CNPs-Based Theranostic

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Biological and Medical Applications of Materials and Interfaces

HP-beta-CD Functionalized Fe3O4/CNPs-Based Theranostic Nanoplatform for pH/NIR Responsive Drug Release and MR/NIRFL Imaging-Guided Synergetic Chemo/Photothermal Therapy of Tumor Saijie Song, Yu Chong, Han Fu, Xinyu Ning, He Shen, and Zhijun Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09999 • Publication Date (Web): 14 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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ACS Applied Materials & Interfaces

HP-beta-CD Functionalized Fe3O4/CNPs-Based Theranostic Nanoplatform for pH/NIR Responsive Drug Release and MR/NIRFL Imaging-Guided Synergetic Chemo/Photothermal Therapy of Tumor

Saijie Song, a,b,c Yu Chong, d Han Fu, a Xinyu Ning, a He Shen, a Zhijun Zhang a,c,*

a

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine, CAS Center for

Excellence in Nanoscience, Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China

b

National & Local Joint Engineering Research Center of Biomedical Functional Materials, Jiangsu

Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Engineering Research Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Bio-functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, China

c

School of Nano Technology and Nano Bionics, University of Science and Technology of China,

Hefei 230026, China

d

State Key Laboratory of Radiation Medicine and Protection, School for Radiological and

interdisciplinary Sciences (RAD-X) and Collaborative Innovation Centre of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, China 1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

*Corresponding author: E-mail address: [email protected] (Z. Zhang)

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Abstract: The combination of chemotherapy and photothermal therapy has aroused great interest for its better antitumor effect than either single therapy alone. Herein, we report on development of hydroxypropyl-beta-cyclodextrin functionalized Fe3O4/carbon nanoparticles (HFCNPs) for pH/near infrared (NIR)-responsive drug release, magnetic resonance/NIR fluorescence (MR/NIRFL) imaging-guided combined chemo/photothermal therapy. The high doxorubicin (DOX) loading capacity (61.2 %) and controlled drug release by NIR irradiation and weak acid microenvironment render HFCNPs a good vector for DOX delivery and controlled release. Moreover, the MR/NIRFL dual-modal imaging was used to define the tumor location, size and boundary, and to track the tumor accumulation of HFCNPs and their biodistribution. The efficient accumulation and prolonged retention-time of the nanoparticles in tumor are beneficial to the tumor therapy. Taking advantage of the NIR laser induced heating and hence promoted drug permeation, remarkable tumor inhibition was realized by synergetic chemo/photothermal therapy. In conclusion, the current work offers a promising approach to the development of smart and efficient multimodal cancer targeted nanotheranostics.

Keywords: carbon nanoparticles; theranostic nanoplatform; chemo/photothermal therapy; dual-modal imaging; pH/NIR responsive drug release.

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1. Introduction

Chemotherapy is among the most commonly used anticancer strategies at present.1, 2 However, the lack of tumor-targeting and cell specificity often leads to low anticancer efficiency of the chemical drugs and serious adverse effect.3 Therefore, to increase the bioavailability of the drugs and decrease the disadvantages of chemotherapy, various nanomaterials, including polymers, inorganic mesoporous materials, metal-organic frameworks, and carbonaceous nanomaterials, are developed as carriers for smart and targeted drug delivery.4-7 Carbonaceous nanomaterials, e. g. carbon nanotubes, carbon nanoparticles (CNPs), fullerene, and graphene oxide (GO), are widely explored as promising candidates for drug delivery systems due to their good biocompatibility, excellent physical and chemical properties, high drug payload and low synthesis cost.8,9 Among various carbonaceous nanomateials, GO has been proved to be an excellent carrier with with ultrahigh drug loading ratio.10,11 Recently, Tu et al. have developed small sized (5-10 nm) CNPs for drug delivery, which possess high loading rate of drugs, outstanding photothermal conversion efficiency, and good biocompatibility.3

Drug release performance is of great importance for safe and high-efficiency therapy. Many nanocarriers suffer from uncontrollable drug release and low accumulated drug release amount, which often leads to poor chemotherapy efficiency.12 Stimuli-responsive drug release is regarded as a feasible strategy against those problems.13 In consideration of the endogenous stimulus of acid microenvironment in tumor, pH-response is widely used to promote controlled drug release.14,15 Functional molecules, such as hydroxypropyl-beta-cyclodextrin (HP-beta-CD), exhibit pH dependent drug release behavior, are often used as a pH responsive switch in drug delivery 4


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systems.16,17 However, use of pH responsive drug release alone is still not enough to realize efficient and controllable drug release, due to the passive drug diffusion governed by complicated tumor microenvironment.18,19

Temperature also has great effect on drug release. At higher temperature, the thermal motion of drug molecules becomes more active, which helps the drugs escape from the carriers. As many studies have demonstrated, significant temperature rise occurs when nanomaterials (e. g. GO, CNPs, black phosphorus) are exposed under NIR laser irradiation.3,20-22 Therefore, the external NIR light-induced thermal stimulus can further promote drug release in a controllable way.23-26 The synergetic pH/NIR responsive drug release leads to enhanced cellular permeability and uptake, and thus improved the chemotherapy efficacy.1 Besides, the hyperthermia (above 42 oC) generated by the nanocarriers under NIR laser can kill cancer cells, contributing to combined PTT and chemotherapy.27-30 For example, to realize pH- and NIR-responsive drug release, Yang and colleagues designed and synthesized CoFe2O4@PDA@ZIF-8 nanocomposites based smart drug delivery system with degradable property and NIR/pH-responsive drug release behavior. Their study showed that the synergistic chemotherapy and PTT exhibited much better anticancer effect than either chemotherapy or PTT alone.27 In another example, Xu et al. have developed HA modified gold nanorods for pH- and NIR-responsive doxorubicin (DOX) release and combined chemotherapy and PTT.28 Since excessive heat may cause severe damage to normal cells and burn the tissue, mild temperature below 48 oC is regarded as the suitable temperature for synergetic chemo/photothermal therapy, as was demonstrated recently.29

To make the most use of anticancer drug and realize safe and effective treatment, in this work, we 5



have developed HP-beta-CD functionalized Fe3O4/CNPs (HFCNPs) as a pH/NIR responsive nanotheranostic platform for MR/NIRFL dual modal imaging, controlled drug delivery and synergetic chemo/photothermal therapy of tumor (Scheme 1). The HFCNPs possess good biocompatibility and strong heat-generating ability. Under NIR irradiation, we expect, promoted drug release will occur at acid environment. Both MR and NIRFL imaging techniques are then employed to investigate the bio-distribution and the accumulation of HFCNPs in tumor region. The cellular and animal experiments have demonstrated the synergetic chemothermal/PTT of HFCNPs, and therefore hold great promise in cancer targeted theranostic nanomedicine. Compared with traditional agents, our strategy holds significant advantages: (1) simple synthesis and low-cost, (2) smart drug delivery and release by taking advantage of tumor microenvironment and noninvasive NIR light, (3) enhanced chemotherapy combining with PTT at mild temperature.

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Scheme 1. Synthesis of HFCNPs and its application for pH/NIR sensitive drug release and MR/NIRFL imaging guided synergistic chemo/photothermal therapy.

2. Experimental Section

2.1 Chemicals and reagents

Doxorubicin hydrochloride, indocyanine green (ICG) and 4, 6-diamino-2-phenylindoe (DAPI) were obtained from Sagon Biotech. WST-1 cell proliferation assay kit was obtained from Beyotime Biotechnology Institute. HP-beta-CD, FeCl2·4H2O, H2SO4 (98 %), HNO3 (95 %), NH3·H2O (25 %), and activated carbon (AC), were purchased from Sinopharm Chemical Reagent Company and used as received.

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2.2 Instrumentation

The instrumentation information and test conditions are the same as our previous work, including TEM, HRTEM, XRD, FTIR, UV-Vis-NIR, DLS, ICP-OES, MRI system, NIRFL imaging system, etc.3,31

2.3 Synthesis of HFCNPs

Firstly, 1 g AC was added in 15 mL 95 % nitric acid and 45 mL 98 % sulfuric acid at 125 oC for 8 min to prepare CNPs.3 The obtained CNPs were purified through dialysis and ultrafiltration. Fe3O4/CNPs nanocomposites were then obtained by coprecipitation of Fe3O4 on the CNPs.31 In brief, 25 mg FeCl2·4H2O and 0.5 mL NH3·H2O were added into 50 mL 0.1 mg/mL CNPs solution at 80 oC for 2 h. The obtained Fe3O4/CNPs were purified by dialysis (10 k Da cutoff) for 2 d. At last, HFCNPs were prepared by self-assembly of HP-beta-CD on Fe3O4/CNPs, similar to the approach described previously for the preparation of beta-CD modified Fe3O4/GO.32 Briefly, 60 mg HP-beta-CD was added into 100 mL Fe3O4/CNPs (0.1 mg/mL) at 65 oC for 3.5 h under mild stirring. After cooling down, thus-obtained HFCNPs were dialyzed (10 k Da cutoff) for 24 h to remove the free HP-beta-CD, and then concentrated by spin dialysis.

2.4 Adsorption and release performance of DOX on HFCNPs 8


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To investigate the adsorption behavior of DOX on HFCNPs, different amount of DOX (50-400 µg) was added into 0.1 mg/mL HFCNPs aqueous solution (with a final volume 1 mL) and put in a shaker at 200 rpm/min at 25 oC for 2 h. After centrifuging at 13,000 rpm for 10 min, the concentration of the DOX remained in the supernatant was calculated according to the maximum absorption of DOX in the UV-vis spectra. The loading capacity of DOX on HFCNPs was evaluated according to the following equations:

LE =

We ×100 W0

(1)

LC =

We ×100 WH

(2)

Where LE is the drug loading efficiency of DOX (%), We is the weight of DOX loaded on HFCNPs (mg), W0 is the initial weight of DOX (mg), LC is the loading capacity of HFCNPs (%), WH is the weight of HFCNPs (mg).

For convenient calculation and application, by adjusting the initial amount of DOX, we used DOX/HFCNPs with suitable components (DOX:HFCNPs, w/w=1:2) for the following experiments.

The DOX release from HFCNPs was investigated to evaluate the drug release performance of HFCNPs. Dialysis bags containing 1 mL DOX/HFCNPs (DOX, 1 mg/mL) were added in 30 mL PBS solution (pH 5.5 or 7.4) and treated with 200 rpm stirring in a shaker in the dark, respectively. During the following 48 h, 2 mL out of the 30 mL dialysate was collected at various time points, and 2 mL fresh PBS solution was replenished to maintain the total volume of the dialysate. 9



2.5 Cellular experiments

Cell Culture

4T1 cells were obtained from Shanghai Institutes for Biological Science, Chinese Academy of Sciences, and cultured under standard condition (37 oC, 5 % CO2).

Cell Viability Assay

The cytotoxicity of the nanomaterials was tested by WST-1 assay. In brief, 96-well plates were used to seed 4T1 cells (8, 000 cells/well). After 24 h, the old culture media were replaced by fresh ones containing DOX, or DOX/HFCNPs ([DOX]=0.3, 0.6, 1.2, 2.5, 5 and 10 µg/mL). After incubating for another 24 h, the old culture media were discarded and the cells were washed twice by PBS. Finally WST-1 method was used to assess the cellular viability.

Cellular Uptake

35 mm confocal dishes were used to seed 4 T1 cells (5×104 cells/well). The cells were treated with free DOX or DOX/HFCNPs (10 µg/mL of DOX) for 1, 6, 24 h under standard culture condition. Then, the cells were washed twice by PBS, and DAPI was added to label cell nucleus. Finally, a Nikon A1 confocal laser scanning microscope was used to record the cell images.

To semi-quantitatively analyze the cellular uptake of HFCNPs, 4T1 cells were incubated with HFCNPs for 6, 12, and 24 h. Then the cells were collected and ICP-OES was employed to determine Fe concentration inside cells at each time point. 10


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In vitro chemotherapy efficiency of DOX loaded nanoparticles

4T1 cells were incubated with DOX/HFCNPs or free DOX ( 0.3, 0.6, 1.2, 2.5, 5, and 10 µg/mL) for 24 h. After washing by PBS, the cell viability was estimated via WST-1 assay.

In vitro photothermal and combination therapy efficiency

4T1 cells were incubated with DOX/HFCNPs or free DOX ([DOX]=0.3, 0.6, 1.2, 2.5, 5, and 10 µg/mL) and treated by NIR irradiation (5 min, 808 nm, 1 W/cm2).. Then cell viability was tested by WST-1 to evaluate the therapy efficacy.

2.6 Animals and tumor model

Healthy BaLb/c mice (female, 6 weeks age) and nude mice (female, 6 weeks age) were purchased from Suzhou Industrial Park Animal Technology Co., Ltd. The authors guarantee that all animals were carefully fed and treated following the Rule of Laboratory Animals.

The tumor model was established by subcutaneous injecting 2×106 4T1 cells into the right hind leg of mice subcutaneously. The tumor volume was defined by: ௟×௪ మ

V=

(3)

ଶ

Where V represents tumor volume (mm3), and l and w refer to tumor length and

2.7 In vivo imaging 11


width (mm).


MR imaging

A 0.5 T MRI scanner was used to measure the T2 relaxation time of HFCNPs. The measurement parameters were set as our previous report.31

To analyze the MRI effect in vivo, tumor-bearing mice were injected with DOX/HFCNPs (0.1 mL, [Fe]=1 mg/mL) via tail vein. MR images were then recorded via 1.5 T MRI scanner at 0, 4, 8, 24 h post injection (p. i.). The measurement parameters were set the same as in our previous work.31

NIRFL imaging

To avoid the distraction of self-fluorescence of mice, indocyanine green (ICG), an NIR fluorescent dye, was used to label HFCNPs. In a typically process, 1 mL 1 mg/mL ICG was dropped in 10 mL 0.2 mg/mL HFCNPs and incubated for 6 h, and then the obtained dark green solution was washed by D. I. water for 3 times to remove free ICG. Tumor-bearing nude mice were injected with ICG/ HFCNPs (2.5 mg/kg of ICG) via tail vein. The animal imaging system (ex: 704 nm, filter: 735 nm) was used to collect in vivo fluorescence images at 0, 1, 2, 4, 8, 12, 24, 48 h p.i., respectively. The NIRFL images of main organs were also collected at 1, 8, 24, and 48 h p.i., respectively.

Thermal imaging

Tumor-bearing mice were injected with DOX/HFCNPs (10 mg/kg, [HFCNPs]) via tail vein. The tumor site of the mouse was put under an NIR light (5 min, 808 nm, 1 W/cm2,) at 8 h p. i., and the thermal images were recorded using an infrared thermal imaging camera.

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2.8 In vivo combination therapy

25 mice were equally assigned to five groups: (1) PBS; (2) free DOX (5 mg/kg); (3) DOX/HFCNPs (DOX: 5 mg/kg, HFCNPs: 10 mg/kg); (4) HFCNPs (10 mg/kg) + 808 nm laser; (5) DOX/HFCNPs (DOX: 5 mg/kg, HFCNPs: 10 mg/kg) + 808 nm laser. Then, the mice were injected with 100 µL aqueous solution of different samples by tail vein. For photothermal therapy groups, the mice were exposed under NIR laser (1 W/cm2, 808 nm, 5 min) at 8 h p.i. Tumor sizes were measured by digital caliper and the weight of tumors were monitored during the therapeutic period. After all experiment, the mice were treated by mercy killing.

3 Results and Discussion

3.1 Synthesis and characterization of HFCNPs

CNPs were synthesized by rapid oxidation of AC in fuming HNO3 and concentrated H2SO4 mixture.3,31 To obtain uniform-sized CNPs, the preliminary CNPs were separated by ultrafiltration (10 and 100 kDa), and the filtrate between 10 to 100 kDa was collected for further use (Figure S1). Then, Fe3O4/CNPs were obtained by co-precipitation of Fe3O4 onto the CNPs, according to the same method of synthesis of Fe3O4/GQD.32 HP-beta-CD was then modified on Fe3O4/CNPs via layer by layer self-assembly subsequently, similar to the method described previously.33 The introduction of HP-beta-CD did not only significantly improve the colloidal stability and biocompatibility of Fe3O4/CNPs, but rendered them pH-responsive drug release feature.16,17,34

The composition of HFCNPs was examined by EDS measurement. As shown in Figure S2, 13



HFCNPs were composed of C, O, and Fe. The morphology and microstructure of HFCNPs were characterized by TEM and HRTEM. As shown in Figure 1a, HFCNPs exhibit monodispersed spherical particles with size of 8-10 nm. The HRTEM image (Figure 1b) indicates that the lattice spacing of 0.32 nm and 0.23 nm observed for HFCNPs are (002) plane and (100) plane for graphite carbon from graphitized surface of CNPs,35,36 and 0.29 nm and 0.25 nm are (220) plane and (311) plane for ferroferric oxide crystal.37 The simultaneous existence of characteristic lattice planes of CNPs and Fe3O4 crystal solidly verifies the composite structure of HFCNPs formed via growth Fe3O4 nanocrystals on CNPs. XRD spectra further evidence the crystallographic information of HFCNPs (Figure 1c). The 2θ value at 30.3o, 35.6o, 43.1o, 57.2o and 62.7o corresponds to the (220), (311), (400), (511) and (440) planes of Fe3O4 nanocrystalline (JCPDS No.19-0629), respectively.38 The chemical structure of HFCNPs was characterized by FTIR spectra (Figure 1d). The peak at 3420 cm-1 is attributed to the stretching mode of –OH, the peak at 1730 cm-1 is assignable to the stretching mode of C=O, while the peak at 1604 cm-1 is due to the stretching of C-O, and the peak at 1405 cm-1 is ascribed to the stretching vibrations of aromatic group. The characteristic peak at 1162 cm-1 is attributable to typical C-O-C stretching/O-H bending vibrations, and the peaks at 1076 cm-1 and 1033 cm-1 are attributed to the coupled O-H bending/C-O/C-C stretching vibration of HP-beta-CD, while the characteristic peak at 580 cm-1 is assignable to Fe-O stretching vibration of Fe3O4 nanocrystal.3,33 As shown in Figure 1e, the hydrate particle sizes of CNPs, Fe3O4/CNPs, and HFCNPs are 8.4 nm, 18.1 nm, and 31.0 nm (Figure 1e), respectively. The larger hydrodynamic diameter of HFCNPs than that observed from TEM image is mainly due to the hydration effect, according to the previous report.36 The zeta

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potential of CNPs, Fe3O4/CNPs, and HFCNPs at various pH values are shown in Figure 1f, respectively. HFCNPs possess higher electronegativity than CNPs and Fe3O4/CNPs at pH 3-11. The storage stability of HFCNPs was further studied by monitoring the change of hydrate particle size and zeta potential during 4 weeks (Figure S3 and S4). The slight fluctuation of the size and zeta potential values indicates that HFCNPs are quite stable in typical storage condition (room temperature and bright ambient light condition). In addition, HFCNPs were found stable in various solutions, including D. I. water, PBS, serum, and culture media (Figure S5). To further confirm the stability of HFCNPs in physiological condition, HFCNPs were incubated in PBS, serum, and culture media for 24 h, and then treated by centrifugation (12,000 rpm 10min) and redistributing in D. I. water before preparing of TEM samples. No obvious aggregation was observed for all samples. (Figure S6).

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Figure 1. (a) TEM image, (b) HRTEM image and (c) XRD spectrum of HFCNPs. (d) FTIR spectra of HP-beta-CD, Fe3O4/CNPs, and HFCNPs. (e) Hydrate particle size of CNPs, Fe3O4/CNPs, and HFCNPs in D. I. water. (f) Zeta potential of CNPs, Fe3O4/CNPs, and HFCNPs in aqueous solution at pH 3, 5, 7, 9, 11.

3.2 Anticancer drug loading and release performance

HFCNPs possess high adsorption capacity, being a promising vehicle for drug delivery. Figure 2a showed that the typical peak of DOX at 490 nm appeared in DOX/HFCNPs, suggesting the successful loading of DOX onto the composite nanoparticles. The saturation level of DOX loaded onto HFCNPs was evaluated by adding different amount of DOX in HFCNPs solution. From Figure 2b it is inferred that the amount of DOX loaded on the HFCNPs increased from 11.84 % to 61.21 % (w/w, DOX/HFCNPs). .

3.3 In vitro photothermal effect and pH/NIR-responsive drug release performance

As demonstrated in our previous work, CNPs exhibit strong photothermal conversion efficiency, which is essential for PTT of tumor.39 Besides, Fe3O4 NPs also possess certain photothermal 16


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conversion effect.40 It is therefore expected that the formation of HFCNPs could enhance, to a large extent, the photothermal effect for cancer ablating. To verify the photothermal effect of HFCNPs, NIR light (808 nm, 5 min, 1 W/cm2) was used to irradiate HFCNPs (Figure 2c). An electronic infrared thermometer was employed to record the temperature every 30 s. As shown in Figure 2c, the temperature of HFCNPs solution increased dramatically under the NIR irradiation, with a temperature difference (∆T) of 32.4 oC (0.34 mg/mL HFCNPs). On the contrary, the temperature rising of PBS (∆T=4.1 oC) is much lower than HFCNPs under the same condition (Figure 2c). The temperature change curves in Figure 2d clearly reveal that the photothermal conversion effect of HFCNPs (black colored curve) is significantly better than those of the Fe3O4 NPs (blue colored curve) and CNPs (green colored curve). The excellent photothermal effect of HFCNPs is mainly inherited from CNPs, which can transform light energy to thermal energy.3 The Fe3O4 NPs grew on CNPs also contribute, to some extent, to the photothermal performance of HFCNPs. The photothermal conversion efficiency of HFCNPs was calculated to be 27.5% (Figure S7). To further confirm HFCNPs as a good and stable photothermal agent, the photothermal performance of HFCNPs was studied via detecting the cyclic heating effect. As shown in Figure 2e, the photothermal capacity of HFCNPs kept quite stable, with no obvious ∆T change during the 4 heating-cooling cycles, which is beneficial to the treatment of tumor by PTT.

To further evaluate the drug release performance of HFCNPs, DOX loaded HFCNPs were put in the dialysis system with PBS (pH 7.4) or PBS (pH 5.5), respectively. DOX release amount was calculated according to UV-vis spectra of the dialysate at different time points. Figure 2f showed that, 33.1% DOX was released from HFCNPs at pH 7.4 during 24 h, which was 47.5% at pH 5.5. 17



The higher cumulative DOX release amount at pH 5.5 is mainly caused by DOX protonation in acidic condition, which accelerates the escape of DOX from HFCNPs.41 To further understand the influence of NIR irradiation on the DOX release performance, DOX/HFCNPs were irradiated by 808 nm light (1 W/cm2, 5 min) at each time point. The significantly improved cumulative DOX release at pH 7.4 (43.5 %) and pH 5.5 (68.3 %) was obtained, as shown in Figure 2f. The drastic release of DOX from HFCNPs is mainly due to the rapid heating and local hyperthermia of HFCNPs under NIR irradiation, which facilitate the molecular thermal motion and the escape of DOX molecules from the surface of HFCNPs. The much higher release rate at pH 5.5 than that at pH 7.4 indicates that DOX is easier to fall off from HFCNPs at acid environment. The above results clearly indicate that DOX/HFCNPs exhibit both pH- and NIR-responsive drug release behavior, while NIR laser irradiation-induced photothermal effect plays an important role in further promoting the release of drug.

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Figure 2. (a) UV-Vis-NIR spectra of DOX, HFCNPs, and DOX/HFCNPs in D. I. water. (b) Drug loading capacity of HFCNPs at different initial DOX concentrations (0.05-0.50 mg/mL). (c) Photothermal effect of HFCNPs in D. I. water at different concentrations (0, 0.02, 0.04, 0.08, 0.17, 0.34 mg/mL.). (d) Photothermal effect of CNPs, Fe3O4 NPs, Fe3O4/CNPs, and HFCNPs in D. I. water (0.17 mg/mL, 808 nm, 1 W/cm2, 5 min). (e) Photothermal stability of HFCNPs under 1 W/cm2 808 nm NIR laser. (f) DOX release performance at pH 5.5, pH 7.4, pH 7.4 + NIR laser, and pH 5.5 + NIR laser.

3.4 Cellular uptake

The effective accumulation of therapeutic agents in desired regions is vital to realize precise and 19



efficient tumor treatment. Since its strong intrinsic red fluorescence, DOX was used directly as a fluorescent label to monitor the cellular uptake behavior of HFCNPs in 4T1 cells. The in vitro cellular uptake of DOX/HFCNPs at different time points was evaluated by a confocal fluorescence microscopy. The two types of fluorescence signals, blue color from DAPI (for cell nucleus staining) and red color from DOX (for labeling of HFCNPs), were collected. The fluorescence intensity of DOX at 1 h post incubation was weak, and increased gradually with the incubating time (Figure 3a). This is mainly due to the slower uptake of the nanoparticles than small molecule, and the sustained release of DOX on HFCNPs.42 To further understand the cellular uptake of HFCNPs by 4T1 cells, the concentration of Fe was tested by ICP-OES at various time points (Figure 3b). About 4.8 µg/106 cells (intracellular Fe concentration) were uptaken by 4T1 cells during 24 h incubation. The increased [Fe] with time further revealed the efficient uptake of HFCNPs by 4T1 cells.

3.5 In vitro combined therapy

To assess the biosafety of HFCNPs, in vitro toxicity experiment was carried out. PBS, CNPs, HP-beta-CD, Fe3O4/CNPs, and HFCNPs were incubated with 4T1 cells for 24 h, respectively. Figure 3c showed that the relative cell viability for each group was higher than 90 %. The result indicates that HFCNPs exhibit no obvious cytotoxicity.

Encouraged by the excellent drug loading property and pH/NIR stimuli controlled drug release performance and high photothermal efficiency of HFCNPs, the in vitro experiment on combined 20


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chemo/photothermal therapy was further conducted to assess the anticancer performance of DOX loaded HFCNPs (Figure 3d). For single chemotherapy of 4T1 cells, DOX/HFCNPs exhibits weaker toxicity than free DOX at various concentrations. This phenomenon is mainly caused by the adsorption of DOX on HFCNPs and sustained release behavior of DOX, which may lead to the delayed and abated therapeutic efficacy.42 For single photothermal therapy, HFCNPs+NIR exhibits enhanced damage to 4T1 cells with the concentration increase, mainly due to the good photothermal property of HFCNPs. For in vitro chemo/photothermal therapy, under the irradiation of NIR laser, DOX/HFCNPs exhibit negligible damage to 4T1 cells at low concentration (lower than 2.5 µg/mL), and significantly enhanced damage at high concentration (higher than 5 µg/mL). The poor inhibition of 4T1 cells at low concentration is mainly due to the ignorable photothermal effect of HFCNPs. On the contrary, the remarkable inhibition effect at high concentration is benefited from the obvious heating caused burst release of DOX and synergetic PTT effect.42 The above results clearly demonstrated that the combined chemo/photothermal therapy is of good feasibility, taking full advantages of the inherent tumor microenvironment and non-invasive NIR induced stimuli responsive drug release and enhanced therapeutics.

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Figure 3. (a) Intracellular uptake of DOX/HFCNPs observed by confocal microscope, with DOX concentration of 10 µg/mL (scale bar=100 µm). (b) The concentration of Fe in 4T1 cells with 6, 12, 24 h incubation. (c) In vitro cytotoxicity of CNPs, Fe3O4 /CNPs, HP-beta-CD, and HFCNPs at various concentration (3, 6, 12, 25, 50, 100 µg/mL). (d) In vitro cytotoxicity of DOX, DOX/HFCNPs, HFCNPs + NIR laser, and DOX/HFCNPs + NIR laser at various concentration (0.3, 0.6, 1.2, 2.5, 5.0, 10.0, and 20.0 µg/mL).

3.6 In vivo imaging

NIRFL imaging

The in vivo accumulation and biodistribution of HFCNPs is of significant importance to the treatment of tumor because such data obtained at various time points could guide the precise and effective cancer therapy. Considering the distraction of self-fluorescence of mice, NIR fluorescent 22


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dye indocyanine green (ICG) was used as labeling molecule for NIRFL imaging. The fluorescence signals were recorded at 0, 1, 2, 4, 8, 12, 24 and 48 h p.i., respectively. At the first 1 h, NIRFL signal was clear and strong over the whole mice body (Figure 4a and 4b), in which process ICG/HFCNPs were spread through the blood circulation. During the initial 8 h p.i., the tumor site fluorescence intensity increased gradually, verifying the successful accumulation of HFCNPs. The fluorescence intensity in other regions decreased rapidly with time, due to rapid elimination of ICG in vivo through metabolism. Meanwhile, the fluorescence intensity at tumor site decreased much slower than that in other regions, mainly due to the widely recognized enhanced permeability and retention (EPR) effect.43

,At 1, 8, 24 and 48 h p.i., we collected the NIRFL images of major organs and tumor to further understand the biodistribution of HFCNPs. For tumor, at 8 h, the maximum fluorescence intensity was observed, and then decreased gradually (Figure 4b). The accumulation of HFCNPs in tumor was also obvious at 24 h, further confirming the long retention time of HFCNPs in tumor. On the contrary, the fluorescence intensity in normal organs was quite weak, compared with that in tumor. As Figure 4c and 4d showed, the NIRFL intensity in tumor is 10.73-fold to heart, 6.13-fold to liver, 5.29-fold to spleen, 4.90-fold to lung, and 3.50-fold to kidney, respectively, at 24 h p.i. The low residue of the nanomaterials in major organs indicated their rapid removal from the body.

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Figure 4. (a) NIRFL images of tumor-bearing mice injected with ICG/HFCNPs recorded at 0, 1, 2, 4, 8, 12, 24, 48 h p.i., respectively. Tumors were marked by blue circle. (b) Average fluorescence intensity in tumor site at 0, 1, 2, 4, 8, 12, 24, and 48 h p.i. (c) NIRFL images and (d) average NIRFL intensity of heart, liver, spleen, lung, kidneyand tumors (from left to right).

MR imaging

Magnetic characterization of HFCNPs was performed by PPMS (Figure 5a). HFCNPs possesses good superparamagnetic property without obviouscoercivity or remanence in M-H loops.The saturation magnetization (Ms) value was calculated to be 18.09 emug/g. Considering of the poor penetration depth and low resolution rate of fluorescence imaging, magnetic resonance imaging could be employed to make up the flaw of optical imaging and get high spatiotemporal resolution image in vivo. The magnetic property was studied before use of HFCNPs as a MRI contrast agent 24


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in cancer imaging. The transverse proton relaxation time (T2) of HFCNPs was measured on a 0.5 T MRI machine, and the r2 value (transverse relaxivity) was obtained according to the linear fitting of 1/T2 vs Fe concentration (Figure 5b). The r2 value of HFCNPs is calculated as 115.82 mM-1s-1, similar to that of Fe3O4/GQD, and much higher than that of the Fe3O4 nanoparticles of the similar size.32

To further explore the feasibility of HFCNPs as an excellent MRI contrast agent in cancer diagnosis, in vivo MRI performance was studied by 1.5 T in vivo MRI machine. Figure 5c shows that at 8 h p.i., the enhancement of MRI contrast in tumor was most significant, and decreased subsequently afterwards. The intensity of MR signal decreased 41.3 % at 8 h p.i., compared with that at 0 h p. i. (Figure 5d). Combining with NIRFL imaging, MRI provides higher spatiotemporal resolution of the tumor site.

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Figure 5. (a) Magnetization curve of HFCNPs at 300 K. (b) Plot of 1/T2 vs CFe. (c) In vivo MR images of 4T1 tumor-bearing mouse after tail intravenous injection of HFCNPs. (d) Relative signal intensity of the tumor in (c).

3.7 In vivo combined chemo/photothermal therapy of tumor

Inspired by the excellent accumulation of HFCNPs in tumor site, the in vivo temperature elevation of tumor was then studied to verify HFCNPs as a promising PTT agent. A thermal imaging camera was used to record the temperature of the tumor. After injecting HFCNPs, the tumors of the mice were irradiated by NIR light at 8 h p.i. The temperature in tumor site could be controlled by changing laser power, irradiation time, as well as injected HFCNPs concentration. To realize mild PTT effect and reduce the pain that mice may suffer from excessively high temperature, the local temperature of tumor is advised to be below 48 oC.29 In our strategy, the tumor temperature reached ~45 oC after 5 min irradiation (Figure 6a and 6b).The therapeutic experiments were performed to assess the combined chemo/photothermal therapy effect of DOX loaded HFCNPs. In a typical protocol, the tumor-bearing mice were randomly divided into 5 groups (n=5): (1) PBS group, (2) DOX group, (3) HFCNPs+IR group, (4) DOX/HFCNPs group, and (5) DOX/HFCNPs+IR group. For (3) and (5) group, the tumor sites of the mice were irradiated by 808 nm laser at 8 h p.i. The temperature in tumor sites was controlled in the range of 42-45 oC and monitored by thermal imaging camera.

The mice weight and tumor volume were measured carefully in the treatment period. Figure 6c 26


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shows slight change of the weight of mice, indicating that each of the treatment imposes no significant influence on the growth of mice. The tumor volume of groups (1), (2), (3), and (4) increased 11.36-fold, 10.65-fold, 8.03-fold, and 6.01-fold, respectively, during the 18 d treatment period, while that of group (5) is only 1.77-fold, as shown in Figure 6d. The better tumor inhibition of group (4) than that of group (2) is caused by the efficient accumulation of HFCNPs in tumor site by EPR effect, on which DOX could be delivered and underwent sustained release to the tumor site. For group (3), the growth speed of tumor is slower than group (1). This is possibly due to the weak lethality of mild heat, which is slightly higher than the stagnation temperature (42 o

C) for solid tumor. The significant inhibition effect against cancer of group (5) indicates that the

combined chemo/photothermal therapy is effective. The mild heating not only accelerate the release of DOX in tumor site, but also exhibited a synergistically enhanced PTT effect to tumor.

After various treatments, all of the mice were executed at 18 d post therapy, and the main organs and solid tumors were dissected and stained by hematoxylin and eosin (H & E) staining for histological analysis (Figures 6e and S5). The obviously decreased cellularity and nuclear shrinkage and fragmentation of tumor tissue for DOX/HFCNPs+NIR group were clearly observed, indicating the severe damage of tumor by DOX/HFCNPs under NIR irradiation. Conversely, the damage of tumor tissue in other groups was not remarkable, compared with the DOX/HFCNPs+NIR group. The above data further confirm the successful combined chemo/photothermal therapy in histological level. Meanwhile, in the main organs of all groups, no significant pathological changes were observed, indicating that HFCNPs showed no remarkable side effects to normal tissues and organs (Figure S8). 27



The weight of tumor and normal tissues were weighed after dissection at 18 d post therapy (Figure 6f). In group (1)~(4), the spleen weight increased obviously, while the change in group (5) is negligible. The similar phenomenon was also observed previously by others.3 Spleen, the important immune organ, would appear abnormal when the animalbecame unhealthy.3 The normalization of the spleen of the mice in the combined therapy group further demonstrated the feasibility of our strategy.

Figure 6. (a) In vivo Thermal images of PBS or DOX/HFCNPs group at 1, 2, 3, 4, and 5 min, respectively. (b) In vivo temperature rising curve. (c) The curve of body weight in the treatment

28


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period. (d) Tumor volume in the treatment period. (p

CNPs-Based Theranostic

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