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Porphyrin-implanted Carbon Nanodots for Photoacoustic Imaging and in Vivo Breast Cancer Ablation Fengshou Wu, Huifang Su, Yuchen Cai, Wai-Kwok Wong, Wenqi Jiang, and Xunjin Zhu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00029 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 9, 2018

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ACS Applied Bio Materials

Porphyrin-implanted Carbon Nanodots for Photoacoustic Imaging and in Vivo Breast Cancer Ablation Fengshou Wu,a,c† Huifang Su,b† Yuchen Cai,b* Wai-Kwok Wong,a Wenqi Jiang,b* and Xunjin Zhua* a

State Key Laboratory of Environmental and Biological Analysis and Department of Chemistry, Hong Kong

Baptist University, Waterloo Road, Hong Kong, P. R. China. b

Department of Medical Oncology, Sun Yat-Sen University Cancer Center, State Key Laboratory of Oncology

in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P. R. China. c

Key Laboratory for Green Chemical Process of the Ministry of Education, School of Chemical Engineering

and Pharmacy, Wuhan Institute of Technology, Wuhan, P. R. China.

†These authors contributed equally to this work. * Corresponding author E-mail addresses: [email protected] (X. Zhu); [email protected] (Y. Cai); [email protected] (W. Jiang).

Abstract The incorporation of intensive light absorbing porphyrins macrocycles with biocompatible nanoparticles would lead to new nanomaterials with multiple imaging and therapeutic modalities. Herein, a facile synthetic strategy has been applied to prepare porphyrin-implanted carbon nanodots (PNDs) by partial and selective pyrolysis of 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP) and citric acid (CA) at an appropriate temperature. As-prepared PNDs exhibit not only the excellent stability and biocompatibility characteristic of carbon nanodots, but the unique properties of porphyrin macrocycle such as strong UV-visible and nearinfrared absorption, specifically, high photodynamic therapy efficiency. More importantly, the PNDs with near-infrared absorption could act as a contrast agent for photoacoustic molecular imaging with deep tissue penetration and fine spatial resolution. The Cetuximab-conjugated porphyrin-based carbon nanodots (C225PNDs) have been further prepared to precisely target the cancer cells (HCC827 and MDA-MB-231 cells) with over-expression of EGFR, leading to highly efficient photodynamic therapy upon two-photo excitation at 800 nm. A complete ablation of tumour together with an enhanced photoacoustic contrast ability for C225-PNDs have been further validated in mice bearing MDA-MB-231 breast cancer.

Key words: porphyrin, nanomaterials, photoacoustic imaging, photodynamic therapy, two-photon irradiation

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1. Introduction Cancer already presents a great challenge to public health globally.1 However, treatment options are limited, and accurate diagnosis and therapy are difficult in many cases. Therefore, tremendous efforts in biomedical research have been devoted to developing new approaches for early stage detection, diagnosis and therapy of cancer, which is now commonly referred to as “theranostics”.2,

3

Photodynamic therapy (PDT) is an

extraordinary theranostic modality for a number of malignant and nonmalignant diseases.4 At the same time, the fluorescence generated upon light activation would be used for molecular imaging in PDT.4, 5 However, the spatial resolution of the fluorescence imaging is low due to the limited penetration depth. Compared with conventional optical imaging modalities, photoacoustic (PA) imaging is a newly developed noninvasive imaging system, which combines good spectral selectivity of laser light with high resolution and deep penetration of ultrasound imaging.6, 7 Thus, the fabrication of integrated photosensitizers for fluorescence and PA dual-modality imaging-guided photodynamic therapy was highly desirable. Porphyrin-based molecules are the most commonly used photosensitizers (PSs). However, many of them are limited in clinical applications because of prolonged cutaneous photosensitivity, poor water solubility, inadequate selectivity and low fluorescence quantum yield. One of the most effective ways to overcome these shortcomings is to create porphyrin-containing nanoparticles through supramolecular self-assembly of lipid or peptide-porphyrin conjugates.8, 10-14

biomedical applications,

Recently, carbon nanodots were found as new fluorescence agents for

because of their excellent photostability, small size, biocompatibility, and highly

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tunable photoluminescence.

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Pyrolysis or carbonization of small organic molecules by heating above their

melting point leads to condensation, nucleation, and subsequent formation of larger carbon dots. It could also serve as drug delivery nanoplatform for photosensitizers through surface functionalization or loading techniques for simultaneous diagnosis and therapeutics.4,19,20 Although these porphyrin-based nanomaterials exhibit excellent biocompatibility and efficient photodynamic or photothermal antitumor properties, the fabrication process involves multiple and complex chemical reactions, such as the synthesis of amphilic porphyrin conjugates and the self-assembling process. In this contribution, we applied a simple synthetic strategy to prepare porphyrin-implanted carbon nanodots for fluorescence and PA dual-modality imaging-guided photodynamic therapy. The partial and selective pyrolysis of TAPP and CA at an appropriate temperature produced the PNDs in a reasonable yield. With the effort to develop new nanomaterials for precise theranostics in clinical tumor therapy,21-23 a well-known targeting moiety (Cetuximab) was subsequently appended on the surface of PNDs to afford C225-PNDs. The Cetuximab is capable of recognizing and targeting, with high binding affinity, the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase highly expressed on many human malignancies, such as head and neck, colorectal, non-small cell lung, and gastric cancers.24 As expected, the targeting ability and subcellular localization of C225-PNDs were confirmed by confocal laser scanning microscope in HCC827 and MDA-MB-231 cells (over-expression of EGFR), and H23 and HBL-100 cells (low expression of EGFR).25 More importantly, intense PA signals were observed after intratumoral administration of C225-PNDs and NIR


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laser irradiation. Meanwhile, in vivo photodynamic cancer treatment with C225-PNDs indicates that low laser power density and short irradiation time could effectively kill tumor cells incubated with C225-PNDs with no

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Fig. 1. The proposed formation pathway of PNDs and the synthetic routes of C225-PNDs.

2. Results and Discussion The PNDs was prepared by a selective pyrolysis of TAPP in the presence of CA (Fig. S1).26, 27 As shown in Fig. 1, the porphyrin macrocycle was successfully implanted into the formed carbon nanodots without structural collapse when the reaction temperature gradually increased from room temperature to 200 oC. When temperature increased higher than 220 oC, the porphyrin macrocycle would decomposed gradually. The amount of porphyrin encapsulation was calculated to be 232 µg per milligram of PNDs from the standard curve of the UV-Vis absorbance of TAPP. The aqueous solution of PNDs did not show any precipitation or aggregation and any change of UV-Vis absorbance after storage at ambient condition for 60 days, suggesting the excellent colloidal stability of PNDs. Subsequently, the surface of PNDs was functionalized with PEG diamine (PEG1500N) to afford the PEG-coated nanodots (NH2-PNDs) with terminated amine groups. The Cetuximab was then covalently bonded to NH2-PNDs using a modified EDC–NHS reaction, yielding the C225-PNDs. The as-prepared PNDs were fully characterized by X-ray diffraction (XRD), Zeta potential measurement, and X-ray photoelectron spectroscopy (XPS). As shown in Fig. S2a, the typical XRD profiles for PNDs showed the 2θ diffraction peaks centered at 22° (0.34 nm), which is attributed to the highly disordered carbon atoms, similar to the graphite (002) lattice spacing.28 Zeta potential is widely used as the indicator of the magnitude of the charge and the stability of nanomaterials. The zeta potential of PNDs was recorded as −5.90 mV at pH 7.0 (Fig. 2f), attributed to the existence of hydroxylic and carboxylic groups on the surface of porphyrin-based nanodots.4 In contrast, the zeta potential of NH2-PNDs became positive with a value of 5.33 mV, suggesting the loss of the carboxylic acid groups upon the surface functionalization of PNDs with PEG diamine. The further conjugation of Cetuximab decreased the zeta potential to 2.84 for C225-PNDs. The elemental composition and type of chemical bonds in PNDs were confirmed by XPS. As shown in Fig. S3a, the peaks at 285.2, 400.8 and 532.7 eV are attributed to the C 1s, N 1s and O 1s emissions, respectively.29 The highresolution C 1s XPS spectra of PNDs show three peaks at 285.1, 286.4 and 288.8 eV (Fig. S3b), which



attribute to C-C/C=C, C-N/C-O and C=O/C=N bonds, respectively. And the high-resolution N 1s spectra of PNDs (Fig. S3c) can be fitted to three peaks of pyridinic N (397.8 eV), pyrrolic N (399.2 eV) and quaternary N (400.7 eV).30 The presence of oxygen-containing functional groups offers options for various types of surface modifications.

Fig. 2. (a) TEM image of PNDs with a corresponding size distribution histogram; (b, c) HRTEM images of PNDs; (d) TEM image of C225-PNDs; (e) Dynamic light scattering (DLS) of C225-PNDs in water; (f) Zeta potential of PNDs, NH2-PNDs and C225-PNDs. The morphology of PNDs was characterized using transmission electron microscopy (TEM) and atomic force microscopy (AFM). As shown in Fig. 2a, the PNDs are spherical nanocomposites and well dispersed with an average diameter of 3.3 nm. The high-resolution TEM images clearly indicate well-resolved lattice fringes (Fig. 2b) with d spacing values of 0.28 and 0.32 nm, which match to the (020) and (002) planes of graphitic carbon, respectively (Fig. 2c). The AFM images of PNDs (Fig. S4) show that the topographic height of PNDs is distributed in the range of 1.2 to 4.5 nm with an average height of 3.7 nm. Specifically, the sizes of PNDs from TEM are somewhat smaller than the overall dot profiles estimated from the height analysis of AFM images, indicating the latter may also include contributions of the porphyrin molecules on carbon particle surface that survived during the process of thermal carbonization.31 The average size of C225-PNDs from TEM is valued around 11 nm (Fig. 2d), which is well consistent with dynamic light scattering (DLS) analysis (12.1 ± 3.5 nm) (Fig. 2e). The increase in size of the nanoparticle after treatment with C225 indicates the antibody molecules were successfully conjugated on the surface of PNDs.


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Fig. 3. UV-vis absorbance spectra (a) and emission spectra (λex = 440 nm) (b) of PNDs, C225-PNDs and TAPP (The inset right is the photograph of PNDs under 365 nm excitation in comparison to the blank control left). The presence of porphyrin ring in PNDs was clearly confirmed by the 1H NMR and UV-vis spectroscopy. As shown in Fig. S5, the 1H NMR signals in the range of 7–9 ppm are indicative of aromatic (sp2) hydrogen atoms from porphyrin macrocycle. The absorption of PNDs (Fig. 3a) in water shows a broad signal in the range of 200–400 nm similar to the carbon nanodots reported in our previous work,32 and two peaks at 450 nm and around 650 nm characteristic of the Soret band and Q bands of porphyrin macrocycle. Compared with free base porphyrin, both Soret and Q-bands of the implanted porphyrin rings in PNDs are red-shifted and the signal of Q-bands become intensified, mainly due to the protonation of inner amine groups of porphyrin rings by carboxylic acid groups on the surface of PNDs.33 In addition, the aqueous solution of PNDs could be easily adjusted into a slightly alkaline environment upon the addition of aqueous ammonia, and the Soret band will shift back to 423 nm together with the change of color from green to red. The equilibrium nature of the protonation mechanism in PNDs has ever been observed for those carbon nanotube-porphyrin system reported previously.33 Upon excitation at 440 nm, the aqueous solution of PNDs at pH = 7.0 displays a NIR emission with a maximum peak at 750 nm (Fig. 3b), while PNDs in PBS buffer at pH = 7.4 displays an intensity stronger red emission with a maximum peak at 678 nm. Obviously, the pH value of solution has a significant effect on the intensity of photoluminescence of PNDs, and the emission maximum peak of PNDs blue-shifted with the increase of the pH value of aqueous solution, which is also consistent with the equilibrium nature of the protonation mechanism of porphyrin.33 After the functionalization with poly(ethylene glycol)diamine and subsequent conjugation with Cetuximab, the C225-PNDs show stable absorption and emission spectra in water and alkaline buffer due to the surface functionalization on carboxylic acid groups. The fluorescence quantum yield of PNDs and C225-PNDs were calculated to be 0.0854 and 0.0301, respectively, in an air atmosphere.



Fig. 4. (a) PA absorption signals of the aqueous solution of PNDs (7 mg/mL) and C225-PNDs (7 mg/mL) at wavelength from 680 to 760 nm; (b) PA absorption signals of the aqueous solution of PNDs and C225-PNDs with different concentrations (0-7 mg/mL); (c) PA imaging of the aqueous solution of PNDs with different concentrations (0-7 mg/mL) under 686 nm; (d) PA imaging of the aqueous solution of C225-PNDs with different concentrations (0-7 mg/mL) under 686 nm; (e) PA imaging of PNDs dispersed in buffer solution (1.75 mg/mL) with different pH values (pH = 6.0, 7.0 and 8.0); (f) PA signal intensity of PNDs dispersed in buffer solution (1.75 mg/mL) with different pH values (pH = 6.0, 7.0 and 8.0).


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Since the PNDs and C225-PNDs exhibit strong absorption in the near infrared region, the PA response was expected and investigated with these two materials. As shown in Fig. 4a, the PNDs and C225-PNDs exhibited significant PA absorption from 680 to 760 nm with the peak around 686 nm, which is consistent with their optical absorption spectrum (Fig. 3a). In addition, the PA signals of both PNDs and C225-PNDs in water increased with their concentrations, as shown in Fig. 4b-d, while the PA signals of C225-PNDs are linearly dependent on the concentrations (R2 = 0.99) from 0 to 7 mg/mL (Fig. 4b). Owning to the strongly pHdependent NIR absorption of PNDs, PA responses of PNDs dispersed in buffer solutions with different pH values were also investigated. As shown in Fig. 4e and 4f, the PNDs displayed strong PA signals at 686 nm in a neutral or slightly acidic environment, and the PA intensity showed a slight decrease with pH in a range of 6 to 7. However, the PA signal amplitude of PNDs decreased by 7 times along with the increase of pH from 7 to 8, probably due to the deprotonation of the porphyrin inside of PNDs in an alkaline environment. Therefore, the pH-responsive nanoprobe based on PNDs might be suitable for PA imaging of tumor acidic microenvironment. The results clearly demonstrate that both PNDs and C225-PNDs could be used as photoacoustic imaging reagents. Meanwhile, the production of reactive oxygen species (ROS) via photoinduced energy transfer from PNDs was quantified using the dichlorofluorescin (DCFH) reagent. The green fluorescence (λem = 525 nm) of DCFH is known to increase quantitatively when it reacts with ROS generated from the porphyrin.34, 35 As shown in Fig. S6, the fluorescence intensity of DCFH’s exhibits a time-dependent enhancement after the reaction with ROS, generated from PNDs upon irradiation with 808-nm laser. The results further demonstrate that the ROS was mainly produced by PNDs. Without doubt, the PNDs could be promising PDT reagents.

Fig. 5. Laser scanning confocal microscopy images (excited at 488 nm laser) of HCC827 and H23 cells (a); and MDA-MB-231 and HBL-100 cells (b) incubated with C225-PNDs at a concentration of 0.5 mg/mL in the cell culture medium for 8 h at 37 ºC (The scale bar is 20 µm).

To investigate the cellular uptake and subcellular localization of PNDs, the fluorescence imaging was performed on human HeLa cells using a laser scanning confocal microscope (LSCM) (Fig. S7). After



incubation with 0.5 mg/mL PNDs for over 24 hours, the HeLa cells displayed a red fluorescence in cytoplasm, indicating the accumulation of PNDs in cells. Consequently, the PNDs displayed a notable photocytotoxicity towards HeLa cells due to the efficient generation of ROS (Fig. S8). Next we evaluated the targeting ability of C225-PNDs. C225-PNDs was incubated with EGFR-positive cell lines (HCC827 and MDA-MB-231 cells) and EGFR-negative cell lines (H23 and HBL-100 cells) at 0.5 mg/mL, respectively, then analyzed by LSCM. As shown in Fig. 5, the HCC827 and MDA-MB-231 cells (the first row of Fig. 5a and 5b) displayed strong red fluorescent signals after 8 hour’s incubation with C225-PNDs. In contrast, very weak red fluorescent signals was observed in H23 and HBL-100 cells under the same condition (the second row of Fig. 5a and 5b). To further confirm the targeting property of C225-PNDs, the HCC827 cells were cultured with control sample (NH2-PNDs) for 8 hours and then analyzed by LSCM. As shown in Fig. S9, no red fluorescence of NH2-PNDs was observed in HCC827 cells, indicating the C225PNDs could target specifically the cancer cells with over-expression of EGFR via EGFR mediated endocytosis. Moreover, the high photostability of C225-PNDs was demonstrated through continuous irradiation using LSCM. As shown in Fig. S10, the fluorescence intensity of C225-PNDs in HCC827 cells did not show any obvious reduction even after 45 continuous scans excited at 488 nm. To further figure out the localization of C225-PNDs in HCC827 and MDA-MB-231 cells, Hoechst 33342 and LysoTracker® Green DND-26 staining were applied to visualize cell nuclei and lysosome, respectively. As shown in Fig. S11, the blue emission from Hoechst 33342, a nuclei probe, is surrounded by red fluorescence from C225-PNDs, which are almost overlaps with that of green fluorescence of DND-26, indicating the C225PNDs are localized primarily in the lysosomes of cells. Moreover, we have also explored the application of C225-PNDs in two-photon imaging of cancer cells. Fig. S12 shows the images of cells incubated with C225-PNDs using bright-field imaging, one-photon (440 nm excitation) fluorescence imaging and two-photon fluorescence imaging excited by a wavelength (800 nm) outside of the absorption range. It can be seen that the red emission of C225-PNDs located at organelle was clearly identified with femtosecond laser (λex = 800 nm) as the excitation source, indicating the cells can be optically monitored by confocal microscopy with two-photon excitation fluorescence of porphyrin-based nanodots. Afterwards, the photodynamic cytotoxicity of C225-PNDs was investigated through an in vitro CCK-8 assay at different concentrations (0, 5, 10, 20, 50, and 100 µg/mL). In addition, cell viability was normalized to control cells (no drug and nonirradiated). A concentration-dependent cytotoxicity was observed when tumor cells HCC827 and MDA-MB-231 with high-expression of EGFR were exposed to C225-PNDs for 8 hours followed by white light irradiation. As shown in Fig. 6, the cell viabilities of C225-PNDs (100 µg/mL) with irradiation were determined to be 22.3% and 15.2% for HCC827 and MDA-MB-231 cells, respectively. However, over 85% of the two cells lines were still survived upon exposure to C225-PNDs (100 µg/mL) without irradiation, indicating the C225-PNDs alone had minor cytotoxicity against cancer cells. In contrast, when H23 or HBL-100 cells was incubated with C225-PNDs at the concentration range from 0-100 µg/mL for


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8 hours and exposed to the light irradiation, the result is almost same as the non-irradiated controls without significant cytotoxicity. The results clearly indicate there is no obvious uptake of C225-PNDs in these cells with low-expression of EGFR, which is also consistent with the LSCM images of C225-PNDs in H23 and HBL-100 cells as shown in Fig. 5a and 5b. Hence, the selective uptake of C225-PNDs in HCC827 and MDAMB-231 tumor cells due to the over-expression of EGFR, allows the targeted drug delivery to tumor cells and image-guided tumor ablation. 120

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Fig. 6. HCC827 (a), H23 (b), MDA-MB-231 (c), HBL-100 (d) cells viability at different concentrations (0, 5, 10, 20, 50, and 100 µg/mL) of C225-PNDs for 8 hours at 37 ºC without or with irradiation for 30 min with a white light (6.5 mW/cm2 ).

Since C225-PNDs showed an absorption spectrum extended into NIR region, its feasibility of for photoacoustic imaging in vivo was further evaluated, using MDA-MB-23 breast tumor mode mice. The mice were treated with intratumoral injections of C225-PNDs (7 mg/mL, 100 µL) when the tumor volume reached 500 mm3. The photoacoustic images of the tumor were measured at different time intervals. The results indicated that during the whole imaging process, C225-PNDs maintained a relatively constant PA signals after long-term blood circulation (Fig. 7a). The intensities of the mean signal of the region of interest (ROI) (Fig. 7b) clearly demonstrated that the signal with strong intensity lasted at least 24 hours, suggesting the feasibility of C225-PNDs for photoacoustic imaging contrast enhancement and diagnostic applications.



Fig. 7. (a) PA Imaging of C225-PNDs in tumor at 0, 5, 20, 60 min and 24 h after intratumoral injection (7 mg/mL, 100 µL); (b) Relative PA intensity of C225-PNDs in tumor at 0, 5, 20, 60 min and 24 h after intratumoral injection (7 mg/mL, 100 µL); (c) Relative tumor volume of the tumor-bearing mice of the different groups after treatment (n = 3, P < 0.05 for each group); (d) Photographs of the tumor-bearing mice after different days (5, 22, and 30 days) of treatments with C225-PNDs+laser (λex = 808 nm); (e) Tumor size of the different groups after treatment for 30 days; (f) Hematoxylin and eosin (H&E)-stained slices of the tumor, heart, liver, spleen, lung, kidney and intestines in mice after treatments. Scar bar: 100 µm.

The in vivo anti-tumor efficiency of C225-PNDs was validated in mice bearing MDA-MB-231 breast cancer. In this experiment, four groups (three mice in each group) of mice bearing tumor were tested. The mice in the treatment group were intratumoral injected with C225-PNDs (7 mg/mL, 100 µL) first and then irradiated with 808 nm laser (2 W/cm2) for 5 minutes. Mice in the control experiment included three groups: PBS only, C225PNDs only, and PBS+2 W/cm2 laser groups. The therapeutic efficiency was evaluated through the


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measurement of the volume of tumors. As shown in Fig. 7c, all the tumor tissues grew rapidly after the sole treatment of C225-PNDs, PBS or irradiation with NIR laser. One of the three mice in PBS plus irradiation group died of tumor burden at the 30th treatment day. After the treatment for 40 days, the average volume of the tumor is about 25 times that of the initial volume, indicating that C225-PNDs or laser irradiation alone had no effect on the tumor growth. However, the mice treated with C225-PNDs+808 nm laser showed obvious empyrosis in the tumor area, resulting in significant inhibition of tumor growth (Fig. 7d). Among three mice, two mice were completely recovered after 30 days (Fig. 7e). That is to say, although the porphyrin macrocycle was implanted inside the carbon nanodots, the 1O2 could be still generated upon laser irradiation to damage the tumor. In addition, no significant body weight loss of mice was found in all groups after 40 days’ treatment (Figure S13), indicating the injection of C225-PNDs and laser irradiation had no adverse effect on mice. After 40 days of treatment, the mice were sacrificed and the tissue slice of kidney, spleen, liver, heart, lung and tumor were acquired and stained with hematoxylin and eosin. As shown in Fig. 7f, the cells in the tumors were damaged significantly after treatment of C225-PNDs +laser irradiation while the normal tissues of mice treated with C225-PNDs (irradiation or not) were in good condition and well preserved, indicating no obvious damage were observed in the organs, suggesting that C225-PNDs had no evident side effects comparing with the PBS group. These results indicate that C225-PNDs, with the excellent photodynamic therapeutic effect and the enhanced photoacoustic contrast ability, can provide an important and promising platform for cancer theranostics.

3. Conclusions In this contribution, we developed new porphyrin-implanted nanodots by selective pyrolysis of porphyrin macrocycles and citric acid. The PNDs exhibited an intense near-infrared absorption and emission, good aqueous dispersibility and favorable biocompatibility. And the PNDs displayed strong PA signals at 686 nm in a neutral or slightly acidic environment, and weaker signals with pH in a range of 7 to 8. The facile surface modification with Cetuximab afforded C225-PNDs which could target specifically the cancer cells with overexpression of EGFR (HCC827 and MDA-MB-231cells) via EGFR mediated endocytosis. Accordingly, the C225-PNDs could accumulated in HCC827 and MDA-MB-231cells, leading to the remarkable photodynamic therapeutic effect under 808 nm laser irradiation The high PDT efficiency of the C225-PNDs is mainly due to its significant two-photon absorption capability and the depth of light penetration through tissue with 808 nm laser irradiation. Moreover, the excellent photodynamic therapeutic effect and the enhanced photoacoustic contrast ability of C225-PNDs were further validated in mice bearing MDA-MB-231 breast cancer. While technical and conceptual challenges still need to be addressed in practical clinical applications, this new class of porphyrin-implanted carbon nanodots should be very promising to be used in numerous and expanding biological applications.

Acknowledgements



The work was supported by the National Natural Science Foundation of China (NSFC) (grant no. 21601142), Natural Science Foundation of Hubei Province (2017CFB689), Hong Kong Research Grants Council (HKBU 22304115). Zhu thanks the financial support from the Faculty Research Grants (FRG2/14-15/034 and FRG1/14-15/058), the Inter-institutional Collaborative Research Scheme (RC-ICRS/15-16/02E, RCICRS/1617/02C-CHE), and the Interdisciplinary Research Matching Scheme (RC-IRMS/16/17/02CHEM). Cai thanks the support of the Innovative Scientific Research Team Introducing Project of Zhongshan City (2015224). All animal experiments were performed according to the guidelines of the Animal Ethics Committee of Sun Yet-Sen University Cancer Center (Approval No. L102042016110H).

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Porphyrin-Implanted Carbon Nanodots for ... - ACS Publications

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