@mSiO2 NPs can be effectively taken up by cancer cells and drugs can be quickly triggered by NIR light.

3.11. In vivo combined therapy We also performed in vivo experiments to test the effect of combined PTT and chemotherapy using the obtained PDA@UCNP@mSiO2 NPs. The nude mice were purchased from the Changchun Institute of 9

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Fig. 9. The H&E staining of major organs.

tumor growth (Fig. 8E, F). The representative pictures of mice under various treatments were demonstrated in Fig. 8D. To further study the tumor damage degree after different treatments, we carried out Hematoxylin&Eosin (H&E) staining to examine tumors from mice. The group of HCPT/DOX-NPs + laser exhibited the most severe nuclear shrinkage, fragmentation and absence, which was possibly the reason that caused the obvious declination of the HCC tumors. Western blot analyses of the levels of caspase-3 and Bcl-2 were performed to further study the apoptotic effects of HCC after vairous treatments (Fig. 8G), which were consistent with the above results. The combination of the PTT and multidrug therapy could effectively inhibit the growth of the HCC in comparison with the single therapy. As shown in Fig. 9, no evident damage or inflammatory lesion appeared in all of the organs, further demonstrating no noticeable toxicity of the NPs in vivo. The antitumor results manifest that the PDA@UCNP@mSiO2 NPs can act as the excellent dual-drug carriers for multi-modal imaging-guided multidrug chemo-phototherapy with no detectable toxicity, which showed the great potential for biomedical applications.

Biological Products and all of the animal procedures were approved by the University Animal Care and Use Committee. Fig. 8A revealed that the high-contrast real-time IRT images can be realized by the I.V. administration of PDA@UCNP@mSiO2 NPs in vivo. There was only a slight tumor temperature increase (~2.5 °C) for the PBS + laser group. In comparison, the tumor temperature of PDA@UCNP@mSiO2 + laser group rapidly increased. After 10 min laser irradiation, the tumor temperature reached a plateau of ~49.3 °C (Fig. S6), which is sufficient to lead hyperthermia and eradicate the tumor immediately. The HCC tumor-bearing nude mice were randomly divided into nine groups with five mice per group including (1) PBS, (2) PDA@UCNP@mSiO2 NPs , (3) free HCPT, (4) free DOX, (5) HCPT-NPs, (6) DOX-NPs, (7) cocktail, (8) HCPT/DOX-NPs, (9) HCPT/DOX-NPs + laser. There existed no obvious difference in average weight between control group and PDA@ UCNP@mSiO2 group, which suggested the drug delivery carriers without affect on weight growth of the mice (Fig. 8B). The tumor volumes were monitored by a caliper every other day (Fig. 8C). Compared to control treated with PBS or PDA@UCNP@mSiO2, tumors on mice after chemotherapy with HCPT-NPs or DOX-NPs revealed only partially reduced growth speed. Notably, the same doses of free DOX and free HCPT resulted in unsatisfactory therapeutic effects, likely due to the poor permeability and retention effect after I.V. injection. The higher inhibition appeared in dual-drug loaded NPs treated group compared with the other group because of their accumulation in the desire tumor site through the enhanced permeability and retention (EPR) [44–46] effect once intravenously administered. In marked contrast, mice after treatment with HCPT/DOX-NPs + laser showed greatly inhibited

4. Conclusions In summary, we have designed and synthesized the multifunctional PDA@UCNP@mSiO2 yolk-like NPs by a facile and novel synthetic strategy. PDA@UCNP@mSiO2 yolk-like NPs using small particles of UCNPs around PDA as core and mesoporous silica as shell with hollow cavities have been served as storage space and passage for the hydrophilic/hydrophobic anticancer drugs. Double drug-carrying in separate 10

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rooms exhibited a higher anticancer effect than the single drug loaded one. PDA@UCNP@mSiO2 yolk-like NPs provide superior multi-modal imaging-guided multidrug chemo-phototherapeutic effectiveness. The development procedure exhibits outstanding performance with photothermal conversion efficiency, pH/NIR dual-responsive properties, low toxicity together with high dual drug loading capability. These studies demonstrate that this multifunctional nanomaterial has potential applications in the fields of imaging, drug delivery and anticancer therapy.

[14]

[15]

[16]

Declaration of Competing Interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

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X.J. Chen and L.X. Song contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant No. 21573040, 21872024), the Education Department of Jilin Province ”13th Five-Year” Science and Technology Research (JJKH20190270KJ, JJKH20190272KJ), the Fundamental Research Funds for the Central Universities (2412018ZD009, 2412019FZ009), the Jilin Provincial Research Foundation for Basic Research (20160519012JH, 20190303100SF) and Jilin Provincial Key Laboratory of Advanced Energy Materials.

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Appendix A. Supplementary data

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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2020.124416.

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