Exceptionally High Payload of IR780 Iodide on Folic Acid

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Exceptionally High Payload of IR780 Iodide on Folic Acid-Functionalized Graphene Quantum Dots for Targeted Photothermal Therapy Shuhua Li, Shixin Zhou, Yunchao Li, Xiaohong Li, Jia Zhu, Louzhen Fan, and Shihe Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07267 • Publication Date (Web): 23 Jun 2017 Downloaded from http://pubs.acs.org on June 25, 2017

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ACS Applied Materials & Interfaces

Exceptionally High Payload of IR780 Iodide on Folic Acid-Functionalized Graphene Quantum Dots for Targeted Photothermal Therapy Shuhua Li,† Shixin Zhou,‡ Yunchao Li,† Xiaohong Li,† Jia Zhu,*,† Louzhen Fan,*,† and Shihe Yang§ †

Department of Chemistry, Beijing Normal University, Beijing, 100875, China

‡

Department of Cell Biology, School of Basic Medicine, Peking University Health Science

Center, Beijing, 100191, China §

Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water

Bay, Kowloon, Hong Kong, China

ABSTRACT: IR780 iodide (IR780) is recognized as an effective theranostic agent for simultaneous near-infrared (NIR) fluorescence imaging and photothermal therapy (PTT). However, the rigid chloro-cyclohexenyl ring makes IR780 insoluble in almost all pharmaceutically acceptable solvents, which inevitably limits its clinical application. We report folic acid (FA)-functionalized graphene quantum dots (GQDs-FA) containing a large and intact sp2-domain with carboxyl groups around the edge. Such GQDs-FA possess exceptionally high loading capacity for IR780 via strong π-π stacking interactions, and the water solubility of IR780 is improved by over 2400-fold after loading onto GQDs-FA (IR780/GQDs-FA). IR780/GQDsFA with improved photostability, enhanced tumor targeting ability and high photothermal conversion efficiency of 87.9% were capable of producing sufficient hyperthermia to effectively

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kill cancer cells and completely eradicate tumors upon 808 nm laser irradiation. The present IR780/GQDs-FA may open up great opportunities for the effective PTT to treat cancer.

KEYWORDS: exceptionally high payload of IR780, folic acid-functionalized graphene quantum dots, large and intact sp2-domain, NIR fluorescence imaging, photothermal therapy

INTRODUCTION Photothermal therapy (PTT), which employs light-induced heating to destroy cancer cells/tissues, has been increasingly regarded as an attractive and noninvasive alternative to traditional cancer therapies including surgery, chemotherapy and radiotherapy because of its improved selectivity, low systemic toxicity and remote controllability.1-3 Due to the low absorption and scattering of light by tissue in the so-called transparency window (650-950 nm),4 a variety of near-infrared (NIR) radiation-active photothermal conversion agents (PTCAs), such as polypyrrole nanoparticles (NPs), polyaniline NPs, metal (Ag, Au, Pd, and Ge) NPs, tungsten oxide nanocrystals (NCs) and copper chalcogenide (Cu2-xE, with E = S, Se, Te) NCs,5-21 have shown potential applications in in vivo PTT. Unfortunately, most of these PTCAs are immunogenic, non-biodegradable, pharmacokinetically poor, or potentially long-term toxic,22 limiting their clinical application. IR780 iodide (IR780), a heptamethine dye with a strong absorption at about 780 nm, can image tumor in vivo to monitor therapeutic response in real time and induce hyperthermia to cause cancer cell apoptosis and tumor necrosis at irradiation sites, resulting in remarkable therapeutic efficacy for cancer.23,24 IR780 is even regarded as a more effective theranostic agent than indocyanine green (ICG), the only NIR fluorescence dye approved by the United States Food and Drug Administration (FDA). However, the existence of a rigid chloro-cyclohexenyl


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ring makes IR780 insoluble in almost all pharmaceutically acceptable solvents,25,26 which inevitably compromises its phototherapeutic effect. Moreover, the poor photostability of IR780 from light-induced decomposition also restricts its application. Much research has been conducted to overcome the drawbacks by encapsulating IR780 into the core-shell polymeric micelles, liposomes and serum albumin NPs.24,27-31 The fluorescence of IR780 was self-quenched in the cores of these carriers and the loading content of IR780 was low due to the structure mismatch between the planar IR780 molecules and spherical carriers. The improvement in the water solubility of IR780 is far from sufficient to meet the clinical requirements. Furthermore, since IR780 was encapsulated by the thick shells, the light absorption efficiency of IR780 was really quite low, seriously affecting the photothermal effect of IR780. Consequently, the development of carriers which can improve the solubility and photostability of IR780 without influencing the optical and thermal properties of IR780 is highly desirable. Longstanding interest in the search of a superior alternative has triggered the recent development of graphene quantum dots (GQDs) as a new class of carriers.32,33 GQDs are generally composed of sp2- and sp3-hybridized carbon clusters, and the existence of sp2 clusters allows the integration of highly aromatic drug molecules through π-π stacking interactions.34,35 In GQDs reported previously, however, a large fraction of carbon is sp3 hybridized, and the remaining small sp2 clusters are isolated within the sp3 carbon matrix,36-40 thereby adversely impacting the loading capacity of GQDs. Here in this work, GQDs containing a large and intact sp2-domain with carboxyl groups around the edge were prepared by pyrolysis of citric acid (CA)41 and subsequent functionalization with acetic acid moieties (Figure 1a). Such GQDs possess exceptionally high loading capacity for IR780 via π-π stacking interactions, while the hydrophilic carboxyl groups around the edge of GQDs could covalently conjugate folic acid (FA)


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by a EDC/NHS condensation reaction to enhance the tumor targeting ability and avoid the photodamage to surrounding normal tissues during PTT.42 After loading onto the FA-functioned GQDs (GQDs-FA) with a high loading content of 33.19%, the water solubility of IR780 is increased by over 2400-fold. More importantly, IR780-loaded GQDs-FA (IR780/GQDs-FA) exhibit improved photostability, enhanced tumor targeting ability and high photothermal conversion efficiency of 87.9%.5-17 Such IR780/GQDs-FA were capable of producing sufficient hyperthermia to effectively kill cancer cells and completely eradicate tumors upon 808 nm laser irradiation (Figure 1b).

Figure 1. Schematic illustrating the preparation of IR780/GQDs-FA (a) and their application for targeted PTT (b).


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EXPERIMENTAL SECTION Materials and Reagents. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 98.0%), IR780 iodide (IR780, 98.0%), N-Hydroxysuccinimide (NHS, 98.0%), 4,6-diamidino-2phenylindole (DAPI, 90.0%), propidium iodide (PI, 95.0%) and cell counting kit-8 (CCK-8) were purchased from Sigma-Aldrich. Other reagents used in this work including citric acid (CA, 99.8%), folic acid (FA, 98.0%), hydrochloric acid (HCl, 37.5%), sodium hydroxide (NaOH, 96.0%), sodium chloroacetate (ClCH2COONa, 98.0%), sodium bicarbonate (NaHCO3, 99.8%) and dimethyl sulfoxide (DMSO, 99.8%) were purchased from Aladdin Industrial Corporation and used without further purification. All the female BALB/c nude mice were purchased from Beijing Vital River experimental animal technical co., LTD, and the weight of each mouse was 18-20 g. Preparation of GQDs, GQDs-FA and IR780/GQDs-FA. In this case, 2 g CA was put into a 25 mL beaker and heated to 200 oC for 20 min. Then 5 g NaOH and 5 g sodium chloroacetate were added into 100 mL above solution (1.0 mg/mL), followed by ultrasonic stirring for 5 h. After neutralized with 0.1 M HCl solution to pH 7.0, the aqueous solution was dialyzed for 3 days and dried under vacuum for 24 h. The obtained brown powder was abbreviated to GQDs. GQDs-FA were prepared by mixing FA (10 mM), EDC (10 mM), NHS (10 mM) and GQDs aqueous solution (10 mL, 1.0 mg/mL) saturated with NaHCO3, followed by magnetic stirring for 18 h. The mixture was then dialyzed for 3 days. For IR780 loading onto GQDs-FA, the same amount of IR780 (1 mg/mL in DMSO) was mixed with different concentrations of GQDs-FA (0.05-10 mg/mL), followed by magnetic stirring for 12 h and filtering through polyvinylidene difluoride syringe filters (0.22 µm). The mixture solution was then dialyzed for 3 days. The loading content and encapsulation efficiency of IR780 were measured according to the following


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formulas: Loading content (%) =

W (IR780 in IR780/GQDs-FA) × 100% W (IR780/GQDs-FA)

Encapsulation efficiency (%) =

W (IR780 in IR780/GQDs-FA) × 100% W (Total added IR780)

Where W (IR780 in IR780/GQDs-FA) , W (IR780/GQDs-FA) and W (Tatol added IR780) are the weights of IR780 in IR780/GQDs-FA, IR780/GQDs-FA and the total added IR780, respectively. Preparation of GOs, rGOs, GOs-FA and rGOs-FA. GOs were synthesized using a modified Hummer’s method43,44 at a gram scale from graphitic flakes. rGOs were synthesized according to the following method. 100 mg GOs and 100 mL water were put into a 250 mL round-bottom flask, followed by ultrasonic treatment for 1 h. Then 1 mL hydrazine hydrate was added into the above GOs solution, and the solution was heated to 80 oC for 30 min. GOs-FA and rGOs-FA were synthesized by mixing FA (10 mM), EDC (10 mM), NHS (10 mM) and GOs or rGOs aqueous solution (10 mL, 1.0 mg/mL) saturated with NaHCO3, followed by magnetic stirring for 18 h and dialysis for 3 days. Characterization of GQDs, GQDs-FA and IR780/GQDs-FA. Transmission electron micrographs (TEM) images were obtained by JEOL JEM 2100 transmission electron microscope (FEI). Atomic force microscopic (AFM) were taken on MultiMode V SPM (VEECO). Raman spectrum was performed with the Laser Confocal Micro-Raman Spectroscopy (LabRAM Aramis). Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) were obtained using a Nicolet 380 spectrograph and ESCALab 250Xi electron spectrometer from VG Scientific using 300 W Al K radiation, respectively. Fluorescence and UV-vis absorption spectra were collected by PerkinElmer-LS55 fluorescence spectrometer and UV-2450 spectrophotometer, respectively. The particle size distribution was measured by particle


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size analyzer (90Plus Zeta). Cellular imaging. HeLa cells (1×105 cells/well) were firstly seeded in a 6-well plate. After overnight incubation in RPMI1640 medium, HeLa cells were treated with IR780/GQDs-FA or free IR780 for 12 h. Then HeLa cells on the plate were washed three times with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde to image using a confocal fluorescence microscope. To confirm the folate receptor-mediated cell uptake, HeLa cells were pretreated with excessive FA for 2 h. Cellular toxicity test. HeLa cells (1×105 cells/well) were seeded into a 12-well plate. After overnight incubation, HeLa cells were cultured with PBS or IR780/GQDs-FA (10 µg/mL for IR780) for 12 h. After 808 nm laser irradiation (1 W/cm2) for 5 min, the cells were washed three times with PBS and stained with PI for 15 min. Fluorescence images were collected at 600-680 nm using confocal fluorescence microscope at an excitation of 488 nm. HeLa cells (1×105 cells/well) were seeded into a 24-well plate and incubated with different concentrations of IR780/GQDs-FA (0-30 µg/mL) for 12 h. After the treatment with or without 808 nm laser irradiation (1 W/cm2) for 5 min, the plates were washed three times with PBS. The fresh medium (100 µL) containing CCK-8 (10 µL) was then added into the 24-well plate, and the cells were incubated for 2 h. Finally, microplate reader was used to measure the absorbance at 450 nm. NIR fluorescence imaging of IR780/GQDs-FA in vivo. Subcutaneous tumor xenografts were established by injecting HeLa cells into the armpits of the female BALB/c nude mice. When the subcutaneous tumors grew to about 100 mm3, IR780/GQDs-FA (2 mg/kg) were injected into the tumor-bearing mice via the tail vein. The NIR fluorescence images were taken at 1, 2, 6, 12, 24 and 48 h after injection using an IVIS Lumina III system with excitation at 740


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nm and emission at 790 nm. Evaluation the phototherapeutic efficacy and systemic toxicity of IR780/GQDs-FA in vivo. The female BALB/c nude mice with tumor volumes of 100-200 mm3 were intravenously injected with saline or same volume of IR780/GQDs-FA (2 mg/kg, n = 5), respectively. After 24 h post-injection, the tumors in mice were illuminated with 808 nm laser irradiation (1 W/cm2, 5 min). The change in body weight and tumor volume (V) were measured every three days. V was calculated as D × d2/2 (D and d are the longest and shortest diameters of tumor measured by a caliper). V/V0 (V0 is the initial tumor volume before PTT) was determined as relative V. After the treatment for 15 days, the mice were sent to Beijing Laboratory Animal Research Center for biochemical and histological analysis.

RESULTS AND DISCUSSION GQDs were synthesized by pyrolysis of citric acid (CA) at 200 oC for 20 min and subsequent functionalization with carboxyl groups via conjugation of acetic acid moieties. CA is a weak organic

tricarboxylic

acid.

During

the

pyrolysis,

inter-molecular

dehydration

and

decarboxylation of CA happened and the intact sp2 graphene nanosheets with abundant hydroxyl and carboxyl groups around the edge formed (Figure 1a).40 Sodium chloroacetate were then added and reacted efficiently with hydroxyl groups under strongly basic conditions to convert hydroxyl groups to carboxyl groups. After neutralized with HCl solution to pH 7.0 and dialyzed for 3 days, GQDs were obtained. The TEM image (Figure 2a) reveals that GQDs are well-dispersed and have a relatively narrow size distribution ranging from 7.5 to 9.5 nm with an average diameter of 8.5 nm (Figure 2a inset). The AFM images (Figure S1) show a typical topographic height of 1.356 nm,


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corresponding to ca. 2-3 graphene layers. The high-resolution TEM (HRTEM) image (Figure 2b) indicates that most of GQDs exhibit uniform atomic arrangements with high degree of crystallinity, and the lattice spacing of 0.242 nm is attributed to the (1120) lattice fringes of graphene.45 It is noteworthy that carbon hexagon structures are arranged orderly and the wellcrystallized carbon areas almost occupy the whole dot (Figure 2c and Figure S2), which demonstrates that GQDs may consist of a large and intact sp2-domain.46 The blurred boundary of GQDs in Figure 2c may be caused by the introduction of oxygenic groups. Raman spectrum of GQDs (Figure S3) shows the crystalline G band at 1601 cm-1 and the disordered D band at 1364 cm-1 with a high intensity ratio IG/ID of ca. 1.52, further indicating fewer defects on GQDs,47 and is well consistent with the high degree of crystallinity of GQDs determined by HRTEM (Figure 2b,c).

Figure 2. (a) TEM image of GQDs. The inset is size distribution of GQDs. The HRTEM image with measured lattice spacing (b) and sp2 domain (c) of GQDs. The chemical composition of GQDs was investigated by XPS and FT-IR measurements. The XPS survey spectrum of GQDs shows a predominant graphitic C1s peak at 284 eV and an O1s peak at 532 eV (Figure 3a). The C/O atomic ratio for GQDs is about 2:1 (Table S1), which is higher than that of CA (about 1:1), suggesting that the inter-molecular dehydration and decarboxylation of CA happened during the pyrolysis. The peak at 288.82 eV in the highresolution C1s spectrum of GQDs (Figure 3b) reveals the presence of carboxyl groups. The


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characteristic peak at 1710 cm-1 in the FT-IR spectrum of GQDs (Figure S4) is assigned to the stretching vibration of C=O in carboxyl groups, while the peak attributed to the C-OH group does not appear, confirming that oxygenic groups around the edge of GQDs exist mainly as carboxyl groups. Folic acid (FA) was conjugated with GQDs by an EDC/NHS coupling reaction between amine groups of FA and carboxyl groups around the edge of GQDs to form FA-functionalized GQDs (GQDs-FA). In the UV-vis absorption spectrum (Figure S5), two characteristic peaks corresponding to FA at approximately 280 and 360 nm appear after the conjugation, which indicates the presence of FA in GQDs-FA.48 In the FT-IR spectrum of GQDs-FA (Figure S4), three new vibrating peaks at 1400, 1605, and 1690 cm-1 are attributed to C-N, N-H and -NH-COrespectively.49 The formation of amide groups in GQDs-FA suggests the EDC/NHS coupling reaction happened. In accordance with the above results, the peaks at 287.03 and 400.97 eV in the high-resolution C1s and N1s spectra of GQDs-FA (Figure 3c, d) are ascribed to -NH-CO-, further confirming the successful functionalization of FA onto GQDs surface. In addition, the presence of the peak assigned to O-C=O at 288.52 eV in the C 1s spectrum of GQDs-FA (Figure 3c) reveals that there remain carboxyl groups around the edge of GQDs after modification with FA, ensuring the water solubility of GQDs-FA.


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Figure 3. (a) XPS survey spectra of GQDs and GQDs-FA. C1s spectra of GQDs (b) and GQDsFA (c). (d) N1s spectra of GQDs-FA. Inset: chemical composition of GQDs and GQDs-FA determined by high-resolution XPS. IR780 was then loaded onto the surface of GQDs-FA (IR780/GQDs-FA) simply by mixing the same amount of IR780 (1 mg/mL in DMSO) with different concentrations of GQDs-FA aqueous solutions (0.05-10 mg/mL) with the aid of slight stirring for 12 h in the dark. After removing the free IR780 and DMSO by dialysis, the loading amount of IR780 was determined by the standard curve of the UV-vis absorbance of IR780. As shown in Figure S6a and Table S2, with the increase of GQDs-FA concentrations, the water solubility of IR780 after loading onto GQDs-FA increases significantly and reaches a plateau (990 µg/mL) when the concentration of


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GQDs-FA is 2 mg/mL. The water solubility of IR780 is increased by over 2400-fold,24 and the encapsulation efficiency of IR780 is as high as 99.36%. The loading content of IR780, another crucial factor in developing drug delivery vesicles, is up to 33.19%, which is much higher than the value when IR780 was loaded onto polymeric micelles (4.47, 7.3 or 14%), silica matrix (1.2%), human serum albumin NPs (1.6%) and heparin-folic acid NPs (4.71%) reported in the literature (Table S3).24, 27-31 We deduce that the unique intact sp2-domain of GQDs in GQDs-FA may play a crucial role in the exceptionally high loading capacity for IR780, which is verified by control experiments. First, GQDs-FA were changed to GOs-FA, where GOs (graphene oxide) were prepared by acid oxidation graphite using a modified Hummer’s method.41,44 The loading content of IR780 in GOs-FA was only 3.08% (Figure S6b and Table S2), which was much lower than that of GQDsFA (33.19%). Next, when GOs in GOs-FA were reduced (rGO) by hydrazine monohydrate to obtain rGOs-FA, the loading content of IR780 in rGOs-FA could be increased to 9.01%. It has been reported that numerous small and isolated sp2 domains were formed in rGOs during the reduction process.50,51 It is obviously that the increased number of newly formed sp2 domains benefit the integration of IR780 through π-π stacking interactions. Taking a step further, we speculaste that GQDs-FA which contain a large and intact sp2-domain are certainly correlated with the much higher loading capacity for IR780. The UV-vis absorbance spectrum of IR780/GQDs-FA shows two NIR absorption peaks centered at 720 and 798 nm (Figure 4a and Figure S7), while the absorption peaks of IR780 appear at 710 and 780 nm, respectively. The obvious redshift of the absorption peaks may be due to the strong π-π stacking interaction between IR780 and GQD-FA.52 The NIR fluorescence emission peak of IR780/GQDs-FA centered at 800 nm could also be observed in Figure 4b,


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indicating that loading the lipophilic IR780 onto GQDs-FA dose not quench the NIR fluorescence of IR780, and hence GQDs-FA/IR780 could be used for NIR fluorescence imaging. The particle size of IR780/GQDs-FA remains consistent (about 9 nm) after storage for one week at room temperature (Figure S8), suggesting that IR780/GQDs-FA can stably disperse in water without aggregation. Furthermore, IR780/GQDs-FA also exhibit high photostability (Figure S9), which are expected to be used for further cells and animal experiments.

Figure 4. (a) UV-vis absorbance spectra of IR780 and IR780/GQDs-FA. (b) Fluorescence emission spectrum of IR780/GQDs-FA under varying excitation wavelengths. Since IR780/GQDs-FA has a strong absorption in the NIR region, the NIR photothermal performance of IR780/GQDs-FA aqueous solution was investigated under 808 nm laser irradiation. The pure water, GQDs-FA and free IR780 were used as controls. From Figure 5a, the temperature of IR780/GQDs-FA with a concentration of 30 µg/mL (10 µg/mL for IR780) increases rapidly during 808 nm laser irradiation and reaches approximately 50 oC after 5 min, while only a slight temperature rise for GQDs-FA and water could be seen. The remarkable temperature rise of IR780/GQDs-FA could cause irreversible damage to cancer cells.53 The temperature of free IR780 with the same concentration of IR780 (10 µg/mL) loaded onto the GQDs-FA increases by 17 oC rapidly in 3 min and then decreases gradually (Figure 5a) due to


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the photo-degradation of the free IR780 after exposure to 808 nm laser irradiation (Figure 5b). Besides, IR780/GQDs-FA also exhibit a higher temperature rise than that of the free IR780 in the first 3 min. The observation may be ascribed to the obvious redshift of the characteristic absorption peak at 780 nm upon IR780 loading onto GQDs-FA (Figure 4a), which increases the absorption of IR780/GQDs-FA for 808 nm laser and resulting in the high mass extinction coefficient (62.8 L⋅g-1⋅cm-1) calculated from the absorbance value of IR780/GQDs-FA at 808 nm.54 The pseudo-color signals for IR780/GQDs-FA (Figure 4a inset) are strengthened significantly with the increase of irradiation time, which directly shows the rapid temperature rise of IR780/GQDs-FA within only 5 min. Taken together, these results demonstrate that IR780/GQDs-FA exhibit even higher photostability and better photothermal performance as compared with free IR780. On the basis of the photothermal curve (Figure 5c) and time constant (τs = 110 s) for heat transfer of the system (Figure 5d), the photothermal conversion efficiency of IR780/GQDs-FA was calculated to be 87.9% (see the Supporting Information for the detailed calculations), which is higher than that of the previously reported PTCAs, such as Au nanocages (64%), Au nanorods (55%), dopamine-melanin nanospheres (40%), carbon dots (38.5%), Cu2-xSe NCs (22%), Cu9S5 NCs (25.7%), and so on (Table S4).5-17


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Figure 5. (a) The photothermal profiles of water, free IR780, GQDs-FA and IR780/GQDs-FA with laser irradiation. The inset shows IR thermal images of IR780/GQDs-FA under 808 nm laser irradiation (1 W/cm2) recorded every 1 min. (b) The influence of the irradiation time on the concentration of IR780 (CIR780) in IR780 or IR780/GQDs-FA aqueous solution, respectively. The inset shows the corresponding color changes of free IR780 and IR780/GQDs-FA. (c) Photothermal curve of IR780/GQDs-FA when illuminated with 808 nm laser (1 W/cm2) for 5 min and then cooled naturally. (d) Plot of cooling time versus negative natural logarithm of the temperature driving force obtained from the cooling stage. The cellular uptake behavior of IR780/GQDs-FA was investigated by the laser confocal scanning microscopy (LCSM) with HeLa cells as a model. DAPI staining was used to visualize nuclei. As illustrated in Figure 6a, HeLa cells incubated with IR780/GQDs-FA present a stronger NIR fluorescence signal in the cytoplasm than that of free IR780, indicating the higher cellular uptake rate of IR780/GQDs-FA. However, when HeLa cells are firstly pretreated with excessive FA before adding IR780/GQDs-FA, NIR fluorescence signal decreases remarkably. The free FA competitive inhibits the specific combination between IR780/GQDs-FA and FA receptor overexpressed on the surface of the HeLa cells,55,56 which results in the decreased cellular uptake


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of IR780/GQDs-FA. Based on the high cellular uptake and photothermal conversion efficiency (87.9%) of IR780/GQDs-FA, propidium iodide (PI) staining and CCK-8 cell viability assays were carried out to evaluate the phototherapeutic effects of IR780/GQDs-FA in vitro. From Figure 6b, dead cells shown in red fluorescence could only be observed in the IR780/GQDs-FA plus laser irradiation group, indicating that IR780/GQDs-FA exhibit prospective abilities to kill HeLa cells upon 808 nm laser irradiation (1 W/cm2, 5 min), while they have almost no toxicity without laser irradiation. The cytotoxicity of IR780/GQDs-FA in HeLa cells with or without laser irradiation were also determined by the quantitative CCK-8 cell viability assays. As the concentration of IR780/GQDs-FA increases from 0.5 to 30 µg/mL, the cell viabilities of IR780/GQDs-FA with laser irradiation group gradually decline and reach the minimum of 2.1% (Figure 6c). The above results reveal that IR780/GQDs-FA alone are relatively harmless and do not cause cell death without laser irradiation, but they display significant toxicity to HeLa cells under 808 nm laser irradiation. In addition, we further explored the cytotoxicity of IR780/GQDs-FA with the loading content of IR780 ranging from 5.86% to 33.19% when exposed to 808 nm laser irradiation for 5 min. As expected, IR780/GQDs-FA with higher loading content of IR780 exhibit a higher temperature rise of HeLa cells (Figure S10) when exposed to 808 nm laser irradiation, which induces lower viability of HeLa cells (Figure 6d). Therefore, we conclude that the exceptionally high loading density of IR780 per GQDs-FA benefits the heat production with ultrahigh efficiency within a short time of 5 min due to the collective heating effect of many IR780 molecules on each GQDs-FA,57-59 and IR780/GQDs-FA with the highest IR780 loading content of 33.19% are expected to be used as an effective PTCAs for PTT.


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Figure 6. (a) The LCSM images of HeLa cells incubated with free IR780, IR780/GQDs-FA and IR780/GQDs-FA pretreated with excessive FA (scale bar: 25 µm). Blue fluorescence images of the nucleus stained with DAPI (top row) were collected at 450-470 nm, under an excitation at 405 nm. NIR fluorescence images (middle row) were collected at 750-850 nm (633 nm excitation). (b) The LCSM images of HeLa cells staining with PI after different treatments. (c) Viability of HeLa cells treated with IR780/GQDs-FA at varying concentrations (0-30 µg/mL) before and after laser irradiation. (d) Viability of HeLa cells incubated with PBS or IR780/GQDs-FA (the loading content of IR780 ranges from 5.86% to 33.19%) after laser irradiation. The visualization towards the delivery and distribution of PTCAs is crucial to precisely guide and optimize PTT process in vivo. The feasibility of IR780/GQDs-FA for targeted NIR fluorescent imaging in vivo was investigated on BALB/c nude mice bearing HeLa tumors. As shown in Figure 7a, NIR fluorescence signals of IR780/GQDs-FA in the tumor region gradually increase and reach the highest level at 24 h after the intravenous injection. In particular, NIR fluorescence intensity of IR780/GQDs-FA in the tumor region is apparently higher than that of free IR780 at each time point, indicating high tumor targeting ability of IR780/GQDs-FA. Ex vivo evaluation of dissected organs at 24 h post-injection (Figure 7b and c) shows that fluorescence signals of free IR780 are whole body distribution, and the signal intensity of liver, lung and kidney are quite high, indicating that a large number of IR780 also accumulate in these organs. By contrast, for IR780/GQDs-FA treated mice, the excised tumor tissue shows strong fluorescence intensity, whereas other organs shows very low signals, further confirming that


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IR780/GQDs-FA preferentially accumulate in tumors. Since tumor region can be imaged and located precisely, only tumors will be irradiated during PTT while the surrounding normal tissue is minimally hurt, which can largely improve the therapeutic effect of IR780/GQDs-FA and obviously reduce systemic side effects.

Figure 7. (a) NIR fluorescence imaging of tumor-bearing mice intravenously injected with free IR780 or IR780/GQDs-FA during 48 h. The tumor is circled with a dotted line. (b) Ex vivo NIR fluorescence imaging of dissected organs at 24 h post-injection. (c) Mean fluorescence intensity of the excised organs from tumor-bearing mice treated with free IR780 or IR780/GQDs-FA. Encouraged by the strong NIR absorption and high tumor accumulation of IR780/GQDs-FA, phototherapeutic efficacy of IR780/GQDs-FA in vivo was evaluated. After being intravenously injected with saline or IR780/GQDs-FA for 24 h, tumor-bearing nude mice were anesthetized and irradiated with 808 nm laser for 5 min. As shown in Figure 8a, the temperature at the tumor site rapidly increases to 58.9 °C in the IR780/GQDs-FA group. In contrast, the maximum


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temperature of the tumor surface in the saline group is only approximately 38.2 °C under the same irradiation condition. In vivo antitumor studies (Figure 8b and c) demonstrate that IR780/GQDs-FA in combination with laser irradiation exhibit obvious suppressive effects on tumor growth and the tumor almost disappears on day 15, while the volumes of tumors increase greatly in the saline group. Moreover, IR780/GQDs-FA with the highest IR780 loading content of 33.19% also shows significant superiority in in vivo PTT, including much lower injected dose (2 mg/kg) and shorter treatment time (15 days) shown in Table S5. Notably, the mice treated with IR780/GQDs-FA survive over 60 days without tumor reoccurrence, whereas the life span of mice receiving saline is no more than 20 days due to the fast tumor growth rate (Figure S11). Despite the observed weak fluctuations during the treatment period, there is no significant reduction in body weights of the mice treated with IR780/GQDs-FA (Figure 8d), preliminarily proving that no obvious side effects are induced by the injection of IR780/GQDs-FA at the given dose. Toxicology analysis of IR780/GQDs-FA is further investigated by hematoxylin and eosin (H&E) staining examination and blood tests. As illustrated in Figure 8e, neither inflammatory infiltration nor pathological tissue damage can be observed in IR780/GQDs-FA treated groups. In addition, blood routine (Table S6) and renal/liver function parameters (Table S7) in serum show that there is no statistical observable difference on the levels of these markers between the treatment and control groups, confirming the excellent hemocompatibility of IR780/GQDs-FA. Collectively, the in vivo study suggests that IR780/GQDs-FA can preferentially accumulate in tumors and achieve enhanced therapeutic efficacy with low toxic and side effects.


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Figure 8. (a) Thermal images showing the temperature variations of tumors after intravenous injection of saline or IR780/GQDs-FA with laser irradiation. (b) Representative images of tumorbearing mice after treatments. Tumor volume (c) and body weight (d) curves of tumor-bearing mice from the different treatment groups. (e) Histological evaluation of tissues from mice treated with saline and IR780/GQDs-FA. Each organ was sliced for H&E staining.

CONCLUSION A novel theranostic platform based on IR780/GQDs-FA was successfully synthesized for targeted tumor imaging and PTT in vitro and in vivo. GQDs containing a large and intact sp2domain with carboxyl groups around the edge exhibit excellent water solubility, suitability for conjugation with FA and exceptionally high loading capacity for IR780 (33.19%) due to the strong π-π stacking interaction. The water solubility of IR780 has been improved by over 2400fold after loading onto GQDs-FA. In vivo NIR fluorescence imaging and biodistribution demonstrate that such IR780/GQDs-FA could preferentially accumulate in tumors. Upon 808 nm laser irradiation, IR780/GQDs-FA are capable to produce sufficient hyperthermia to induce


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cancer cell apoptosis and tumor necrosis, resulting in the complete tumor disappearance without relapse. We anticipate that the present IR780/GQDs-FA may open up great opportunities for the enhanced PTT to treat cancer because of their high photostability and water solubility, bioimaging and targeting capability, and therapeutic efficacy.

ASSOCIATED CONTENT Supporting Information Figures S1-S11, Tables S1-7, and calculation of photothermal conversion efficiency of IR780/GQDs-FA (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEGEMENTS This work was supported by NSFC of China (21573019), the Major Research Plan of NSFC (21233003), and the Fundamental Research Funds for the Central Universities.

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Table of Content


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Exceptionally High Payload of IR780 Iodide on Folic Acid

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