Multifunctional Nanosystem Based on Graphene Oxide for Synergistic

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Multifunctional nanosystem based on graphene oxide for synergesitic multistage tumor-targeting and combined chemo-photothermal therapy Huabing Zhang, Yang Li, Zhou Pan, Yilin Chen, Zhongxiong Fan, Haina Tian, Song Zhou, Yubin Zhang, Jiajia Shang, Beili Jiang, Fanfan Wang, Fanghong Luo, and Zhenqing Hou Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b01335 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Multifunctional nanosystem based on graphene oxide for synergesitic multistage tumor-targeting and combined chemo-photothermal therapy Huabing Zhang,1 5

‡

Yang Li,2,6

‡

Zhou Pan,1Yilin Chen,4Zhongxiong Fan,1Haina

Tian,1Song Zhou,5Yubin Zhang,1 Jiajia Shang,1Beili Jiang,1Fanfan Wang,1 Fanghong Luo,3* Zhenqing Hou,1* 1Department

of Biomaterials, College of Materials, Research Center of Biomedical

Engineering of Xiamen & Key Laboratory of Biomedical Engineering of Fujian Province, Xiamen University, Xiamen 361005, China 10

2CAS

Key Laboratory of Design and Assembly of Functional Nanostructures, and

Fujian Provincial Key Laboratory of Nanomaterials, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, China 3

Cancer Research Center, School of Medicine, Xiamen University, Xiamen 361005,

China 15

4School

of Pharmaceutical Science, Fujian Provincial Key Laboratory of Innovative

Drug Target Research, Xiamen University, Xiamen 361005, China 5Department

of General Surgery, The Affiliated Southeast Hospital of Xiamen

University, Zhangzhou 363000, China 6Department

20

of Translational Medicine, Xiamen Institute of Rare Earth Materials,

Chinese Academy of Sciences, Xiamen 361024, P. R. China. Author Contributions: ‡HuabingZhang and Yang Li contributed equally to this work.

ABSTRACT:


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25

Locating nanomedicines at the active sites plays a pivotal role in nanoparticle-based cancer therapy field. Herein, a multifuntionalnanotherapeutic is designed by using graphene oxide (GO) nanosheets with rich carboxyl groups as the supporter for hyaluronic

acid

(HA)-methotrexate

(MTX)

prodrug

modification

via

adipicdihydrazide (ADH)crosslinker, achieving synergisticmultistage tumor-targeting 30

and combined chemo-photothermal therapy. As a tumor-targeting biomaterials, HA can increase affinity of the nanocarrier towards CD44 receptor for enhanced cellular uptake. MTX, a chemotherapeutic agent, can also serve as a tumor-targeting enhancer towards folate receptor based on its similar structure with folic acid. The prepared nanosystems possess a sheet shape with a dynamic size of approximately 200 nm and

35

pH-responsive drug release. Unexpectedly, the physiological stability of HA-MTX prodrug-decorated GO nanosystems in PBS, serum, and even plasma is more excellent than that of HA-decorated GO nanosystems, while both of them exhibit enhanced photothermal effect than GO nanosheets. More importantly, because of good blood compatibility as well as reduced undesired interactions with blood

40

components, HA-MTX prodrug-decorated GO nanosystems exhibited remarkably superior accumulation at the tumor sites by passive and active targeting mechanisms, achieving highly effective synergistic chemo-photothermal therapeutic effect upon near-infrared laser irradiation, efficient ablation of tumors, and negligible systemic toxicity. Hence, the HA-MTX prodrug-decorated hybridnanosystems have a

45

promising potential for synergistic multistage tumor-targeting therapy.

Keywords: graphene oxide, hyaluronic acid, methotrexate, chemo-photothermal therapy, targeting ability

50

1. Introduction Conventional

chemotherapy

for

cancer

suffers

from

weakness

that

chemotherapeutic agents lack specific biodistribution for tumor cells, leading to high toxicity against normal cells and poor therapeutic effect.1 To overcome the problem, many researches have focused on developing targeting delivery systems to transport



55

anticancer drugs specifically to tumor tissues/cells based on active or passive targeting strategy.2-3 Nanoparticles and liposomes are able to reach tumor sites by enhanced permeation and retention (EPR) effects to achieve passive targeting2. As for active targeting, over-expression of many receptors exhibit on the surface membranes of tumor or angiogenic endothelial cells, and thus nanosystems grafted with targeting

60

moieties can specifically recognize and bind to these receptors, realizing selective cell internalization.4-6 However, still light or even equivalent cancer-relevant receptors express on certain healthy cells, leading to unwanted uptake. 7It has been reported that dual-/multi-ligands-based targeting chemotherapy can possibly enhance tumor uptake through increasing the differentiation between normal and tumor cells.8-9 Hence it is

65

possible to design highly efficient and simple targeted drug delivery systems according to passive and dual-/multi-active targeting strategies. Besides, current studies have indicated that anticancer efficacy could be enhanced when chemotherapy was combined with hyperthermia, which would enhance cytotoxicity of some anti-tumor drugs.10 Previous studies have proposed

70

photothermal therapy (PTT) to achieve local hyperthermia using near-infrared (NIR)-absorbing photothermal agents by conversion of absorbed NIR light energy into thermal energy11. Recently, many studies have focused on studying nanosystems including chemotherapeutic drugs and photothermal agents to achieve the combination of targeted chemotherapy and PTT.12-14

75

Graphene oxide (GO), a kind of two-dimensional nanomaterial, has attracted much attention for drug delivery because of its large surface area with both sides. 15-17Besides,

GO possesses a large number of reactive functional groups including

epoxy, hydroxyl, and carboxylic acid groups, providing for modification in order to improve the stability, solubility, and biocompatibility.18-21 Moreover, GO is able to be 80

used for photothermal therapy since it shows high NIR absorbance especially after being reduced by carboxylation.12,

22-23

Therefore, GO has the potential to

simultaneously serve as drug carrier and photothermal agent if it can be reasonably designed. However, high toxicity and low stability limit its application as biomaterial, so poly(ethylene glycol) (PEG) and polysaccharides have been studied to overcome


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85

the weakness.22, 24 Hyaluronic acid (HA), a naturally existing polysaccharides, possesses advantages such as favorable biocompatibility, biodegradability, and non-immunogenicity, showing great success in many biomedical applications, like drug delivery and tissue engineering.25 Besides, it has been demonstrated that HA could enhance stability,

90

water-solubility,

and

biocompatibility

for

carbon-based

two-dimensional

nanomaterials such as fullerene, carbon nanotube, and GO.15, 18 More importantly, it can be specifically internalized by kinds of tumor cells with high-level expression of CD44 receptors.25-27 Hence, grafting HA onto GO can offer not only physiological stability in long-term blood circulation but also active targeting capability into tumor 95

cells. Methotrexate (MTX), a kind of antimetabolite of folic acid and therapeutic agent for immune system suppression, is able to inhibit tumor cell growth and proliferation through the inhibition of dihydrofolatereductase (DHFR).28-29 Moreover, owing to the similar structure to folic acid (FA), MTX is able to achieve effective cell

100

internalization through specific interactions with FA receptors over-expressed by various tumor cells.30-32 Nevertheless, because of its distribution and metabolism throughout the body after chemotherapy, many patients treated with MTX have suffered from severe side effects including hair loss, headache, and nausea, restricting its long-term use and tolerance to a great deal.33-34 Besides, clinical research has

105

approved that MTX possibly could lead to adverse effects like liver fibrosis and pneumonia.35-36 To address these problems, some studies have been focused on designing nanocarriers to deliver MTX specifically targeted to tumor issues. 35-36Consequently,

we expect that nanosystem combined HA with MTX could be able

to integrate dual active targeting effect and anticancer ability. 110

In the present study, GO-ADH-HA-MTX nanosystem was designed to achieve dual-ligands-based targeting chemotherapy combined with PTT, as well as obtain a simple but efficient platform to reduce undesired toxicity. As illustrated in Scheme 1, HA-MTX prodrug was firstly synthesized via esterification reaction to realize pH-sensitive MTX release, and then HA-MTX prodrug was grafted to



115

carboxyl-functionalized

GO

(GO-COOH)

through

Page 6 of 38

adipicdihydrazide

(ADH)

crosslinker via amidation reaction to construct a robust GO-ADH-HA-MTX nanosystem. Herein, GO could be served as not only drug delivery carrier but PTT agent,12 while HA was designed to improve stability and biocompatibility of GO and simultaneously played the part of targeting moiety for CD44 receptors. 120

15, 37Different

from other chemotherapy agent, MTX could also act as targeting moiety for FA receptors. In addition, MTX was introduced onto the surface of nanocarriers through relatively stable chemical linkage, which could decrease drug loss and burst drug release. More importantly, ester bond between HA and MTX was able to keep relatively stable at physiological pH, but could be cleaved to achieve MTX release in

125

acidic endo/lysosomes inside tumor cells (pH 4.5-5.5).38 As shown in Scheme 1B, GO-ADH-HA-MTX nanosystem had the potential to keep relatively stable during blood circulation after intravenous injection, and then accumulated in tumor tissues and within tumor cells via EPR effect-based passive targeting mechanism and CD44/FA

130

receptors-mediated

dual

active

targeting

mechanism.

After

cell

internalization, the nanosystems could release MTX via acid-induced cleavage of ester linkage while inducing local heat triggered by NIR irradiation, eventually leading to highly efficient cell death via the combination of chemotherapy and PTT therapy.

135

2. Materials and methods 2.1. Materials. All chemical reagents were of analytical grade and used without further purification unless otherwise stated. Deionized (DI) water was used throughout. Sodium hyaluronic acid (molecular weights = 8000 Da) was purchased from Shandong Freda Biopharm Co. Ltd. (China). Methotrexate (MTX, purify ≥

140

98.0%) and folic acid (FA, purify ≥ 96.0%) were purchased from Bio Basic Inc. (Markham,

Ontario,

Canada).

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

Adipicdihydrazide (EDC),

(ADH),

N-hydroxysuccinimide

(NHS), and 4-dimethylaminopyridine (DMAP) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Graphite powder and 4-Morpholineethanesulfonic


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145


acid

hydrate

(MES)

were

purchased

from

Macklin

(Shanghai,

China).

1’-dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodide (DiR) and 4’,6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes Inc. (Eugene, OR, USA). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Dulbecco’s modified 150

Eagle’s medium (DMEM) were purchased from M&C Gene Technology (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (AG, Switzerland). 2.2. Synthesis of HA-MTX conjugate.MTX (270 mg) was firstly dissolved in 11 mL DMSO, then EDC (300 mg) was added to the solution, stirring for 30 min. HA (200

155

mg) was dissolved in the mixture solvent of 2 mL distilled (DI) water and 8 mL DMF, and then the HA dispersion was added to MTX dispersion. DMAP (150 mg) was subsequently added to the dispersion, which was then stirred for 24 h in dark at room temperature. After reaction, the dispersion was dialyzed (8000~14000 Da MWCO) against DI water for 48 h, and then lyophilized at -80oC to obtain HA-MTX.

160

2.3. Synthesis of ADH-HA-MTX conjugate. HA-MTX (220 mg) was dissolved in the mixture solvent of 3 mL DI water and 5 mL DMSO, and then EDC (250mg) was added to the dispersion. After stirring for 30 min, ADH (50 mg) and NHS (100 mg) was added to the dispersion. Subsequently, the reaction was stirred at 60°C under dark for another 24 h. After reaction, the dispersion was dialyzed (8000~14000 Da

165

MWCO) against DI water for 48 h followed by dialysis against DI water for 48 h, and then lyophilized at -80oC to obtain ADH-HA-MTX. 2.4. Synthesis of ADH-HA conjugate. HA (100 mg) was dissolved in 3 mL DI water, EDC (250 mg) was dissolved in 11 mL DMSO, and then the two dispersions were mixed together. After stirring for 30 min, ADH (50 mg)and NHS (100 mg) was

170

added to the dispersion. Subsequently, the reaction was stirred at 60°C under dark for another 24 h. After reaction, the dispersion was dialyzed (8000~14000 Da MWCO) against DI water for 48 h followed by dialysis against DI water for 48 h, and then lyophilized at -80oC to obtain ADH-HA. 2.5. Synthesis of GO-COOH nanosheets. GO was prepared from graphite powder



175

Page 8 of 38

by the modified Hummer’s method24. For carboxylation, GO aqueous solution (20 mL, 2 mg/mL) was ultrasonicated for 30 min at 600 W with 10 s intervals in an ice bath using a probetypeultrasonicator to obtain a clear suspension. NaOH (800 mg, 0.02 mol) and ClCH2COONa (2.3 g, 0.02 mol) were added to the suspension and bath sonicated for 3 h to convert the –OH group to –COOH group16b. The obtained

180

GO-COOH solution was purified through repeated centrifugation and dialyzed (8000~14000 Da MWCO) against DI water for 48 h. 2.6. Synthesis of GO-ADH-HA-MTX nanosystems.10 mL of GO-COOH suspension (1 mg/mL) was added to 15 mL of MES solution (0.1 M, pH=6.0). Then 100 mg of EDC and 120 mg of NHS were added to the solution and stirred for 30

185

min. Subsequently, 60 mg of ADH-HA-MTX was added and stirred for another 24 h.The mass ratio of MTX and GO-COOH was 2.7:1 in reaction system. The reaction solution was purified through repeated centrifugation and dialyzed (8000~14000 Da MWCO) against DI water for 48 h. The obtained solution was ultrasonicated for 30 min at 600 W with 10 s intervals in an ice bath using a probetypeultrasonicator to

190

obtain GO-ADH-HA-MTX nanosystems suspension. 2.7. Synthesis of GO-ADH-HA nanosystems. 10 mL of GO-COOH suspension (1 mg/mL) was added to 15 mL of MES solution (0.1 M, pH=6.0). Then 100 mg of EDC and 120 mg of NHS was added to the solution and stirred for 30 min. Subsequently, 33 mg of ADH-HA was added and stirred for another 24 h. The reaction solution was

195

purified through repeated centrifugation and dialyzed (8000~14000 Da MWCO) against DI water for 48 h. The obtained solution was ultrasonicated for 30 min at 600 W with 10 s intervals in an ice bath using a probetypeultrasonicator to obtain GO-ADH-HA nanosystems suspension. 2.8. Characterization of various conjugates. The 1H NMR nuclear magnetic

200

resonance (NMR) spectra were determined on a Bruker AV400 MHz NMR spectrometer (Bruker, Billerica, MA, USA). The Fourier transform infrared spectroscopy (FTIR) spectra were performed on a Bruker IFS-55 infrared spectrometer (ThermoFisher, China). The X-ray diffraction (XRD) patterns were recorded

by

an

X-ray

diffractometer

(Bruker-axs,


Bruker,

USA).

The

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205


ultraviolet-visible (UV-vis) absorption spectra were recorded with a Perkin Elmer Lambda 750 UV-vis-near-infrared spectrophotometer (Perkin-Elmer, Norwalk CT). Differential Scanning Calorimeter (DSC) results were recorded by DSC 204 (Netzsch, German). 2.9. Characterization of GO-ADH-HA-MTX and GO-ADH-HA nanosystems.

210

The hydrodynamic particle size, polydispersity index (PDI), and surface charge were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, U. K.). The zeta potential was determined by electrophoretic light scattering (ELS) using a same equipment. The morphology was visualized by scanning electron microscopy (SEM, LEO1530VP, Carl Zeiss AG,

215

Germany) operated at an acceleration voltage of 20 kV and transmission electron microscopy (TEM, JEM2100, JEOL, Japan) operated at an acceleration voltage of 200 kV. The thickness was detected by Atomic Force Microscopy (AFM, Multimode 8, Bruker, Switzerland). 2.10. In vitro stability. For the stability study, GO, GO-ADH-HA, and

220

GO-ADH-HA-MTX nanosystems were kept standing in different solutions (including DI water, 0.9% NaCl, PBS, FBS, DMEM solutions) at room temperature with concentration at 0.1 M or 0.2 M. And the zeta potential of GO-ADH-HA-MTX nanosystems in PBS was measured at 0, 1, 2, 4, 8, 12, 24, and 48 h. 2.11. Hemolysis analysis. Red blood cells (RBCs) were obtained from 2 mL of rabbit

225

bloodthrough centrifugation (3000 rpm, 10 min), washed by PBS solution for three times, and then diluted to 10% (v/v) by PBS. And then 200 µL of RBCs solutions were respectively added to 1 mL of DI water as positive control (+), 1 mL of PBS solution as negative control (-), and 1 mL of PBS solution of different concentration GO-ADH-HA-MTX nanosystems. The obtained solutions were treated through

230

incubation at 37oC for 5 h and centrifugation (3000 rpm, 10 min). The supernatant was determined at 541 nm by microplate reader, and then hemolysis percentage was calculated through equ (1):

Hemolysis percentage(%) 

AS - AC(-)  100% (1) AC(  ) - AC(-)



Where 235

AS,

AC(+),

and

AC(-)

respectively

Page 10 of 38

represent

the

absorption

of

GO-ADH-HA-MTX nanosystems with RBCs, positive control, and negative control. 2.12. In vitro drug release. The MTX drug release characteristics of GO-ADH-HA-MTX nanosystems were determined by a dialysis method. Briefly, 1 mL of GO-ADH-HA-MTX suspension was placed in a dialysis bag (1000 Da MWCO) dialysis in PBS with or without esterase at pH 7.4 (mimicking physiological

240

pH in blood plasma and normal tissues) or pH 5.0 (mimicking acidic pH in endo/lysosomes of tumor cell) at 37oC with moderate shaking (100 r/min). At predetermined time intervals, 1 mL of the release medium were withdrawn for analysis and an equivalent volume of the release medium was replenished. The amount of MTX released was determined by UV-vis absorption spectroscopy. The

245

cumulative release amount was calculated using equ (2): 1 Cumulative release (%) 

n 1

 Ci  50  Cn i 1

weight of drug in nanosystems

 100% (2)

where Ci means the concentration of HCPT or MTX drug in dialysate at i time. 2.13. In vitrophotothermal heating analysis. The in vitrophotothermal effect of GO, GO-COOH, GO-ADH-HA, and GO-ADH-HA-MTX were analyzed under NIR laser 250

irradiation. GO, GO-ADH-HA, and GO-ADH-HA-MTX solutions with concentration at 0.1, 0.2, 0.5, or 1.0 mg/ml were treated with 808 nm laser irradiation (0.5, 1, or 1.5 W/cm2) for 5 min. The temperature of solutions was recorded by a FOTRIC 220 infrared thermal camera, while DI water was used as control. 2.14. Cell culture. MCF-7 human breast cancer cell lines, mouse embryo NIH-3T3

255

fibroblasts and HeLa (human cervical carcinoma) cancer cell lines with high expression of both CD44 and folate receptors were originally obtained from American Type Culture Collection (ATCC). MCF-7 and HeLa cells were cultured in DMEM medium

supplemented

with

10%

fetal

bovine

serum

(FBS)

and

1%

penicillin-streptomycin in an incubator under an atmosphere of 5% CO2 at 37°C. 260

2.15. In vitro cellular uptake. Cellular uptake behaviors of GO-ADH-HA and GO-ADH-HA-MTX nanosystems by mouse embryo NIH-3T3 fibroblastsMCF-7 or


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HeLa cells were detected by laser confocal microscopy (CLSM, Leica Microsystems, Mannheim, Germany) through detecting the fluorescence. MCF-7 or HeLa cells (1 × 105 cells) were seeded in a 6-well plate and cultured at 37°C for 24 h in a humidified 265

atmosphere with 5% CO2. The cells were incubated with DMEM solutions containing DiR-labeled GO-ADH-HA or GO-ADH-HA-MTX nanosystems for 1 or 5 h at 37°C. Subsequently, the medium was removed and cells were washed carefully with PBS thrice, fixed with 4% paraformaldehyde. DAPI (4’, 6-diamidino-2-phenyl-indole) was used to stain nuclei, which could be observed using CLSM when excited at 405 nm.

270

In addition, to investigate the folate/HA receptor-mediated internalization of GO-ADH-HA or GO-ADH-HA-MTX nanosystems, MCF-7 or HeLa cells were pretreated with the excess of free FA, HA, or FA/HA for 2 h before incubation with nanosystems. 2.16. Flow cytometry analysis. Flow cytometry analysis was used to detect

275

quantitative cellular uptake of GO-ADH-HA or GO-ADH-HA-MTX nanosystems. HeLa or MCF-7 cells were seeded at a density of 2 × 105 cells per well into 6-well plates and further cultured for 24 h. The cells were incubated with DiR-labeled GO-ADH-HA

or

GO-ADH-HA-MTX

nanosystems

at

an

equivalent

DiR

concentration for the same time periods. Then, the culture medium was removed and 280

the cells were washed with PBS for three times. After that, the cells were detached with trypsin/EDTA, suspended in PBS with 10% FBS, harvested by centrifugation at 2000 rpm for 5 min at 4°C, and resuspended in fluorescence-activated cell sorting (FACS) buffer. The data were detected on a FACSC alibur flow cytometer (Becton Dickinson, USA) and the data were analyzed using Cell Quest software.

285

2.17. In vitro cytotoxicity. MTT assay was performed to determine cytotoxicity on HeLa cells when treated with GO, MTX, GO-ADH-HA, or GO-ADH-HA-MTX. A 96-well plate was used to seed HeLa cells with 10, 000 cells each well. Cells grew in specific cell culture medium for overnight at 37oC with 5% CO2. After that, the culture medium was replaced by culture media with GO, MTX, GO-ADH-HA, or

290

GO-ADH-HA-MTX at concentration of GO ranging from 1 to 50 μg/mL or concentration of MTX ranging from 3 to 150 μg/mL. One group was chosen to be



treated with 808 nm laser irradiation (1 W/cm2) for 5 min, while another group was treated with nothing, and then cells were incubated for 24 h. After that, 10 μL of MTT solution (5 M, PBS as the solvent, pH 7.4) was added to each well, cells kept 295

incubation for further 4 h. Subsequently, the media was replaced by 150 μL of DMSO. Finally, the plate was shaken for 10 min to get the formazan crystals dissolved. Absorbance at 570 nm was quantified using the spectrophotometer (Biotek, USA). 2.18. In vivo fluorescence imaging. The BALB/c nude mice bearing HeLa tumor

300

model was used to determine the in vivo biodistribution. Briefly, 2×106HeLa cells were inoculated subcutaneously into the right armpit of the nude mice. When the tumor exceeded 200 mm3, the HeLa tumor-bearing mice were randomly divided into 3 groups, and intravenously injected with 200 µL PBS solutions of DiR-labeled GO, GO-ADH-HA and GO-ADH-HA-MTX nanosystems (1 mg/mL) at an equivalent DiR

305

concentration, respectively. The in vivo distribution and tumor accumulation of DiR-labeled GO, GO-ADH-HA, and GO-ADH-HA-MTX nanosystems were recorded at the pre-scheduled post-injection time intervals using an in vivo imaging system (IVIS Spectrum 200, Perkin-Elmer Co., MA, USA). Mice were sacrificed at 36 h after post-injection, and the tissues were excised and observed by the imaging system. The

310

fluorescence intensity of different tissues was quantified as the sum of all the detected photon counts within the region of interest (ROI) in the unit of [photo/cm2/s]. 2.19. In vivo anti-tumor effect. In vivo anti-tumor activity was evaluated using H22 tumor-bearing mice models. Briefly, 0.2 mL of cell suspension containing 2× 106 H22 cells was inoculated subcutaneously into the right armpit of the mice (4-6 weeks,

315

16-22 g). When the tumor reached to around 200 mm3, the tumor-bearing mice were randomly divided into six groups, and 200 µL of PBS solutions of 0.9% NaCl (control group), GO-ADH-HA, and GO-ADH-HA-MTX were intravenously injected to two groups of mice, respectively. To investigate in vivophotothermal effect, 808 nm laser irradiation was used to treat one group HeLa-tumor bearing mice at 12 h injection of

320

0.9% NaCl, GO-ADH-HA, and GO-ADH-HA-MTX. The temperature and images of treated site in mice were recorded by a FOTRIC 220 infrared thermal camera at


Page 12 of 38

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designed time. The tumor volume and body weight of the six groups mice were observed and recorded for 22 days, and mice images were also recorded. On the 22th day, all groups’ mice were dissected, and the tumors and major 325

organs (including liver, heart, spleen, kidney, and lung) were harvested, fixed with 4% paraformaldehyde for 12 h, embedded in paraffin, and stained by hematoxylin and eosin (H&E). The stained tumor and major organs sections were observed by an optical microscopy (DM5500B, Leica). 2.20. Statistical analysis. All quantitative data were expressed as the means

330

plus/minus standard deviation (mean ± SD). P value was calculated by Student’s t-test or SPSS 19.0 software. A value of P< 0.05 was considered as statistically significant. A value of P< 0.01 was considered as highly statistically significant.

3. Results and discussion 335

3.1. Synthesis of HA-MTX and ADH-HA-MTX conjugate As shown in Scheme 1, HA-MTX conjugate was obtained through an esterification reaction between HA and MTX, and ADH-HA-MTX (or ADH-HA) conjugate was synthesized via the formation of amide linkage between ADH and HA-MTX (or HA). A series of physicochemical characterizations were carried out to

340

prove the successful synthesis. The 1H nuclear magnetic resonance (1H NMR) spectra of pristine materials and chemical conjugates were shown in Figure 1A, B. The characteristic peak of MTX was observed around δ (ppm) 6.8, 7.5, and 8.6, which was ascribed to pteridine ring and p-phenyl ring protons. In addition, the characteristic peak of HA at δ (ppm) 2.0 and 3.0-4.0 could respectively identify the existence of

345

N-acetyl (-NHCOCH3) group and glucosidic proton of methylene and hydroxyl groups. The 1H spectrum of HA-MTX displayed the characteristic peak of both MTX and HA, demonstrating the successful synthesis of HA-MTX. Besides, the successful graft of ADH to HA-MTX was indicated by the existing signals around δ (ppm) 2.2 and 1.5 in the spectrum of ADH-HA-MTX, corresponding to the methylene group of

350

ADH. The Fourier transform infrared (FT-IR) spectrum of pristine materials, physical



mixtures, and chemical conjugates were shown in Figure 1C, D. Different from the spectrum of MTX, HA, and their physical mixture, the new absorption peak at 1699 cm-1 appeared in the spectrum of HA-MTX, which was ascribed to C=O stretching 355

vibrations of ester bond, implying the successful formation of ester bond between HA and MTX. Compared with the spectrum of ADH or ADH/HA-MTX physical mixture, the narrow absorption peak at 3274 cm-1 and 3185 cm-1 corresponding to the N-H stretching vibrations of amino group disappeared in the spectrum of ADH-HA-MTX. The result indicated the formation of amide linkage between ADH and HA-MTX.

360

The ultraviolet-visible spectrophotometer (UV-vis) absorption spectra of pristine materials and chemical conjugates in deionized (DI) water were detected (Figure 1E). Compared with the spectrum of MTX, the maximum absorption peak at 302 nm shifted to 305 nm in the spectrum of HA-MTX. After the grafting of ADH to HA-MTX, an additional 5 nm red-shift was observed in the spectrum of

365

ADH-HA-MTX. The microstructure of pristine materials, physical mixtures, and chemical conjugates was also analyzed by X-ray diffraction (XRD) experiments. Compared to the spectrum of HA/MTX or ADH/HA-MTX mixture, the crystalline signals of MTX, HA, and ADH disappeared in the spectrum of HA-MTX or ADH-HA-MTX conjugate (Figure 1F, G), implying the disruption of crystalline

370

structure. The above UV-vis and XRD results demonstrated HA-MTX or ADH-HA-MTX was obtained through chemical reactions between HA and MTX or between ADH and HA-MTX. In addition, the successful conjugation of ADH-HA was confirmed by the disappearance of narrow absorption peaks at 3274 cm-1 and 3185 cm-1 in FT-IR spectra of ADH-HA and the disappearance of crystalline signals

375

in XRD spectra of ADH-HA (Figure S2). 3.2. Characterization of GO-ADH-HA-MTX and GO-ADH-HA nanosystems HA-MTX prodrug (or HA) was grafted to carboxyl-functionalized GO (GO-COOH) through adipicdihydrazide (ADH) crosslinker via amidation reaction to construct a stable and robust GO-ADH-HA-MTX (or GO-ADH-HA) nanosystem.

380

FTIR and UV-vis analyses were firstly used to detect the successful conjugation of GO-ADH-HA-MTX and GO-ADH-HA. For the analysis of FT-IR spectra, the


Page 14 of 38

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absorption peak at 1716 cm-1 disappeared in GO-ADH-HA-MTX (or GO-ADH-HA) spectrum, which existed in GO-COOH spectrum owing to C=O stretching vibrations of carboxylic groups (Figure 2A, B).Regarding the analysis of UV-visspectra (Figure 385

2C), the absorption peak at 225 nm in the spectrum of GO significantly shifted to 215 nm in that of GO-ADH-HA. Different from the maximum absorption peak at 305 nm in the spectrum of ADH-HA-MTX, a 3 nm red-shift could be observed in that of GO-ADH-HA-MTX. According to the FT-IR and UV-vis analyses, the successful synthesis of GO-ADH-HA-MTX (or GO-ADH-HA) could be firstly confirmed. In

390

addition, the surface charge of GO-COOH was about -25.6 mV by determination of zeta potential (Figure 2D). After conjugation with ADH-HA or ADH-HA-MTX, the surface charge respectively changed to be -17.8 mV or -15.7 mV (Figure 2E, F). The reason for the changes might be that carboxyl groups on the surface of GO decreased after amide reaction with ADH-HA-MTX or ADH-HA.

395

The morphology of GO, GO-ADH-HA, and GO-ADH-HA-MTX nanosystems was observed using transmission electron microscopy (TEM) and atomic force microscopy (AFM). TEM images (Figure 3A, B, and C) showed that the size of GO was around 100 nm, while the size of GO-ADH-HA, and GO-ADH-HA-MTX nanosystems was around 200 nm. As shown in AFM images, the height of GO

400

nanosheets was less than 2 nm (Figure 3D), while the height of GO-ADH-HA-MTX (Figure 3E) or GO-ADH-HA (Figure 3F) nanosystems increased to 9.3 nm or 7.8 nm, respectively. 3.3. Stability of GO-ADH-HA-MTX and GO-ADH-HA nanosystems The size distribution of GO, GO-ADH-HA, and GO-ADH-HA-MTX

405

nanosystems was in unimodal model with polydispersity index (PDI) of 0.294, 0.303, and 0.214, respectively (Figure S3). And it could be observed in TEM images that GO-ADH-HA-MTX nanosystems exhibited impressive dispersity (Figure 3C). These results indicated GO-ADH-HA-MTX nanosystems possessed the ability to keep stable. To observe their stability, different nanosystems kept standing in different

410

physiological

solutions.

As

shown

in

Figure

4A,

GO-ADH-HA

and

GO-ADH-HA-MTX nanosystems with concentration at 0.1 M could keep stable for



48 h in NaCl, PBS, FBS, but only GO-ADH-HA-MTX nanosystems could keep for 48 h in DMEM. The considerable stability of GO-ADH-HA-MTX nanosystems was also affirmed in different physiological solutions (including NaCl, PBS, FBS, 415

DMEM) with different concentrations at 0.2 M (Figure S4). Besides, the surface charges of GO-ADH-HA-MTX nanosystems were negatively with zeta potential of -15.6 mV (Figure 2F) and could keep nearly unchangeable for 48 h (Figure 4B), which might be able to prevent the potential aggregations via electrostatic repulsion. Thus, the excellent physiological stability of GO-ADH-HA-MTX nanosystems in

420

water, PBS buffer, cell medium, and serum was beneficial to deliver them to tumor sites in intact from during blood circulation. 3.4. Hemolysis assay The considerable blood compatibility was vital for drug delivery systems, which could prevent their hemolysis during blood circulation after intravenous injection. To

425

estimate the blood compatibility, the hemolysis assay of GO-ADH-HA-MTX nanosystems was carried out. As shown in Figure 4C, the hemolysis percentage and representative images indicated that no obvious red cell membranes damage associated with hemolysis was existed in GO-ADH-HA-MTX group in a certain range of high concentration (0.1-0.3 mg/mL) compared to negative control group, revealing

430

the excellent blood compatibility of GO-ADH-HA-MTX nanosystems. 3.5. In vitrophotothermal effect The in vitrophotothermal effects of GO, GO-COOH, GO-ADH-HA, and GO-ADH-HA-MTX nanosystems were analyzed under NIR laser irradiation. As shown in Figure 5A, the temperature of GO-COOH, GO-ADH-HA, and

435

GO-ADH-HA-MTX dispersion with the same concentration at 0.2 mg/mL respectively increased 27.1oC, 33.2oC, and 33.9oC under 808 nm laser irradiation (1 W/cm2) for 5 min, while GO dispersion and PBS solution could only increase 14.1oC and 9.5oC under the same condition. The photothermalconversion efficiency of GO-ADH-HA-MTX was calculated to be 69.89% (calculation details in Supplemental

440

Information, Figure S7).Besides, temperature was also improved along with increase of nanosystems concentration (Figure 5B, S5) and NIR laser intensity (Figure 5C).


Page 16 of 38

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Temperature changes of different dispersions (with concentration at 0.1, 0.5, or 1.0 mg/mL) also implied the stronger absorption at 808 nm of GO-COOH, GO-AHD-HA, and GO-ADH-HA-MTX nanosystems than GO nanosheets (Figure S5). The 445

enhanced photothermal effects could explained by the reduction of GO through converting epoxide and hydroxyl groups to carboxyl group, or the introduction of aromatic clusters after chemical reaction, either simply or in combination37. In addition, Solutions of GO-ADH-HA-MTX nanosystem was irradiated with NIR laser for 5 min (808 nm, 1W/cm2, LASER ON, Figure S6), followed by naturally cooling to

450

room temperature without NIR laser irradiation for 15 min (LASER OFF, Figure S6). This cycle was repeated five times in order to investigate the photostability of GO-ADH-HA-MTX nanosystem.As shown in FigureS6, no significant decrease for the temperature elevation was observed for GO-ADH-HA-MTX nanosystem. 3.6. In vitro release behavior

455

The release rate of MTX fromGO-ADH-HA-MTXnanosystems was explored through dialysis in PBS with or without esterase at pH 7.4 (mimicking physiological pH in bloodstream and normal tissues) or pH 5.0 (mimicking physiological pH in intracellular acidic microenvironment) at 37oC. As shown in Figure 5D, under condition of pH 7.4 without or with esterase, ~12% or ~22% of MTX was released

460

from GO-ADH-HA-MTX nanosystems within 48 h, while ~28% or ~46.8% of MTX was released under condition of pH 5.0 with or without esterase. MTX release curves indicated the release of MTX from GO-ADH-HA-MTX nanosystems was dependent on pH and esterase, which was accorded with the report that the break of ester linkage would be accelerated under environment with acid and esterase.

465

3.7. In vitro cellular uptake To observe the selective cellular uptake of GO, GO-ADH-HA, or GO-ADH-HA-MTX nanosystems, DiR as a lipophilic near-infrared fluorescent dye was selected to be loaded within GO, GO-ADH-HA or GO-ADH-HA-MTX (designed as GO@DiR, GO-ADH-HA@DiR, or GO-ADH-HA-MTX@DiR) nanosystems

470

through hydrophobic and π-π stacked interaction for near-infrared fluorescence imaging. HeLa and MCF-7 cell lines with over-expression of CD44/folate receptors



were investigated using confocal scanning laser microscopy (CLSM) after incubation with different nanosystems. As shown in Figure 6A, HeLa cells treated with GO-ADH-HA-MTX nanosystems exhibited stronger fluorescent signals than that 475

treated with GO-ADH-HA nanosystems, implying that the decoration of dual kinds of ligands onto nanosheets significantly improved cellular uptake because of the much higher affinities of GO-ADH-HA-MTX nanosystems towards dual CD44/folate receptors. Besides, the fluorescent signals increased at 3 h compared with 0.5 h, implying gradual accumulation of nanosystems over time. However, the cellular

480

uptake of GO-ADH-HA-MTX nanosystems was dramatically decreased when HeLa cells was pretreated with free form of HA or FA, which could be explained by the block of HA or FA receptors-mediated endocytosis pathway. These results indicated that dual-targeting GO-ADH-HA-MTX nanosystems could achieve tumor cell uptake and internalization through dual CD44/folate receptors more efficiently than

485

single-targeting GO-ADH-HA nanosystems. Experiments on MCF-7 cell lines showed similar result with those on HeLa cell lines (Figure 6B). Besides, the selectivity of GO-ADH-HA-MTX on tumor cells could be further proved through normal cells. As shown in in Figure S6, only weak fluorescent signals could be seen and there is no obvious difference among no pre-treating, pretreated with HA, and

490

pretreated with FA on mouse embryo NIH-3T3 fibroblasts. The remarkable targeting ability of dual-targeting GO-ADH-HA-MTX nanosystems was further confirmed by flow cytometry analysis. As shown in flow cytometric histogram, HeLa cells incubated with GO-ADH-HA-MTX nanosystems exhibited an obvious right shift compared with those incubated with GO-ADH-HA

495

nanosystems (Figure 6C). Besides, HeLa cells incubated GO-ADH-HA-MTX nanosystems showed a prominent blue shift, which were pretreated with free form of FA or HA. These quantitative results and mean fluorescence intensity analysis (Figure 6D) were well consistent with the qualitative results determined by CLSM (Figure 6A), which proved that both introduction of HA and MTX could

500

synergistically elevate the targeting efficacy. 3.8. In vitro cytotoxicity


Page 18 of 38

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The in vitro anti-tumor activity was investigated using HeLa cell lines. MTT assay was used to evaluate the cell viability of free MTX, GO-ADH-HA, and GO-ADH-HA-MTX with or without laser treatment for 24 h incubation. All drug 505

formulations presented concentration-dependent cytotoxicity behaviors towards HeLa cells (Figure 7A). Pristine GO exhibited high cytotoxicity (Figure 7A) because of its undesirable biocompatibility39-40, implying its limitation for drug loading application. The cytotoxicity of GO-ADH-HA nanosystems was much weaker than that caused by GO nanosheets, making it possible for biomedical use. After cell internalization,

510

MTX could be rapidly released from GO-ADH-HA-MTX nanosystems due to the cleavage of ester linkage under environment with acid and esterase inside endo/lysosomes of tumor cells. Besides, the cell inhibition effect of free MTX was lower than that of GO-ADH-HA-MTX nanosystems because of some extent of hydrophilic property and low cellular uptake of free form of MTX (easily soluble in

515

PBS at pH 7.4) (Figure 7B). Moreover, it could be seen that the cytotoxicity of GO-ADH-HA and GO-ADH-HA-MTX nanosystems significantly increased after cells being treated with 808 nm NIR laser for 5 min owing to photothermal effect. Consequently, GO-ADH-HA-MTX nanosystems treated with laser demonstrated highest in vitro anti-tumor activity as a result of the combination of chemotherapy and

520

photothermal therapy. 3.9. In vivotargetability and photothermal effect To determine in vivo tumor targetability of GO-ADH-HA-MTX nanosystems, the lipophilic near-infrared fluorescence dye DiR with strong NIR absorbance was doped

525

within

GO-ADH-HA-MTX

(designed

as

GO-ADH-HA-MTX@DiR)

nanosystems for in vivo near-infrared fluorescence imaging. In addition, the GO@DiR and

GO-ADH-HA@DiRnanosystems

biodistribution

of

were

GO@DiR,

used

as

control

groups.

GO-ADH-HA@DiR,

The and

GO-ADH-HA-MTX@DiRnanosystems was tested on HeLa tumor-bearing BALB/c nude female mice. Different formulations were intravenously administrated into nude 530

mice via tail vein, and different time points at 1, 4, 12, and 24 h were selected to observe the distribution of nanosystems in whole body and their tumor accumulation.



Page 20 of 38

As shown in Figure 8A, GO nanosheets showed rare accumulation at the tumor sites at 12 and 24 h after injection resulted from EPR effect, while GO-ADH-HA nanosystems exhibited weak fluorescence signals at 0.5 h and gradually became 535

stronger with the time being. Besides, the fluorescence signals at tumor sites of GO-ADH-HA nanosystems than that of GO nanosheets, which was likely due to the additional CD44 receptors-mediated active targeting effects. However, both GO and GO-ADH-HA nanosystems exhibited much stronger fluorescence signals at liver tissues than other sites including tumor tissues. Interestingly, GO-ADH-HA-MTX

540

nanosystems accumulated in tumor and liver tissues at beginning, but most of them achieved highly effective tumor accumulation with obviously lower signals around liver tissues at 12 and 24 h after injection. At 24 h injection, the mice were sacrificed and the normal and tumor tissues were excised for ex vivo fluorescence imaging. As shown

545

in

Figure

8B

the

fluorescence

signals

at

the

tumor

sites

of

GO-ADH-HA-MTX nanosystems group were significantly stronger than that of GO-ADH-HA nanosystems, confirming its more effective tumor targeting abilities. Besides, no obvious signals were observed in heart, lung, and kidney organs, while only weak signals in liver and lung organs, indicating the low side effects of GO-ADH-HA-MTX nanosystems, which was in accord with the ROI analysis results

550

(Figure

8C).

These

results

indicated

strong

tumor-targeting

ability

of

GO-ADH-HA-MTX nanosystems, which could be explained by the collaborative effect of passive targeting mechanism (relied on EPR effect) and active targeting pathway (relied on CD44/folate receptors-mediated endocytosis). In vivo PTT effects were also performed on HeLa tumor-bearing mice. At 12 h 555

injection of GO-ADH-HA or GO-ADH-HA-MTX nanosystems, tumor areas were treated by NIR laser irradiation (808 nm, 1 W/cm2) for 5 min. The temperature of 0.9% NaCl group only increased to 38oC. By contrast, the temperature of GO-ADH-HA group increased to 51oC from 37oC after treated with irradiation for 5 min, while that of GO-ADH-HA-MTX group could increase to 59oC, implying the

560

much more effective in vivo PTT effects of GO-ADH-HA-MTX nanosystems (Figure 9).


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According to above-mentioned discussion, the GO-ADH-HA-MTX nanosystems exhibited strong tumor accumulation while reducing the accumulation in normal tissues, and thus MTX could be highly delivered to tumor sites with extremely 565

decreased damage for healthy tissues. More importantly, the GO-ADH-HA-MTX nanosystems could perform excellent in vivo PTT effects at the tumor sites based on the excellent tumor targeting capacity. Hence, the sheet-like GO-ADH-HA-MTX nanosystems was possibly able to achieve combination therapy of chemotherapy and PTT.

570

3.10. In vivo anti-tumor effect The in vivo anti-tumor efficacy of GO-ADH-HA-MTX and GO-ADH-HA nanosystems was investigated on H22 tumor-bearing Kunming mice. The results of tumor suppression were analyzed through tumor volume and body weight with saline as a negative control. Tumor volume changes in Figure 10B demonstrated

575

GO-ADH-HA-MTX nanosystems exhibited obvious decrease of tumor volume the first 4 days, but then anti-tumor effect disappeared owing to the metabolism of MTX drugs. The anti-tumor efficacy of PTT was investigated on mice treated with laser at 12 h injection of GO-ADH-HA or GO-ADH-HA-MTX nanosystems. The tumor gradually decreased over time without proliferation until the 21st day, revealing the

580

highly efficient killing ability for tumor tissues of PTT effect based on GO-COOH. Notably, tumor sites treated with laser (GO-ADH-HA and GO-ADH-HA-MTX groups) appeared scabs which would then desquamate from mice, the appearance of scabs

might

result

from

hyperthermia.

Above

all,

mice

treated

with

GO-ADH-HA-MTX nanosystems combined with laser resulted in the most effective 585

anti-tumor results compared to all other groups, owing to the combination of chemotherapy and efficient PTT based on its high tumor targeting ability. The optical pictures (Figure 10A) were also recorded to investigate the tumor changes, the results of which accorded well with tumor volume changes. Afterward, the outstanding anti-tumor ability of GO-ADH-HA-MTX nanosystems with laser was also affirmed

590

by the histological analysis of the hematoxylin and eosin (H&E) staining on tumor. As shown in Figure 10D, the tumor tissues in 0.9% NaCl, free MTX, GO-ADH-HA,



Page 22 of 38

and GO-ADH-HA-MTX groups consisted of tightly packed tumor cells, while those exhibited different degrees of remission of cancer cells in GO-ADH-HA with laser and GO-ADH-HA-MTX with laser groups. 595

Besides, no obvious changes were observed for body weight of Kunming micein all formulation groups after treatment (Figure 10C), meanwhile no mice death was found during investigation after accepting treatment. Hematoxylin and eosin (H&E) staining of normal organs such as kidney, lung, spleen, liver, and heart was performed to evaluate the potential systemic toxicity. As shown in Figure 10D, no severe

600

inflammation and organ damage was observed in the GO-ADH-HA-MTX groups. These could reveal that the treatment of GO-ADH-HA-MTX nanosystems caused no significant adverse effects. All these results implied that the combination therapy of chemotherapy and PTT based on our designed dual-targeting GO-ADH-HA-MTX nanosystems possessed the ability to eliminate tumors efficiently and cause no

605

obvious side effect for mice.

4. Conclusion We developed a multifunctional nanosytemsbased on GO-ADH-HA-MTX conjugates 610

for

synergesiticselected

tumor-targeting

and

combined

chemo-photothermal therapy. Herein, HA and MTX were firstly connected through esterification reaction, and then HA-MTX prodrug was grafted to GO-COOH through ADH

crosslinker

via

amidation

reactions.The

obtained

GO-ADH-HA-MTXnanosystemsexhibited considerable dispersibility, high plasma stability, and excellent blood compatibility with size around 200 nm and height 615

around 7 nm. Furthermore, drug release results indicated MTX could release from GO-ADH-HA-MTX via pH-responsiveness within tumor environments. The in vitro and in vivo results revealed that GO-ADH-HA-MTX nanosystems possessed enhanced tumor accumulation ability via multilevel targeting (EPR effect-based passive targeting mechanism and CD44/FA receptors-mediated dual active targeting

620

mechanism). Meanwhile it could also achieve favorable photothermal effect under 808 nm laser irradiation based on high NIR absorbance of GO and then enhanced


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tumor accumulation. Combination therapy was subsequently carried out, proving remarkable tumor elimination without reappearance based on chemo-photothermal therapy 625

of

GO-ADH-HA-MTX

nanosystems

compared

with

single

therapy.Furthermore, no obvious side effects were observed during treatment period. Taken together, the selected-targeting GO-AHD-HA-MTX nanosystems held great promise for cancer treatment with integration of chemotherapy and PTT. ASSOCIATED CONTENT

630

Supporting Information The Supporting Information is available free of charge on theACS Publications website at DOI: More characterization data including DSC spectra, FTIR spectra, DLS determination, representative optical photographs, temperature variation curves, laser ON/OFF

635

temperature evaluation cycles, and photothermal response. (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected].

640

Notes The authors declare no competing financial interest. Acknowledgments The work is supported by the National Natural Science Foundation of China (31771029, 81472458), Science and Technology Foundation of Fujian Province,

645

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of Methotrexate and Mitomycin C with Synergistic Anticancer Effect. Molecular Pharmaceutics 2015,12, 769-782.



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Scheme 1. Schematic illustration of synthesis route of GO-ADH-HA-MTX. HA-MTX conjugate was obtained through esterification reaction between HA and MTX, and then HA-MTX was grafted to GO-COOH through ADH as a linker via amidation

reaction.



Page 28 of 38

780

Scheme 2. (A) Synthesis of GO-ADH-HA-MTX nanosystems. (B) Schematic illustration of GO-ADH-HA-MTX nanosystems for combination therapy of chemotherapy and PTT through dual-active targeting delivery: I) enhanced accumulation of nanosystems within tumor sites through EPR effects; II) improved 785

tumor cellular internalization of nanosystems through CD44/FA receptors-mediated dual active targeting mechanisms by specific recognition for HA or MTX); III) controlled release of MTX from nanosystems through acid-induced cleavage of ester linkage. IV) combination therapy of chemotherapy and PTT achieved via DHFR enzyme inhibition induced by MTX and local heat triggered through NIR irradiation

790

working

on


nanosystems.

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Figure 1. Characterization of HA-MTX and ADH-HA-MTX polymer. (A) 1H NMR spectra of MTX (DMSO-d6 as solvent), HA (D2O as solvent), and HA-MTX (D2O and DMSO-d6 as solvent). (B) 1H NMR spectra of HA-MTX (D2O/DMSO-d6 as 795

solvent), ADH (D2O as solvent), and ADH-HA-MTX (D2O as solvent). (C) FT-IR



spectra of MTX, HA, HA/MTX mixture, and HA-MTX. (D) FT-IR spectra of HA-MTX, ADH, HA-MTX/ADH mixture, and ADH-HA-MTX. (E) UV-vis absorption spectra of MTX, HA, HA-MTX, ADH, and ADH-HA-MTX (DI water as solvent). (F) XRD spectra of MTX, HA, HA/MTX mixture, and HA-MTX. (G) XRD 800

spectra of HA-MTX, ADH, HA-MTX/ADH mixture, and ADH-HA-MTX.

Figure 2. Characterization of GO-ADH-HA and GO-ADH-HA-MTX. (A) FT-IR spectra of GO-COOH, ADH-HA, GO/ADH-HA mixture, and GO-ADH-HA. (B) FT-IR spectra of GO-COOH, ADH-HA-MTX, GO/ADH-HA-MTX mixture, and 805

GO-ADH-HA-MTX. (C) UV-vis absorption spectra of GO-COOH, ADH-HA, ADH-HA-MTX, GO-ADH-HA, and GO-ADH-HA-MTX (DI water as solvent). (D) Zeta potential of GO. (E) Zeta potential of GO-ADH-HA. (F) Zeta potential of GO-ADH-HA-MTX.


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810


Figure

3.Morphology

of

GO,GO-ADH-HA,

and

GO-ADH-HA-MTX

nanosystems.(A) TEM image of GO nanosheets. (B) TEM image of GO-AHD-HA nanosystems. (C) TEM image of GO-ADH-HA-MTX nanosystems. (D) AFM image and height distribution of GO nanosystems. (E) AFM image and height distribution of GO-ADH-HA 815

nanosystems.

(F)

AFM

image

and

GO-AHD-HA-MTX nanosystems.


height

distribution

of


Figure 4. (A) Representative images for stability of GO (a), GO-ADH-HA (b), and GO-ADH-HA-MTX (c) nanosystems (0.1 M) in DI water, 0.9% NaCl, PBS, FBS, and DMEM during storage for 48 h. (B) Zeta potential changes of GO-ADH-HA-MTX 820

nanosystems in DI water. (C) Hemolytic activity of GO-ADH-HA-MTX nanosystems samples with RBCs at different concentrations, DI water as positive control (+), 1 mL PBS solution as negative control (-).


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Figure 5. (A) Temperature variation curves of aqueous dispersion of H2O, GO, 825

GO-COOH, GO-ADH-HA, and GO-ADH-HA-MTX nanosystems under 808 nm laser irradiation (1 W/cm2) at a concentration of 0.2 mg/mL. (B) Temperature variation curves of aqueous dispersion of GO-ADH-HA-MTX nanosystems under 808 nm laser irradiation with different laser power density. (C) Temperature variation curves of aqueous dispersion of GO-ADH-HA-MTX nanosystems at different concentrations

830

under 808 nm laser irradiation (1 W/cm2). (D)Drug release profiles of MTX from GO-ADH-HA-MTX nanosystems at pH 5.0 or 7.4 with or without esterase.



Figure 6. In vitro cell uptake. (A) CLSM images of HeLa cells (high expression of CD44/folate receptors) incubated with nanosystems of DiR-labeled GO-ADH-HA, 835

GO-ADH-HA-MTX, or GO-ADH-HA-MTX pretreated with free HA or FA for 0.5 h or 3 h. (B) CLSM images of MCF-7 cells (high expression of CD44/folate receptors) incubated with nanosystems of DiR-labeled GO-ADH-HA, GO-ADH-HA-MTX, or GO-ADH-HA-MTX pretreated with free HA or FA for 0.5 h or 3 h. (C) Flow cytometry profiles of HeLa cells incubated with GO-ADH-HA, GO-ADH-HT-MTX,


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840


or GO-ADH-HA-MTX pretreated with free HA or FA for 6 h. Error bars indicate SD (n = 3). *P< 0.05. **P< 0.01

Figure 7. In vitro cytotoxicity.(A) In vitro cell viability of HeLa cells incubated with GO for 24 h treated with or without 808 nm laser irradiation for 5 min. (B) In vitro 845

cell

viability

of

HeLa

cells

incubated

with

MTX,

GO-ADH-HA,

and

GO-ADH-HA-MTX for 24 h treated with or without 808 nm laser irradiation for 5 min. Error bars indicate SD (n = 3). *P< 0.05. **P< 0.01.

Figure 8. (A) In vivo fluorescence imaging of HeLa tumor-bearing nude mice at



850

Page 36 of 38

different time point after intravenous injection of DiR-labeled GO, GO-ADH-HA, GO-ADH-HA-MTX nanosystems. (B) Ex vivo fluorescence images of major organs and tumors at 24 h injection of DiR-labeled GO-ADH-HA and GO-ADH-HA-MTX nanosystems.

(C)

ROI

analysis

of

DiR-labeled

GO-ADH-HA

and

GO-ADH-HA-MTX nanosystems from major organs and tumor. Error bars indicate 855

SD (n = 3). *P< 0.05. **P< 0.01.

Figure 9. (A) In vivo IR thermal images of HeLa tumor-bearing mice upon 808 nm laser irradiation at differenttimepointsafter injection of0.9% NaCl, GO-ADH-HA, and GO-ADH-HA-MTX nanosystems. (B) Temperature variation curves of tumor region 860

recorded by the IR thermal camera during NIR laser irradiation. Error bars indicate SD (n = 3). *P< 0.05. **P< 0.01.


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Figure 10.In vivo therapeutic efficacy of H22 tumor-bearing mice after intravenous injection of 0.9% NaCl, GO-ADH-HA, and GO-ADH-HA-MTX 865

nanosystems treated with or without 808 nm laser irradiation for 5 min. (A) Representative images of H22 tumor-bearing mice with different treatment for



different time periods. (B) Tumor volume change curves of mice with different treatment. (C) Body weight change curves of mice with different treatment. (D) H&E staining of tumor, heart, liver, spleen, lung and kidney of mice with different 870

treatment. Error bars indicate SD (n = 3). *P< 0.05. **P< 0.01.


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Multifunctional Nanosystem Based on Graphene Oxide for Synergistic

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