Triple Stimuli-Responsive Magnetic Hollow Porous Carbon-Based

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Biological and Medical Applications of Materials and Interfaces

Triple Stimuli-Responsive Magnetic Hollow Porous Carbon-Based Nano-Drug Delivery System for Magnetic Resonance ImagingGuided Synergistic Photothermal/Chemo-Therapy of Cancer Fan Wu, Ming Zhang, Hanwen Lu, Dong Liang, Yaliang Huang, Yonghong Xia, Yuqing Hu, Shengqiang Hu, Jianxiu Wang, Xinyao Yi, and Jun Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07213 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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ACS Applied Materials & Interfaces

Triple Stimuli-Responsive Magnetic Hollow Porous Carbon-Based Nano-Drug Delivery System for Magnetic Resonance Imaging-Guided Synergistic Photothermal/Chemo-Therapy of Cancer Fan Wu,†‡§ Ming Zhang, ‡§ Hanwen Lu,† Dong Liang,† Yaliang Huang,† Yonghong Xia,† Yuqing Hu,† Shengqiang Hu,† Jianxiu Wang,† Xinyao Yi*,†, and Jun Zhang*,‡§ †

College of Chemistry and Chemical Engineering, Central South University,

Changsha 410083, PR China ‡

Jiangsu Collaborative Innovation Center for Biological Functional Materials, College

of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, PR China §

Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Engineering Research

Center for Biomedical Function Materials, Nanjing 210023, PR China

Abstract: The premature leakage of anticancer drugs during blood circulation may damage immune system, normal cells and tissues. Constructing targeted nanocarrier with pH, glutathione (GSH) and NIR triple-responsive property can effectively avoid the leakage of anticancer drugs before it arrives targeted site. In this paper, magnetic hollow porous carbon nanoparticles (MHPCNs) were successfully fabricated as nanocarrier. Poly(γ-glutamic acid) (PGA) was used to cap the pores of MHPCNs.

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Utilizing photothermal conversion property of carbon and iron oxide (Fe3O4) nanomaterials

to

perform

photothermal

therapy

(PTT)

to

overcome

multidrug-resistance (MDR) produced by chemotherapy. The biodistribution of nanoparticles was investigated by magnetic resonance (MR) imaging. Experiments in vivo confirm the efficient accumulations of nanoparticles at tumor sites. Meanwhile, tumor growth was effectively inhibited via synergistic photothermal/chemo-therapy with minimal side effect.

KEYWORDS: triple-responsive, MHPCNs, PGA, MR imaging, synergistic photothermal/chemo-therapy

1. INTRODUCTION Cancer is one of the leading causes of death worldwide.1,2 At present, the cancer treatment mainly relies on chemotherapy technology.3-5 However, anticancer drugs may cause some side effects on immune system, normal cells, and tissues.6,7 Thus, ideal nanotechnology-based drug delivery systems (DDSs) should keep a steady encapsulation before nanoparticles arrive tumor region during blood circulation, and accomplish efficient release of anticancer drugs in the targeted cancer cells. A nanovehicle system equipped with stimuli-responsive capability can control the release of drugs at specific case.8-11 On the basis of previous research, the tumor microenvironment has lower pH (6.0-7.0) in contrast to blood and normal tissues (∼7.4), and the pH value of intracellular organelles will decline further, including lysosomes (pH < 5.5) and endosomes (pH = 5.5).12-14 In addition, the glutathione (GSH) concentration of extracellular environment was about 0.1-1% of that in intracellular compartments. And the GSH concentration in normal cells is about one-third of that in cancerous cells.15-17 Based on these internal features, porous nanoparticles with high pore volume and large surface area are often used as pH- and GSH-responsive (internal stimuli) nano-drug carriers to inhibit leakage of drug in the period of blood circulation and acquire burst release in the targeted tumor site to


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improve the tumoricidal ability of DDSs.18-20 However, single medical chemotherapy is restricted by multidrug-resistance (MDR) among half of the patients.4,21-23 MDR is caused by various mechanisms, and the major factor is that Pglycoprotein (P-gp), membrane transporters, accelerate anticancer drug efflux and avoid intracellular accumulation.24-26 In order to overcome MDR, designing multifunctional DDSs for synergistic tumor therapy is necessary. Photothermal therapy (PTT), a major hyperthermia approach for tumor treatment, takes advantage of near infrared (NIR) resonant nanomaterials transforming light into heat for achieving desired efficacy. When NIR irradiation, it can trigger intracellular temperature increasing and the local targeted tumor ablation, and minimize destruction to the surrounding normal cells.27-30 Meanwhile, heat produced by NIR irradiation could offer a supplementary method to regulate the drug release (external stimuli).31,32 When stimuli-responsive chemotherapy and PTT are integrated with targeted delivery, therapeutic effect will be improved significantly. Moreover, the visualization towards the delivery and distribution of nanoparticles plays a guidance role in cancer therapy. And exploiting imaging-guided nanotechnology-based DDSs is an ideal treatment project to improve therapeutic efficiency.33-36 Magnetic resonance (MR) imaging with unlimited tissue penetration and high resolution is one of the most great diagnostic techniques among the multitudinous imaging technology.37,38 Iron oxide (Fe3O4) nanoparticles with good biocompatibility, low toxicity, and high stability in the physiological circumstance are often used as contrast agents for T2-weighted MR imaging for diagnostic and therapeutic applications.39,40 Herein, we report a GSH-, pH- and NIR-responsive magnetic hollow and porous carbon

nanoparticles

(MHPCNs)

for

imaging-guided

synergistic

photothermal/chemo-therapy of cancer (Figure 1). The shell of MHPCNs is consisted of two layers: an inner layer of Fe3O4 and an outer layer of porous carbon buried with fluorescent carbon nanodots. Carbon-based nanomaterials and Fe3O4 nanoparticles,



NIR resonant materials, can convert NIR light into thermal effect for tumor ablation.12,40 The preparation of MHPCNs was performed by a sacrificial template method.41 In this course, the surface of silica nanospheres (SiO2) absorbed ferrocene, and then the mixture was oxidized with H2O2 to obtain SiO2@Fe3O4@C nanoparticles. NH3·H2O was used to etch SiO2 core by hydrothermal process to obtain MHPCNs. Carboxylic groups of MHPCNs were covalently conjugated with cystamine dihydrochloride (MHPCNs-SS), and then the pores of MHPCNs-SS were capped with, a natural, nontoxic, biodegradable and biocompatible polymer, Poly(γ-glutamic acid) (PGA), by covalent interaction (MHPCNs-SS-PGA).42 Folic acid (FA) was covalently conjugated to the MHPCNs-SS-PGA surface (MHPCHs-SS-PGA-FA) to perform targeted therapy.27 Doxorubixin (DOX), an efficient anticancer drug which can treat sorts of human malignancies applied widely in clinic, is selected as a model drug (MHPCNs-SS-PGA-FA/DOX).43 When the nanocarriers turn into cancer cells through endocytosis process, the disulfide bond could be quickly cleaved due to the intracellular high concentration of GSH,15 and then PGA layer detached thus realizing the release of drug. Results of synergetic therapy in vitro and vivo demonstrate that MHPCNs-SS-PGA-FA/DOX has an excellent tumor-suppression capability with low side effect.

Figure 1. Synthetic procedure of stimuli-responsive MHPCNs-based drug delivery system for synergistic photothermal and chemotherapy of tumor.


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2. EXPERIMENTAL SECTION 2.1. Materials. H2O2 (30 %), sodium chloride, disodium hydrogen phosphate, ammonium

hydroxide,

potassium

chloride,

ferrocene,

potassium

phosphate

monobasic, DMSO, tetraethoxysilane and acetone were obtained from Sinopharm Chemical

Reagent

Co.,

Ltd.

(Shanghai,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 4’,6-diamidino-2-phenylindole

(DAPI),

China).

bromide

FA, (MTT),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), DOX, and cystamine dihydrochloride were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). PGA was acquired from Yuanye Biotechnology Co., Ltd. (Shanghai, China). 2.2. Instrumentation. The visualization of MHPCNs and MHPCNs-SS-PGA-FA were performed by employing HITACHI H-7650 transmission electron microscopy (TEM). Energy-dispersive X-ray (EDX) elemental mapping were analysed by utilizing Tecnai G2 F30 S-TWIN TEM. The analysis of zeta potentials and size distributions of nanoparticles was carried out by Malvern Nano-ZS 90 Nanosizer. ASAP2050 system was used to measure the Brunauer-Emmett-Teller (BET) pore size and surface area of samples. The Fourier transform infrared (FTIR) spectra of samples were detected by employing NEXUS670 FTIR spectrometer. Thermogravimetric analysis (TGA) curves were acquired by using the PerkinElmer Instruments (Diamond TG/DTA) in N2 atmosphere with a heating rate of 10 °C/min. X-ray diffraction (XRD) patterns were obtained by employing Rigaku Ultima IV powder diffractometer. Raman spectra were recorded by employing Raman spectrometer (Labram HR800) at 514 nm excitation laser. Magnetic properties of samples were analyzed under an applied magnetic field by using vibrating-sample magnetometer (VSM, HH-15). UV-vis spectra of samples were measured by using UV-vis spectrophotometer (Agilent 8453). 2.3. Synthesis of MHPCNs-SS-PGA-FA. 2.3.1. Preparation of MHPCNs. SiO2 nanospheres with a diameter of 60 nm were prepared according to the Stöber



method.44 The SiO2 nanospheres serve as a template to construct the hollow nanostructures.41 Briefly, 25 mg SiO2 nanospheres and 100 mg ferrocene were dispersed in 30 mL acetone and the mixed solution was sonicated for 20 min. 1.0 mL H2O2 (30 %) was then added into the solution under vigorous stirring for 2 h. After that, the mixture was transferred into a 50-mL Teflon-lined stainless autoclave. The temperature of the autoclave was maintained at 210 °C for 48 h. The resultant SiO2@Fe3O4@C nanoparticles were collected by centrifugation and washed three times with acetone. Afterwards, the nanoparticles were dispersed in 30 mL deionized water and mixed with 15 mL NH3·H2O under stirring for 5 min. The above mixture was again transferred into the teflon-lined stainless autoclave and heated to 150 °C for 6 h. The MHPCNs were obtained by magnetic separation and washed three times with deionized water. 2.3.2. Synthesis of MHPCNs-SS. The activation of the carboxylic acid groups on the MHPCNs was achieved by dissolving 10 mg MHPCNs, 70 mg EDC, and 70 mg NHS in 20 mL H2O under stirring. After 2 h, 100 mg cystamine dihydrochloride was added into the solution and stirred for 24 h. The MHPCNs-SS was obtained by magnetic separation, washed three times with deionized water and dried under vacuum. 2.3.3. Synthesis of MHPCNs-SS-PGA. To activate the carboxylic acid groups on PGA, 100 mg PGA, 700 mg NHS, and 700 mg EDC were dissolved in 50 mL H2O and stirred for 2 h. . Afterwards, 10 mg MHPCNs-SS was added into the solution and stirred for 24 h. The MHPCNs-SS-PGA was obtained by magnetic separation, washed repeatedly with deionized water and dried under vacuum. 2.3.4. Synthesis of MHPCNs-SS-PGA-FA. The carboxylic acid groups on MHPCNs-SS-PGA was activated by dissolving 10 mg MHPCNs-SS-PGA, 70 mg EDC, and 70 mg NHS in 20 mL H2O under stirring for 2 h. 100 mg FA dissolved in 10 mL DMSO was then added and the solution was stirred for 24 h. The MHPCNs-SS-PGA-FA was collected by magnetic separation. The unreacted FA was


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removed by repeatedly washing with deionized water. 2.4. DOX Loading. Before PGA capping, 3 mg MHPCNs-SS was incubated with 30 mL phosphate buffered saline (PBS, pH 7.4) containing 0.3 mg/mL DOX

under

stirring for 24 h. Via centrifugation to remove the unloaded DOX, the MHPCNs-SS/DOX was washed with PBS and dried under vacuum. The MHPCNs-SS with DOX loading was further subject to modifications with PGA and then FA according to the above procedures. The amount of unloaded DOX was measured the supernatant by UV-vis spectrophotometer at 480 nm.31 The DOX loading content in the MHPCNs-SS-PGA-FA was calculated by [(M1-M2)/M3]×100%, where M1 represented the original weight of DOX added, M2 the weight of DOX in the supernatant, and M3 the weight of MHPCNs-SS-PGA-FA/DOX. 2.5. Stimuli-Responsive DOX Release from MHPCNs-SS-PGA-FA/DOX. PBS with different pH values (5.0, 6.8 and 7.4) and GSH concentrations (0-10 mM) were selected as release medium to study the pH- and GSH-responsive release behaviours of

MHPCNs-SS-PGA-FA/DOX.

Briefly,

2

mL

PBS

containing

MHPCNs-SS-PGA-FA/DOX was added to a dialysis tube (MWCO = 14000 Da) immersed in 20 mL release medium under gentle stirring at 37 °C. One mL release medium was collected at the given time intervals and 1 mL fresh medium was supplied to the solution. The collected solution was used to measure the concentration of DOX via UV−vis spectrophotometer at 480 nm. The NIR-responsive DOX release of

MHPCNs-SS-PGA-FA/DOX

was

evaluated

as

follows:

MHPCNs-SS-PGA-FA/DOX was dispersed in 2 mL PBS (pH 5.0) with GSH (10 mM). The mixture was transferred to a dialysis tube (MWCO = 14000 Da) immersed in 20 mL release medium under gentle stirring at 37 °C. At given time intervals, the dialysis tube were irradiated by NIR light (808 nm, 1.5 W/cm2) for 300 s, and then 1 mL release medium was collected and equal volume of fresh medium was added. The collected samples were used to measure the DOX concentration. 2.6. Hemolysis Test and the Shape of Erythrocytes. Erythrocytes were acquired



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by centrifuging 5 mL whole blood with anticoagulation at 3000 rpm for 8 min. Ablution of erythrocytes was used PBS, and then resuspended in PBS at a volume ratio of 1:9. Afterwards, 1.2 mL of MHPCNs-SS-PGA-FA solutions (in PBS) with different concentrations were mixed with 0.3 mL of the attenuated erythrocytes suspension. Positive and negative controls were acquired by mixing 1.2 mL of deionized water and PBS with 0.3 mL of the attenuated erythrocytes suspension, respectively. All mixtures maintained at 37 °C for 3.5 h, and then centrifuged at 3000 rpm for 8 min. Microplate reader (BioTek synergy 2) was used to measure the optical density (OD) of the samples at 541 nm. The calculating equation of hemolysis rate was as follow: hemolysis rate (%) = [(ODsample − ODnegative)/(ODpositive − ODnegative)] × 100%. ODsample, ODpositive, and ODnegative represent the absorption values of the sample, positive control, and negative control, respectively. Light microscopy (Nanjing Jiangnan Novel Optics Co., Ltd, BM2100) was used for observing the shape of erythrocytes. One point two mL of MHPCNs-SS-PGA-FA solutions (in PBS) with different concentrations were incubated with 0.3 mL of the attenuated erythrocytes suspension in tubes for 3.5 h at 37 °C. The mixtures were centrifuged to acquire erythrocytes pellet. Erythrocytes pellet were dispersed in PBS. Dispersion was dropped on glass slides to perform microscopy research. 2.7. Intracellular Drug Release. Intracellular drug release research was performed as follow: HeLa cells were incubated with MHPCNs-SS-PGA-FA/DOX or MHPCNs-SS-PGA-FA (20 µg/mL) for 4 h. After washing with PBS, cells were immobilized with 4% paraformaldehyde. The addition of DAPI was used for nuclei staining. After washing with PBS, cells were observed by using confocal imaging (TI-EA1R, Nikon, Japan). 2.8. Cell Cytotoxicity. MTT assay was used to test cell viability of various samples. In brief, HeLa cells or human umbilical vein endothelial cells (HUVECs) were seeded into 96-well plates at 2 × 104/well. Attaching for 24 h, cells were incubated with different

concentrations

of

MHPCNs-SS-PGA-FA,

DOX,

and

MHPCNs-SS-PGA-FA/DOX for 24 h. Afterwards, each well was added into MTT ACS Paragon Plus Environment

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solution (50 µL, 2 mg/mL). After 4 h, the supernatant was discarded and each well was added into 150 µL DMSO. Microplate reader was used to test the OD at 570 nm. For PTT, some HeLa cells were incubated with MHPCNs-SS-PGA-FA and MHPCNs-SS-PGA-FA/DOX for 1.5 h, and then irradiated with 1.5 W/cm2 NIR light for 5 min. After additional incubation, the cell viability was measured by MTT assay. 2.9. Animal model. All animal operations were carried out in accordance with institutional animal use and care regulations provided by Central South University. Female nude mice (20 g) were inoculated by subcutaneous injection of 2 × 106 cells in 100 µL PBS on the flank of each mouse. 2.10. In Vivo MR Imaging. For imaging test, the tumor-bearing mice were injected with MHPCNs-SS-PGA-FA/DOX (14.26 mg/kg) via the tail vein. T2-weighted images were acquired using the 1T clinical MRI scanner (Bruker Icon, Germany). 2.11. In Vivo Antitumor Efficacy of MHPCNs-SS-PGA-FA/DOX. To estimate the photothermal/chemo-therapy efficacy of MHPCNs-SS-PGA-FA/DOX, the tumor-bearing mice were divided into 5 groups (n = 5, each group). Two groups of mice

were

respectively

injected

with

100

µL MHPCNs-SS-PGA-FA or

MHPCNs-SS-PGA-FA/DOX (2.85 mg/mL, [MHPCNs-SS-PGA-FA]: 10 mg/kg, [DOX]: 4.26 mg/kg). After one day, the tumor regions of mice were irradiated with NIR light (808 nm, 1.5 W/cm2, 5 min). The other three groups of mice were injected with 100 µL of PBS, DOX or MHPCNs-SS-PGA-FA/DOX ([MHPCNs-SS-PGA-FA]: 10 mg/kg, [DOX]: 4.26 mg/kg), but without receiving with NIR light as controls. Tumor sizes and body weight were recorded every 2 days during therapy period. The calculating formula of the tumor volume was as follow: width2 × length/2. After completion of the treatment, the mice were sacrificed and dissected to collect tumors and major organs. Tumors and organs were embedded with paraformaldehyde, stained with hematoxylin and eosin (H&E) for histological analysis. 2.12. In Vivo Biodistribution. The biodistribution of nanoparticles in



tumor-bearing mice was assessed by intravenous injection of MHPCNs-SS-PGA-FA (10 mg/kg). At different time points (1, 12, 24, 48 h), the mice were euthanized. The heart, liver, spleen, lung, kidney and tumor were extracted and mixed with aqua regia (Vnitric acid: Vhydrochloric acid = 1:3) for 24 h to get clear solution and then characterized by inductively coupled plasma atomic emission spectroscopy (ICP-AES) for Fe content analyze. 2.13. Statistical Analysis. The significance of experimental results was analyzed by using the analysis of variance (ANOVA) test. 0.05 was chosen as the significance level, probabilities as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) were marked in each figure. 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Samples. The SiO2@Fe3O4@C with a core–shell-shell structure is shown in Figure S1. The SiO2 core, Fe3O4 layer and carbon layer were clearly observed. SiO2 core, as a template, was removed to construct the hollow nanostructure. TEM images (Figure 2a,b) revealed that MHPCNs and MHPCNs-SS-PGA-FA had a uniform morphology and good dispersibility with particle sizes of approximate 114 nm and 127 nm. For biomaterials with nanoscale (60-400 nm), tumor tissue present enhanced permeability and retention (EPR) effect, which increases the accumulation of nanomaterials at tumor region.45 The hollow and porous nanostructure of MHPCNs could be clearly observed. As shown in Figure S2, some nanodots (< 10 nm) buried in the carbon shell of the MHPCNs was clearly observed, and the lattice spacing of nanodots was about 0.21 nm which accorded with the (100) lattice planes of graphitic (sp2) carbon,32,46 confirming that the shell of MHPCNs contained carbon nanodots. The size distributions of MHPCNs and MHPCNs-SS-PGA-FA are shown in Figure 2c. After MHPCNs were modified by polymer, their hydrodynamic diameter increased, which proved the existence of modified layer on the MHPCNs surface. The hydrodynamic size changes of MHPCNs-SS-PGA-FA in PBS at pH 5.0, 6.8 and 7.4 in a week were shown in Figure


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S3. No significant size changes were observed indicating that MHPCNs-SS-PGA-FA possess good stability. The elemental mapping of MHPCNs-SS-PGA-FA (Figure 2d) indicated the existence and uniform distribution of C, Fe, O, N and S elements. In XRD pattern of MHPCNs (Figure S4), the position of the main peaks confirmed that the existence of Fe3O4.41 The magnetization saturation values of MHPCNs and MHPCNs-SS-PGA-FA were 48.6 and 19.9 emu/g, respectively (Figure S5). The presence of coating layer lead to the decrease of values.

Figure 2. TEM images of MHPCNs (a) and MHPCNs-SS-PGA-FA (b). (c) Size distributions of MHPCNs and MHPCNs-SS-PGA-FA. (d) EDX elemental mapping images of C, O, Fe, N and S elements in MHPCNs-SS-PGA-FA. Figure 3a shows the FTIR spectra of MHPCNs and MHPCNs-SS. MHPCNs and MHPCNs-SS all presented an obvious absorption peak at 3395 cm-1 that was due to the presence of -OH group.47 MHPCNs showed a characteristic absorption peak of carboxylic acid groups at 1710 cm-1.48 At 589 cm-1 a peak was observed due to the Fe-O vibration in Fe3O4 of MHPCNs.49 After modified by cystamine dihydrochloride, the new absorption peak observed at 1630 cm-1 was attributed to the amide carbonyl vibration.48 In addition, the decreased intensity of the absorption peak at 1710 cm-1, indicating the most of carboxylic acid groups had reaction with cystamine dihydrochloride. The Raman spectra of MHPCNs (Figure 3b) showed two characteristic peaks of carbon material, G-band (1576 cm−1) and D-band (1350 cm−1).50 A new peak at 591 cm-1 was observed in the Raman spectra of MHPCNs-SS due to S–S vibrational features.51 The results of Raman was consistent with FTIR



spectra and both proved the successful preparation of MHPCNs-SS.

Figure 3. (a) FTIR and (b) Raman spectra of MHPCNs and MHPCNs-SS. (c) TGA curves and (d) N2 adsorption-desorption isotherms of MHPCNs, MHPCNs-SS, MHPCNs-SS-PGA and MHPCNs-SS-PGA-FA. The inset in panel (d) shows the surface area of the materials. The TGA of samples are shown in Figure 3c. The 46.35% weight loss of original MHPCNs was due to the evaporation of water and the degradation of carbon. The weight loss of MHPCNs-SS increased to 50.28%, which proved the existence of modified-coating. After blocking with PGA, 63.23% weight loss occurred. The additional weight loss was attributed to the presence of PGA. MHPCNs-SS-PGA-FA yielded the highest weight loss (66.52%). TGA data confirmed the successful modification of MHPCNs after each stage. The results of zeta potential analysis are shown in Figure S6 which also proved the successful surface functionalization of MHPCNs. The MHPCNs and MHPCNs-SS owned zeta potential value of -29.9 mV and -16.8 mV, respectively. Amino groups of cystamine dihydrochloride increased the positive zeta potential value of MHPCNs. The zeta potential value of MHPCNs-SS-PGA was decreased to -25.1 mV due to the negatively charged PGA


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indicating the success in MHPCNs-SS coated by PGA. MHPCNs-SS-PGA-FA possess an increase of negative zeta potential value (-29.7 mV), which implied that the surface conjugation of negatively charged FA was successful. Figure 3d shows the N2 adsorption–desorption isotherms of MHPCNs, MHPCNs-SS, MHPCNs-SS-PGA and MHPCNs-SS-PGA-FA. The isotherms of MHPCNs and MHPCNs-SS were type-IV isotherms, which suggested the existence of mesopores. The surface area of MHPCNs suffered a decrease from 146.6 to 103.7 m2 g-1 after coating by cystamine dihydrochloride. However, MHPCNs-SS still had enough volume for drug encapsulation with high drug loading capacity. After MHPCNs-SS capped by PGA, the surface area dramatically decreased to 32.8 m2 g-1, and the surface area of MHPCNs-SS-PGA-FA was 21.6 m2 g-1. In addition, the absence of narrow sharp peak in the pore size distribution of MHPCNs-SS-PGA-FA (Figure S7) suggests that the pores were blocked completely. Therefore, the process of drug encapsulation should be carried out before the PGA capping. The photothermal performance of MHPCNs-SS-PGA-FA is shown in Figure S8 and obvious concentration-dependent temperature elevation was observed. The UV-vis spectra of MHPCNs-SS-PGA-FA suspension with concentration of 0.15 mg/mL was shown in Figure S9 and the absorption observed around 808 nm indicated the potential PTT capability. The photothermal conversion efficiency (η), a key factor to evaluate the photothermal effect, plays a vital role in the PTT and the calculation formula is based on previous literature

(See

calculation

formula

in

supporting

information).52

MHPCNs-SS-PGA-FA suspension (0.15 mg/mL) was exposed in NIR laser (808 nm, 1.5 W/cm2). When a steady-state temperature was achieved, the light source was turned off. The mixture was naturally cooled down to ambient temperature and the temperature variation curve was shown in Figure S10. τ can be determined by linear relationship between cooling time and –ln(θ) (Figure S11) and the photothermal conversion efficiency of MHPCNs-SS-PGA-FA was calculated to be 36%. 3.2. The Shape of Erythrocytes and Hemolysis Rate. When nanoparticles are chosen as biological application in vivo, evaluating hemocompatibility of samples is



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very important. When blood contacts incompatible materials, erythrocytes will suffer from aggregation, aberration and hemolysis. These factors will cause some harmful influence, such as inflammatory reaction and thrombus.42 The shape of erythrocytes observation and hemolysis rate measurement are often used to assess the hemocompatibility of samples. The untreated erythrocytes (Figure 4a, negative control) had a normal biconcave shape and erythrocytes in deionized water (Figure S12,

positive

control)

suffered

destruction.

When

contacted

with

MHPCNs-SS-PGA-FA at different concentrations, the erythrocytes still remain at a normal biconcave shape (Figure 4b-e). MHPCNs-SS-PGA-FA at different concentrations all possessed a lower hemolysis rate (Figure 4f). The hemolysis rate of biomaterials should be under 5%, which is considered as a safety standard.53 Hence, the hemolytic activity of MHPCNs-SS-PGA-FA was inappreciable, indicating that it could be used via intravenous injection.

Figure 4. Erythrocyte shapes after treatment with MHPCNs-SS-PGA-FA with varying concentrations: (a) 0 µg mL-1 (negative control), (b) 50 µg mL-1, (c) 100 µg mL-1, (d) 200 µg mL-1, and (e) 400 µg mL-1. (f) Hemolysis rate in the case of MHPCNs-SS-PGA-FA with varying concentrations. 3.3. Drug Release Study. The DOX loading capacity of MHPCNs-SS-PGA-FA was 29.9%. Two factors made MHPCNs-SS-PGA-FA have high drug loading capacity.


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First, the aromatic DOX had a strong interaction with the graphite carbon-based materials via supramolecular π-stacking.12 Second, enough space was offered for drug storage due to the porous and hollow structure of materials. The negative charges of PGA had abundant carboxyl groups which strongly electrostatic interacted with an amine-carrying structure drug, DOX. Therefore, PGA layer could hamper the drug leakage during the circulation process. The UV–vis absorption spectra of MHPCNs-SS-PGA-FA/DOX showed a characteristic UV-vis absorption peak of DOX at 480 nm (Figure 5a), indicating that drug encapsulation was successful. And the zeta potential value of MHPCNs-SS-PGA-FA had a positive-charged increase after DOX loading (Figure S6), which also proved the successful loading of the DOX into the MHPCNs-SS-PGA-FA. To examine the internal stimulus-response DOX release, the MHPCNs-SS-PGA-FA/DOX was put in PBS (pH = 5.0, 6.8 and 7.4) with various GSH concentrations. The drug release profiles of MHPCNs-SS-PGA-FA/DOX are shown in Figure 5b, c and d. When GSH concentration was 0 mM (pH = 5), the release rates were quite slow. Only 18.7% DOX was released. With the increased GSH concentration, the accelerated release ratios were observed due to the rupture of disulfide bond caused by GSH which leaded to the detachment of PGA layer making the pores unblocked. When GSH concentration was at 10 mM, DOX release amount could reach 63.8% at pH 5.0. The drug release behaviors were then tested in PBS (pH = 6.8), the deceleration of DOX release was observed. Although the release rate was much slower than in PBS (pH = 7.4), the DOX release rate also relied on the GSH concentration. Both the two factors affected the pH-responsive release behaviors.12,54 First,

DOX

molecules

electrostatic

interacted

with

hydroxy

groups

of

MHPCNs-SS-PGA-FA. When the pH turned to acidic, the electrostatic interactions was weakened due to the protonation of hydroxy groups. Second, DOX presented enhanced hydrophilicity in acidic condition. Compared to internal stimuli, external stimuli was also an accurate and easy technology utilized in nanodelivery system. As shown in Figure 5e, MHPCNs-SS-PGA-FA/DOX was incubated in release medium at pH = 5.0 with 10 mM GSH. The mixture was exposed with several pulses of NIR laser irradiation (1.5 W/cm2, every pulse for 5 min). During NIR light irradiation ACS Paragon Plus Environment


Page 16 of 32

process, significant drug burst release phenomenon could be observed. When NIR light was turned off, DOX releasing rate slowed down. This burst release phenomenon was due to the local thermal energy produced by carbon and Fe3O4 under NIR

light

irradiation, which leaded to the dissociation of DOX

from

MHPCNs-SS-PGA-FA.55 The GSH-, pH- and NIR-responsive nanocarrier had a great significance in practical biomedical field. It is well known that pH value in tumor intracellular environment is 5.0−5.5, and the concentration of GSH inside the cancer cells is much higher than that outside cancer cells. In normal physiological conditions, avoiding drug leakage can reduce the adverse effects to normal organs. Compared with mono or dual stimuli-responsive DDSs, the triple stimuli-responsive DDSs could realize a more effective and complete drug release. The accumulation of DOX in tumor tissues could enhance the chemotherapy efficacy.

Figure

5.

(a)

UV-vis

spectra

of

DOX,

MHPCNs-SS-PGA-FA

and

MHPCNs-SS-PGA-FA/DOX. Release curves of MHPCNs-SS-PGA-FA/DOX in PBS at pH=5.0 (b), pH=6.8 (c) and pH=7.4 (d) under different GSH concentrations. (e) NIR-responsive release curve of MHPCNs-SS-PGA-FA/DOX in PBS (pH=5.4) with 10 mM GSH. 3.4. Cell Experiments. The cellular uptake behaviours of MHPCNs-SS-PGA-FA and MHPCNs-SS-PGA-FA/DOX inside HeLa cells could be guided by the


Page 17 of 32 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


fluorescent carbon nanodots. Red fluorescence was from carbon nanodots and DOX. Cell nucleus (blue) were stained by DAPI. Confocal laser scanning microscopy images (Figure 6a) showed that MHPCNs-SS-PGA-FA was mainly distributed in cytoplasm under irradiation with laser wavelength at 543 nm and the fluorescence signal was not observed in cell nuclei due to nuclear membrane buried with numerous nuclear pore complexes (9−40 nm) which could hamper large-sized nanoparticles transporting.45 However, after loading DOX, uniform bright red fluorescence in the cytoplasm and nuclei could be observed which was due to the release of fluorescent DOX entering into the cell nuclei suggesting that the uniform distributions of drug molecules were in the nuclei and cytoplasm. The photothermal/chemo-therapy in vitro of MHPCNs-SS-PGA-FA was studied by MTT assay. The cytotoxicity of pure DOX, MHPCNs-SS-PGA-FA/DOX at different DOX concentrations and the corresponding amount of MHPCNs-SS-PGA-FA were shown in Figure 6b. MHPCNs-SS-PGA-FA at a high concentration remained high cell viabilities (> 95%), suggesting that MHPCNs-SS-PGA-FA itself had mild cytotoxicity to cancer cells. However, it was observed that MHPCNs-SS-PGA-FA treated cells under NIR light showed obvious viabilities of reductions, indicating that MHPCNs-SS-PGA-FA had a good PTT ability. Meanwhile, cell growth inhibition effect could be also observed when cells were incubated with DOX or MHPCNs-SS-PGA-FA/DOX. Cells were incubated with MHPCNs-SS-PGA-FA/DOX under NIR light exposure which had the lowest cell viabilities. Therefore, it could be inferred that the combination of photothermal and chemotherapy could possess an excellent therapeutic effect to cancer cells. In order to investigate the cytotoxicity of MHPCNs-SS-PGA-FA/DOX in normal cell line, the HUVECs was selected to evaluate the in vitro cytotoxicity. As shown in Figure S13, no significant cytotoxicity was observed when MHPCNs-SS-PGA-FA was cultured with HUVECs. Meanwhile, the viability of MHPCNs-SS-PGA-FA/DOX group in HUVECs was higher than free DOX. These results indicated that PGA shell could restrain the leakage of DOX and reduce adverse side effects in the normal cell. ACS Paragon Plus Environment


Page 18 of 32

Figure 6. (a) Confocal fluorescence images of HeLa cells treated with MHPCNs-SS-PGA-FA or MHPCNs-SS-PGA-FA/DOX for 4 h. Scale bar: 10 µm. (b) Cell

viability

of

HeLa

cells

treated

MHPCNs-SS-PGA-FA/DOX,

with

MHPCNs-SS-PGA-FA,

DOX,

MHPCNs-SS-PGA-FA+Laser

and

MHPCNs-SS-PGA-FA/DOX+Laser at different concentrations of DOX and the corresponding amount of drug-free nanocapsules. HeLa cells treated with MHPCNs-SS-PGA-FA was used as control (* p < 0.05, ** p < 0.01, *** p < 0.001). 3.5.

In

Vivo

MR

Imaging.

The

tumor

targeting

ability

of

MHPCNs-SS-PGA-FA/DOX in animal was studied by using MR imaging. MHPCNs-SS-PGA-FA/DOX dispersions at different Fe concentrations were tested their property as MR contrast agents. Figure 7a shows a gradual darkening effect with the dependence of Fe concentration, demonstrating its possible bioapplication in T2-weighted MR imaging. The T2 weighted MR images of the tumor bearing mice before injection and after injection at different points in time (1, 2, 4, 12 and 24 h) were shown in Figure 7b. The tumor region presented a significant darken appearance from 1 to 24 h, indicating that nanoparticles were accumulated in tumor region and possessed the ability for tumor targeting.


Page 19 of 32 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


Figure 7. (a) T2-weighted MR images and T2 relaxation rates (r2) in the case MHPCNs-SS-PGA-FA/DOX at different concentrations. (b) In vivo T2-weighted MR images of a tumor-bearing mouse taken before and after injection at different points in time (1, 2, 4, 12 and 24 h) of MHPCNs-SS-PGA-FA/DOX (14.26 mg/kg). The tumor regions are pointed out in the red circle. 3.6. In Vivo Anticancer Efficiency. The ability of MHPCNs-SS-PGA-FA/DOX nanoplatform to treat the tumor via PTT and chemotherapy was evaluated in HeLa tumor-bearing

mice.

Mice

injected

with

MHPCNs-SS-PGA-FA

and

MHPCNs-SS-PGA-FA/DOX, the temperature in their tumor region increased by about 20 °C after NIR light irradiation (Figure 8a). By comparison, mice treated with PBS, tumor temperature did not show obvious increase under the same NIR light exposure. These results suggested that light energy could be transferred into thermal energy efficiently in the body by MHPCNs-SS-PGA-FA. Figure 8b,c showed the therapeutic

effects

of

different

groups,

MHPCNs-SS-PGA-FA/DOX

and

MHPCNs-SS-PGA-FA+Laser groups had a better antitumous effect in comparison to free DOX group, which is due to the targeting ability of FA and EPR effect enhancing tumor tissues uptake of nanoparticles. In MHPCNs-SS-PGA-FA/DOX+Laser group, the tumor size was the smallest, which indicating that the targeted synergistic photothermal/chemo-therapy had the most excellent therapeutic effect. H&E staining assay was also used to evaluate therapeutic effects (Figure 8d). The majority of cancer cells presented serious apoptosis and necrosis which could be observed in



Page 20 of 32

MHPCNs-SS-PGA-FA/DOX+Laser group. 3.7.

In

Vivo

Biodistribution

and

Toxicity.

The

biodistribution

of

MHPCNs-SS-PGA-FA was investigated through detecting Fe content in primary organs and tumor by ICP-AES. As shown in Figure S14, Fe element primarily existed in the spleen, lung, and liver and the gradual decrease of Fe concentration could be observed in each viscera over time demonstrating the time-dependent clearance of MHPCNs-SS-PGA-FA from major viscera. Meanwhile, the Fe concentration in the tumor had an enhancement from 1 to 24 h due to the targeted FA and EPR effect indicating the good accumulation of MHPCNs-SS-PGA-FA in tumor. Figure S15 shows the variations of weights in different groups during the therapeutic process. Weights of DOX group presented obvious declines, because of the toxic and side effect of free DOX. And there was no apparent weight decrease in other groups. The histological analysis of the major viscera (heart, liver, spleen, lung, and kidney, Figure S16) indicated that no apparent damages or changes of viscera could be observed between MHPCNs-SS-PGA-FA/DOX+Laser group and PBS group. These results demonstrated that MHPCNs-SS-PGA-FA/DOX+Laser could perform effectively in cancer treatment with little side effect.


Page 21 of 32 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


Figure 8. (a) Infrared thermal images of tumor-bearing mice treated with PBS, MHPCNs-SS-PGA-FA and MHPCNs-SS-PGA-FA/DOX via irradiation with 808 nm NIR light for different time periods. (b) Photographs of tumors. (c) Tumor-volume variation curves. PBS group was used as control (** p < 0.01, *** p < 0.001). (d) H&E staining of the tumor. 4. Conclusion In summary, a novel GSH-, pH- and NIR-responsive targeted PGA-gatekeeper nanocarrier based on magnetic hollow and porous carbon-based nanoparticles was successfully

designed

and

fabricated

for

in

vivo

cancer

therapy.

MHPCNs-SS-PGA-FA possessed a high drug loading capacity and avoided the leakage of DOX during blood circulation. Exploiting intrinsic physical nature of Fe3O4, MR imaging was performed on tumor-bearing mice, demonstrating efficient accumulations of nanoparticles at tumor sites via the EPR effect and targeting function.

Tumor

growth

was

effectively

inhibited

via

synergistic

photothermal/chemo-therapy and no apparent toxic and side effect to the treated



animals was observed. Finally, MHPCNs-SS-PGA-FA/DOX could be a promising nanoplatform for MR imaging-guided highly effective treatment. AUTHOR INFORMATION Corresponding Author *Email: [email protected] (J.Z.), [email protected] (X.Y.) ACKNOWLEDGMENTS The authors acknowledge financial support from the Priority Academic Program Development of Jiangsu Higher Education Institutions and Open Program of Jiangsu Key Laboratory of 3D Printing Equipment and Manufacturing (BM 2013006). This work was also supported by the National Natural Science Foundation of China (No. 21705166). REFERENCE (1) Nagai, Y.; Miyazawa, H.; Huqun; Tanaka, T.; Udagawa, K.; Kato, M.; Fukuyama, S.; Yokote, A.; Kobayashi, K.; Kanazawa, M.; Hagiwara, K. Genetic Heterogeneity of the Epidermal Growth Factor Receptor in Non–small Cell Lung Cancer Cell Lines Revealed by a Rapid and Sensitive Detection System, the Peptide Nucleic Acid-Locked Nucleic Acid PCR Clamp. Cancer Res. 2005, 65, 7276-7282. (2) Wu, P.; Deng, D.; Gao, J.; Cai, C. Tube-like gold sphere-attapulgite nanocomposites with a high photothermal conversion ability in the near-infrared region for enhanced cancer photothermal therapy. ACS Appl. Mater. Interfaces, 2016, 8, 10243-10252. (3) Fan, W.; Lu, N.; Huang, P.; Liu, Y.; Yang, Z.; Wang, S.; Yu,G.; Liu, Y.; Hu,J.; He, Q.; Qu, J.; Wang, T.; Chen, X. Glucose-Responsive Sequential Generation of Hydrogen Peroxide and Nitric Oxide for Synergistic Cancer Starving-Like/Gas Therapy. Angew. Chem., Int. Ed. 2017, 56, 1229-1233.


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Table of Contents Triple Stimuli-Responsive Magnetic Hollow Porous Carbon-Based Nano-Drug Delivery System for Magnetic Resonance Imaging-Guided Synergistic Photothermal/Chemo-Therapy of Cancer Fan Wu,†‡§ Hanwen Lu,† Dong Liang,† Yaliang Huang,† Yonghong Xia,† Yuqing Hu,† Shengqiang Hu,† Jianxiu Wang,† Xinyao Yi*,†, and Jun Zhang*,‡§ †

College of Chemistry and Chemical Engineering, Central South University,

Changsha 410083, PR China ‡

Jiangsu Collaborative Innovation Center for Biological Functional Materials, College

of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, PR China §

Jiangsu Key Laboratory of Biofunctional Materials, Jiangsu Engineering Research

Center for Biomedical Function Materials, Nanjing 210023, PR China



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Poly(γ-glutamic acid) was used to cap the pores of porous carbon nanoparticles. Anticancer drug could accomplish efficient release in the targeted cancer cells. Tumor growth

was

effectively

inhibited

via

MR

imaging-guided

photothermal/chemo-therapy with little side effect.


synergistic

Triple Stimuli-Responsive Magnetic Hollow Porous Carbon-Based

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