Photothermally Enhanced Chemotherapy Delivered by Graphene

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Photothermally Enhanced Chemotherapy Delivered by Graphene Oxide-based Multi-responsive Nanogels Weili Zhang, Shulun Ai, Ping Ji, Jiyan Liu, Yulin Li, Yuhong Zhang, and Peixin He ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00611 • Publication Date (Web): 14 Dec 2018 Downloaded from http://pubs.acs.org on December 18, 2018

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ACS Applied Bio Materials

Photothermally Enhanced Chemotherapy Delivered by Graphene Oxide-based Multi-responsive Nanogels Weili Zhang,a,d† Shulun Ai,b† Ping Ji,a Jiyan Liu,c Yulin Li,a Yuhong Zhanga*, and Peixin Hea* a Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Key Laboratory for the Synthesis and Application of Organic Functional Molecules, Ministry of Education, College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China b Department of Chemistry, Wuhan University, Wuhan 430072, China c Key Laboratory of Optoelectronic Chemical Materials and Devices, Ministry of Education, Jianghan University, Wuhan 430056, China d Shangqiu Polytechnic, Shangqiu 476000, China

E-mail:[email protected] (Yuhong Zhang); [email protected] (Peixin He)

† The two authors contributed equally to this work.

ACS Paragon Plus Environment

ACS Applied Bio Materials 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

KEYWORDS:

Graphene

oxide,

Photothermal

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therapy,

Synergistic

effects,

Nanotechnology, Drug delivery ABSTRACT

In order to develop multifunctional anticancer nanomedicines, photothermal nanogels with multi-stimulative properties are fabricated by hybridizing graphene oxide (GO) with poly (N-isopropylacrylamide, PNIPAM) matrix. This technique allows for easy monomer-intercalation between GO sheets, followed by in situ polymerization to promote GO exfoliation as nanoplatelets inside emulsified PNIPAM nanodrops, followed by fixation using a disulfide-containing crosslinker. The resulting nanogels own significantly-improved colloidal stability and biocompatibility as compared to native GO. They can effectively encapsulate an anticancer drug, and accelerate its release under conditions mimicking acidic/reducible solid tumor and intracellular microenvironments. Drug delivery can be further enhanced via remote photothermal treatment. The local photothermal effect and drug release smartness make the nanogels as an ideal nanoplatform for synergistic anticancer therapy upon their arrival at tumor tissue and/or inside cancer cells. Introduction Although several kinds of small chemoptherapeutics (e.g., doxorubicin) has been widely used to treat cancer, the low therapeutic efficacy due to multidrug resistance usually requires injection of large quantities of drugs,1,2 causing toxicity to the normal tissues.3,4 Nanotechnology has been creating great promise to develop nanocarriers (e.g., dendrimers, micelles, nanogels, inorganic nanoparticles and et al) as a therapeutic delivery platform for cancer treatment.5-7 Recently, different kinds of liposomal nanoformulations, such as PEGylated liposomal doxorubicin (DOXil), Genexol-PM and nanoparticle albuminbound paclitaxel (Abraxane) have been employed to administer chemotherapeutic anticancer agents, which can decrease the drug toxicity and prolong its pharmacokinetics.8,9 However, most nanoformulations are non-biodegradable, limiting their access to tumor blood vessels and tumor depth


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through the collagen matrix that passes through the interstitial space.10,11 Furthermore, the current nanoformulations mostly lack active drug delivery property, which led to an ineffective drug release capacity inside the tumor and limited intracellular drug accumulation at a required therapeutic concentration, resulting in poor anti-tumor effects.12,13 Accordingly, it is necessary to design a stimulus-responsive nano-carrier to promote the release of anti-cancer drugs, upon certain pathological triggers (pH, redox conditions) of tumors and/or exogenous stimuli (temperature, electric/magnetic field, light) preferentially in a remotely-manipulated approach.14,15 Phototherapy is a kind of noninvasive medical treatment where cancers can be thermally killed through local introduction of light after nanocarrier arrival at tumor site. This approach can increase the target specificity of anti-cancer drugs and reduce their side-effects.16-18 Among the various photothermal agents, Au is the most popular one owing to their high photothermal efficiency. Therefore, a considerable number of works has been done on gold nanorod19,20. Nowadays, graphene, as a two-dimensional carbon nano-material with polyaromatic surface structure, can effectively convert infrared light energy into heat energy, which makes it as a good candidate for photothermal therapy (PTT).21 It is widely used in the field of antibacterial and anticancer. For example, an “on demand” drug delivery platform involved graphene-mesoporous silica nanosheet (GS) was constructed in Qu’s team, which exhibited extraordinary antibacterial effect.22 As a derivative of graphene, the abundance and low cost of graphene oxide (GO) make it attractive in large-scale applications. Meanwhile, GO obtained by partial oxidation of graphene has strong polarity to improve its drug loading ability through the electrostatic interaction with some anti-cancer drugs. Unfortunately, graphene oxide, as a kind of inorganic materials, is easy to accumulate in small amounts during intravenous injection due to the lack of colloid stability.23 Too strong complexation interactions between native GO and drug may decrease its drug release efficiency.24,25 In this sense, hybridization of GO into hydrophilic nanogels may improve its colloidal stability, where the nanogel flexibility enables a rapid response to external stimuli.26,27 Furthermore, the



similarity of nanogels to soft tissues/cells enable their better tissue penetration,28 and more efficient cell uptake ability.29 Herein, we develop a facile approach to developing biodegradable GO-hybridized nanogels (PG) with good therapeutic drug release

controllability

by

encapsulating

GO

nanoparticles

into

poly(N-

isopropylacrylamide) (PNIPAM, a thermosensitive polymer)-based matrix via in situ polymerizing

N-isopropylacrylamide

(NIPAM)

in

the

presence

of

N,N’-bis(acryloyl)cystamine (BAC, a reducible crosslinker) and sodium dodecyl sulfate (SDS, an emulsifier) (Scheme 1).30

SDS

Crosslinked by BAC

S-S

SS

In situ polymerization

S-S

Emulsification

S-S

DOX Loading PG

S-S

S-S

GO: BAC:

NIPAM:

S-S

PNIPAM: S-S

or DOX:

S-S

PGD

SS

NIR Laser S-S

S-S S-S

S-S

S-S

S-S S-S

S-S

S-S

SS

NIR Laser-sensitivity

S-S

Temperature-sensitivity

S-S S-S S-S S-S

S-S S-S S-S S-S

SS

Nucleus

SH

SH

S-S

Cytoplasm

SH

S-S

SH

S-S

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

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pH-sensitivity

Redox-sensitivity

Scheme 1. Schematic representation on how PG nanogels can be prepared for anticancer drug delivery.

Experimental Materials. N-isopropylacrylamide (NIPAM) of analytical grade was bought from Tokyo Chemical Industry Co., Ltd. Graphene oxide (GO) was acquired from Hengqiu Technology Co., Ltd. N,N’-bis(acryloyl)cystamine (BAC) and dimethylsulfoxide


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(DMSO) were supplied by Sigma-Aldrich. Potassium persulfate (KPS), sodium bisulfite (NaHSO3) and sodium dodecyl sulfate (SDS) were obtained from Sinopharm Chemical Reagent Co.,Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) and glutathione (GSH) were obtained from Amresco. Hoechst 33258 was purchased from Invirogen. Doxorubicin hydrochloride (DOX) was bought from Zibo Ocean International Trade Co., Ltd. NIPAM was recrystallized using n-hexane as solvent. And KPS was recrystallized using distilled water as a solvent under room temperature. In addition, other reagents were all used as received without further purification. Human cervical carcinoma cell line HeLa was purchased from China Center for Typical Culture Collection (Wuhan, China). HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) which was supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin and 2 mg/mL NaHCO3 and then incubated in a humidified atmosphere (37 °C, 5% CO2). Preparation of PNIPAM nanogels (P) and GO-hybridized nanogels (PG). 3.26 mL of aqueous dispersion of GO (0.5 mg/mL) was blended with 50 mL of aqueous solution containing NIPAM (monomer, 0.0648 M), BAC (cross-linker, 0.0016 M), and SDS (emulsifier, 0.0073 M) under vigorously stirring for 0.5 h. The mixture was deoxygenated by nitrogen purge for 0.5 h at 60°C, followed by the addition of KPS (initiator, 0.0200 M) and NaHSO3 (accelerant, 0.0259 M) under stirring. After 6 h reaction, the mixture was purified through dialysis against deionized water employing a dialysis bag (MWCO: 8,000-12,000 Da, Biosharp) for 3 days, followed by lyophilization to obtain PG nanogels. The PNIPAM nanogels (P) was prepared under similar procedures in the absence of GO. Characterizations. Transmission electron microscopy (TEM) was used to observe the morphology. Measurements were performed using JEM-100CXII TEM at an acceleration voltage of 100 kV. The TEM samples (0.5mg/mL) were dispersed in ultrapure water in the presence or absence of 5 mM GSH for 10 min at 37 °C. The water droplets of the samples were then dripped onto a 400 mesh copper grid, and then air-dried before testing. Scanning electron microscopy (SEM) images were



performed on a Hitachi S-5200 SEM at an acceleration voltage of 8 kV. The PG samples (0.5mg/mL) were dispersed in ultrapure water in the presence or absence of 5 mM GSH for 10 min at 37 °C. The GO samples (0.5mg/mL) were dispersed in ultrapure water at 37 °C. SEM samples were prepared by coating the dried samples with gold film. Ultraviolet-visible (UV-Vis) spectra were carried out by a UV-Vis spectrometer (Lambda 35 CA, Perkin Elmer) at a wavelength of 400 - 800 nm with a 1 cm quartz cuvette. X-ray diffraction (XRD) patterns were performed by a X-ray diffractometer (Bruker D8) with nickel-filtered Cu Kα radiation (40 kV, 40 mA) in scan area from 5-25˚ at a scan speed of 5˚/min. The hydraulic radius and the zeta potential of the samples were conducted on Malvern Zetasizer Nano ZS. The samples were dispersed in deionized water (0.5 mg/mL), followed by 10 min sonication before measurement. The efficiency of photothermal conversion was investigated by a NIR diode laser (808 nm, 2 W/cm2) (Hi-Tech Optoelectronics) for 7 min irradiation, and the change of temperature was monitored using a digital datatrack (Apresys). The concentration of GO in both two samples (GO and PG) was 250 μg/mL in the photothermal test. Drug loading study. For drug loading, 50 mg GO, and the P and PG nanogels in 5 mL ultrapure water were mixed with 1 mL of DOX aqueous solution (2 mg/mL) under stirring at room temperature overnight. The free drug was removed through dialysis (MWCO: 8,000-14,000 Da) against deionized water at room temperature overnight. After dialysis, the solution containing the free drugs was analyzed by spectrophotometry at 490 nm, and the encapsulation efficiency (EE) of DOX was determined indirectly. After dialysis and lyophilization, the PNIPAM/DOX (PD) and PG/DOX (PGD) nanogels was obtained. The nanogels were then stored at 4°C for subsequent studies to use. All the drug loading process was performed under dark. In vitro drug release study. The drug release behaviors of the samples were performed by dialysis. Firstly, empty the aqueous solution of 2 mg of DOX-loaded nanogels into 1mL of ultrapure water into a dialysis bag (MWCO: 8000-12.000 Da). Dialysis was then performed against 30 mL PBS solution in the presence or absence


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of NIR light irradiation, under different pH values (7.4, 6.5 or 5.0), at a temperature of 25, 37 or 42°C, at different GSH concentrations (0, 5 mM). At different intervals, take out 3 mL of release media for testing by spectrophotometry at 490 nm, simultaneously replenish 3 mL of new PBS buffer. NIR induced release was performed in the similar way, the only difference was that PGD nanogels under external environment (≈15°C, pH 7.4) against 8 mL PBS buffer was illuminated by an 808 nm NIR light (2 W/cm2) for 3 min at different intervals and the control group without laser treatment was operated in a similar approach. The cumulative release (Cr) of DOX against time was obtained according to the equation: Cr = 100 * Wt/Wtot

(1)

where Wt and Wtot are the total amount of free DOX in the solution at time t and the total amount of loaded DOX existed in the DOX-loaded samples employed for drug release test. In vitro cytotoxicity assay. HeLa cells were seeded in a 96-well culture plate (105 cells/well) with 100 μL of media each well. After incubation in humidified air for 24 h, cells were cultured with DOX-free nanogel solution of different concentrations (31.25 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL) for 12 h. After that, MTT solution (20 μL, 5 mg/mL) was added to each well, followed by incubation for another 4 h. After treatments, the medium was removed, followed by adding 150 μL of DMSO to dissolve the formazan crystals produced by living cells. Finally, the absorbance of formazan was measured by a microplate reader (Bio-rad 550) at 570 nm to determine the optical density (OD) value. Cell Viability % = ODeg/ODcg * 100%

(2)

where ODeg and ODcg are OD values for experimental group and control group, respectively. The cytotoxicity assays of free DOX and DOX-loaded nanogels (PD and PGD) were conducted in a similar way. In addition, in vitro cytotoxicity against HeLa cells cultured with P+laser, PG+laser, PD+laser and PGD+laser were evaluated under NIR light treatment. For NIR light treatment, cells were incubated with the samples



for 2 h, followed by exposure to NIR light for 2 min (808 nm, 2 W/cm2). In the experiments, the DOX concentrations in the samples were 0 mM, 1 mM, 3 mM, 5 mM, 10 mM and 20 mM, respectively. All the data above were produced as mean standard deviation (SD) based on 3 independent measurements. Confocal laser scanning microscope assessments. HeLa cells were cultured in 2 mL of DMEM in a 4-well culture dish (105 cells per well) overnight at 37 °C. DMEM solution was replaced with 2 mL fresh DMEM solutions of PGD nanogels containing DOX concentration (2 μM) and cells were then respectively incubated for 4 and 12 h at 37 °C. Whereafter, the cells were stained with Hoechst 33258 (10 μL) for 15 min at culture temperature followed by washing by PBS trice and fixing with paraformaldehyde (4%) for 15 min. The samples were observed by a Confocal Laser Scanning Microscopy (CLSM, Nikon, TE2000, EZ-C1, Japan). NIS viewer (Nikon) and Photoshop (Adobe CS5) were used to merge the pictures (all images were treated in the same way). For photothermal investigation, cells incubated with sample solution for 2 h were treated by NIR light irradiation (808 nm, 2 W/cm2, 20 min, at the light spot of 3 cm × 3 cm), followed by further incubation. The total incubation time was 4 h. Samples under NIR-light treatment were marked as PGD (L+), and those without NIR-light treatment were marked as PGD (L-). Flow cytometry assessments. In order to conduct the quantitatively study on the accumulation of drugs in cells, HeLa cells were seeded into two-well plates at a density of 4.0 × 105 cells/well in 2 mL of DMEM, and then cultured overnight at 37°C in 5% CO2 atmosphere. After 24 h incubation, the DMEM was displaced with 2 mL PGD nanogels in DMEM solution containing the DOX concentration of 2.0 μM and incubated for another 4 h. Then HeLa cells were handled by using trypsin for enzymatic detachment of cells from plastic substrate and washed with PBS solution thrice. Afterwards, Flow cytometry (FCM) analysis was performed on a flow cytometer (BD, USA) for collection of 10,000 gated events for each sample. For photothermal investigation, cells incubated with sample solution for 2 h were treated by NIR light irradiation (808 nm, 2 W/cm2, 20 min, at the light spot of 3 cm × 3 cm),


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followed by further incubation. The total incubation time was 4 h. Samples under NIR-light treatment were marked as PGD (L+), and those without NIR-light treatment were marked as PGD (L-). RESULTS AND DISCUSSION

This technique allows for a better penetration of NIPAM monomer into GO pellets, where the following in situ polymerization may induce an easy exfoliation of GO into 2D nanoplatelets. Finally, GO nanoplatelets were encapsulated into PNIPAM-based matrix. To check this hypothesis, the structure and morphology of the GO and PG nanogels was examined by SEM. Seen from the images of GO (Figure 1a and 1d), the typical lamellar structure of GO was observed, but it can be seen that the GO sheets were stacked several layers together, not in a single layer. As shown in Figure 1b and 1e, it is obvious that the PG nanogels without GSH-treatment were well dispersed as individual particles with spherical shape and there were no apparent lamellar structure of GO, which was similar to the images in some peers’ work31. After GSH-treatment for 10 min, from the SEM images of the PG nanogels (Figure 1c and 1f), the nanoparticles were sticked together and wrapped by the chiffon-like substance. Compared with the original GO (Figure 1a and 1d), the released GO was thinner in thickness and smaller in size as it could be encapsulated in the nanoparticles. Originally, the GO nanoplatelets were inside the PG nanoparticles. After GSH-treatment, the nanoparticles were degraded by the reducing agent (GSH), and the exfoliated GO was released. To further verify the exfoliation of GO, the samples were underwent X-ray diffraction characterization. As shown in Figure 1g, the typical diffraction peak of pure GO sheets was observed at about 2θ=10.72˚, corresponding to their interlayer space of 0.82 nm.32 The typical amorphous diffraction peaks of pure PNIPAM appeared at 2θ=7.47 and 20.81˚.33 After reaction, the characteristic crystal diffraction peak of GO disappeared, suggesting that GO has been fully exfoliated.34



Figure 1. The structure and morphology of GO and PG nanogels. SEM images of GO (a), d)) and PG nanogels without (b), e)) or with (c), f)) 5 mM GSH-treatment for 10 min at 37 °C, g) X-ray diffraction patterns of PNIPAM, GO and PG.

Figure 2. Physical and morphological properties of PG nanogels. a) Hydrodynamic sizes of GO and PG in PBS buffer (0.5 mg/mL, pH=7.4) as a function of time. b) The photographs of physiological stabilities of PG (10 mg/mL) and GO (1 mg/mL) in PBS buffer (pH=7.4). TEM images of PG nanogels c) in ultrapure water (0.5 mg/mL) for 10 min and d) in 5 mM GSH solution (0.5 mg/mL) for 10 min at 37 °C.

The exfoliated GO can be homogenously distributed through PNIPAM-based nanogels with hydrodynamic size of 93 ± 1 nm, where the presence of PNIPAM may favor the improvement of their colloidal stability. Additionally, GO content in the hybrid nanogels can be estimated to be 0.073% according to TGA measurements in


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Figure S3. As can be seen from Figure 2a-b, PG nanogels maintained a good physiological stability, while native GO formed micro-sized aggregation in physiological conditions. The improvement of colloidal stability may overcome the bottleneck of native GO aggregation, and pose a promising intravenous injection application of the GO-hybridized PG nanogels for a possible photothermal therapy. TEM images (Figure 2c-d) indicated that PG nanogels had a size of 73 ± 2 nm with a sphere shape and a narrow size distribution (PDI: 0.10), which can be easily degraded under reducible conditions mimicking intracellular endo-lysomal compartments via 10 min-treatment with GSH as a reducing model agent. Meanwhile, GO was released as the dark substance around the nanopaticles in the image, which is consistant with the SEM results. As such, GO-hybridized nanogels (PG) with good colloidal stability have been successfully developed. The NIR thermal properties of GO nanosheets may afford the nanogels with photothermal effects, which, together with PNIPAM thermosensitivity, can endow them with smartness in drug delivery. The presence of disulfide-crosslinks may enable redox-stimulative drug release property. Furthermore, GO incorporation is helpful to increase drug loading capacity via its strong interactions (e.g., π–π stacking, electrostatic interaction and hydrogen-bonding interactions) with cationic DOX drug.35,36 To check if GO hybridization can improve drug loading capacity, DOX as a model drug was encapsulated into the nanogels by simply mixing them in aqueous solution. As is shown in Table S1, the loading capacity of PGD (PG/DOX) increased along with the increase of DOX/nanogel feed ratio. However, it has to be mentioned that the increasing drug amount for encapsulation resulted in a rapid increase of the particles sizes from 93±1 nm to 356±29 nm. It is known that ~100 nm nanomedicines can guide chemotherapeutic drugs to reach to tumour sites via enhanced permeability and retention (EPR) effect,37, 38 so PGD with size of 93±1 nm and LC% of ~2.5 wt% was selected for biological study in this report. Furthermore, seen from Table 1, it was found that the hybridized PGD nanogels presented high DOX encapsulation efficiency (EE, 63 ± 1%), which is 1.6 fold of that (38 ± 3%) of pure PNIPAM/DOX



Page 12 of 26

nanogels (PD). According to the variation of the Zeta potential values from -28.1 mV before drug loading to -18.7 mV after drug loading, apparently electrostatic interaction

plays

an

important

role

in

this

process.

Compared

with

temperature-insensitive GO, PGD nanogels displayed an excellent thermo-responsive behavior (Figure S1). Although GO also gave a high EE (85 ± 15%), its poor colloidal stability may be a hindrance to the biomedical applications in vivo. Furthermore, only less than 8% of loaded DOX can be released from GD (GO/DOX) samples, which may be associated with the aggregation of GO nanosheets under physiological conditions that greatly impeded drug release in an effective way (Figure 3a). Since GO nanosheets were fully exfoliated and homogenously distributed in the nanogels, about 85% of DOX (more than 10-fold release of GD) can be released from PGD nanogels in a sustainable way. As comparison, free DOX drug gave a burst release. Table 1 Characterization of DOX- free and loaded Nanogels.

Sample

Size,nm[a]

Zeta, mV[a]

EE, %[b]

LC%[c]

GO

379±4

-40.6

PNIPAM

71±1

-15.2

PG

93±1

-28.1

GD

9331±4619

-31.7

85±15

3.3±0.5

PD

73±1

-17.3

38±3

1.5±0.1

PGD

93±1

-18.7

63±1

2.5±0.0

[a] Size and Zeta potential were determined in ultrapure water at 37°C. [b] Encapsulation efficiency (EE) = 100*W1/W0 (3), where W0 and W1 are the total DOX weight employed for drug loading experiment and the weight of loaded DOX, respectively. [c] Loading capacity (LC) = 100*W1/W (4), where Wt and W are the weight of loaded DOX and the weight of DOX-loaded samples, respectively.


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Figure 3. In vitro drug release profiles of DOX-loaded samples: a) free DOX, GD, PD and PGD nanogels at 37 °C, **P

Photothermally Enhanced Chemotherapy Delivered by Graphene

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