Highly Biocompatible Chlorin e6-Loaded Chitosan Nanoparticles for

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Highly Biocompatible Chlorin e6-loaded Chitosan Nanoparticles for Improved Photodynamic Cancer Therapy Yuanfu Ding, Shengke Li, Lijun Liang, Qiaoxian Huang, Lihui Yuwen, Wen Jing Yang, Ruibing Wang, and Lianhui Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01522 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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ACS Applied Materials & Interfaces

Highly Biocompatible Chlorin e6-loaded Chitosan Nanoparticles for Improved Photodynamic Cancer Therapy Yuan-Fu Ding,1, 2 Shengke Li,2 Lijun Liang,1 Qiaoxian Huang,2 Lihui Yuwen,1 Wenjing Yang,1 Ruibing Wang,2* and Lian-Hui Wang1* 1

Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory

of Biosensors, Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210023, China 2

State Key Laboratory of Quality Research in Chinese Medicine, and Institute of Chinese

Medical Sciences, University of Macau, Taipa, Macau, China. KEYWORDS: Natural chitosan; Drug delivery systems, Biocompatibility, Photodynamic therapy, Cell imaging

ABSTRACT: The photosensitizer Chlorin e6 (Ce6) has been frequently employed for photodynamic therapy (PDT) of cancer, however its non-specific toxicity has limited its clinical applications. In this study, we prepared chitosan nanoparticles (CNPs), with a mean diameter of approximately 130 nm, by a non-solvent-aided counterion complexation method in an aqueous solution, into which Ce6 could be physically entrapped during the preparation process. These

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CNPs

and

Ce6-loaded

CNPs

(CNPs-Ce6)

were

fully

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characterized

by

UV-vis,

photoluminescence, and Fourier transform infrared spectroscopic analysis, as well as dynamic light scattering and TEM measurements. More importantly, the biocompatibility of the otherwise toxic Ce6 was significantly improved upon its loading into the CNPs, as demonstrated by both confocal laser scanning microscopy analysis and MTT assays. Furthermore, the PDT efficiency of Ce6-loaded CNPs was dramatically enhanced, in comparison with that of the free Ce6, as shown by both MTT and flow cytometry assays. This discovery provides a novel strategy for improving the biocompatibility and therapeutic efficacy of PDT agents by using a natural, biocompatible polysaccharide carrier.

1. INTRODUCTION Nowadays, cancer induced mortality and morbidity have become a worldwide issue that constantly challenges the well-being as well as the healthcare system of human society.1 Apart from the traditional cancer therapeutic methods such as surgery, chemotherapy,2-4 radiotherapy, and immunotherapy, other novel strategies have also been extensively developed in recent years.5-7 In particular, photodynamic therapy (PDT) is one of the emerging cancer therapeutic methods that has been recognized as an indispensable tool in oncology,8 as it not only avoids invasive injuries and other side-effects resulting from surgery and radiotherapy,9 but also circumvents potential drug resistance caused by chemotherapy.10-11 During PDT process, cytotoxic reactive oxygen species (ROS) such as singlet oxygen (1O2), free radicals, and peroxides are generated by photoexcited photosensitizers (PSs), which can selectively and irreversibly destroy the surrounding cancer cells and tumor tissues.12 The efficacy of PDT is highly dependent on the oxygen content of the tissues, light intensity and PSs efficiency, among which PSs efficiency holds the crucial role.13-14 Due to the high singlet oxygen generation


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efficiency15 and near infrared light activation that allows deep tissue penetration,16 Chlorin e6 (Ce6), as a FDA approved second-generation PS, has shown promising clinical efficacies.17-18 Additionally, the fluorescent emission of Ce6 or Ce6-based PSs could also serve as imaging agents for potentially tracking targeted therapy.19

Despite these achievements, the high

hydrophobicity and non-selective cytotoxicity of Ce6 and other porphyrin-based PSs has indeed limited their clinical applications. Additionally, the non-specific, low accumulation of Ce6 and Ce6-based PSs in tumor tissues and cells has often compromised their therapeutic efficacy.16 To address these issues, chemical modification of Ce6 with hydrophilic, biocompatible, and/or targeting molecules, and the encapsulation of Ce6 within biocompatible nanocarriers are two main strategies that have been investigated.

For instance, chemical conjugation with

biocompatible molecules such as hyaluronic acid (HA),20 polyethylene glycol (PEG),21 and human serum albumin (HSA),22 have been demonstrated to improve the hydrophilicity, biocompatibility, and efficacy of Ce6 PSs. In addition, liposomes,23 polymers,24 and inorganic nanoparticles25-27 have also been developed as nanocarriers to encapsulate Ce6 for the reduction of its non-specific toxicity and improvement of therapeutic efficacy. However, tedious synthesis and complicated materials engineering are often required during the functional groups grafting and nanoparticles preparation procedures. Additionally, synthetic nanocarriers may also have biocompatibility issues, especially inorganic ones.28-29 Therefore, the employment of naturally derived polysaccharides for facile fabrication of drug delivery carriers would have less safety concerns.20, 30-32 Indeed, Lee and coworkers reported two strategies regarding the utilization of glycol chitosan nanoparticles as carriers for improved delivery of Ce6 for cancer therapy.33 In one strategy, Ce6 was physically loaded into hydrophobically-modified glycol chitosan nanoparticles via self-assembly of amphiphilic conjugates. The other strategy was to chemically


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graft Ce6 onto the backbone of glycol chitosan polymers, therefore Ce6 acted as they hydrophobic moiety of the amphiphilic conjugates, which subsequently self-assembled into nanoparticles. However, polysaccharide derivatization still involved chemical synthesis, which usually adds cost and time, and might introduce new biocompatibility issues. Furthermore, the glycol chitosan nanoparticles bearing directly grafted Ce6 were proven to self-assemble into nanoparticles with uncontrollable particle sizes.

In this study, we utilized natural chitosan

directly for the first time to fabricate Ce6-loaded nanoparticles for improvements of both the biocompatibility and therapeutic efficacy of Ce6. As shown in Fig. 1, chitosan nanoparticles were prepared by a non-solvent-aided counterion complexation method and the photosensitizer Ce6 was encapsulated into chitosan nanoparticles with high loading efficiency.34-36 The physicochemical properties and PDT effects of the therapeutic NPs were fully evaluated in vitro. Our work provides a novel, facile strategy for improving the biocompatibility and therapeutic efficacy of PDT agents.


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Figure 1. Representative scheme depicting Ce6-loaded chitosan nanoparticles for photodynamic therapy and imaging. 2. EXPERIMENT SECTION 2.1. Materials. Low molecular weight chitosan was purchased from Aoxing Biomedical Company (Zhejiang, China) and used without purification. Chlorin e6 (Ce6), glutaraldehyde, ethylene diamine tetraacetic acid (EDTA), 9,10-anthracenedipropionicacid (ADPA), and 2,7duchlorodihydrofluorscein diacetate (DCHFDA) were purchased from Sigma-Aldrich. Hydroxylamine hydrochloride was purchased from Aladdin. Ultra-pure water was prepared via a Milli-Q system water (18.2 MΩ cm) and used for all of the experiments. All reagents were used as received without further purification. 2.2. Preparation of blank and Ce6 loaded chitosan nanoparticles. Blank chitosan nanoparticles (CNPs) were fabricated according to a published method with minor modification.36 Briefly, 20 mg of low molecular weight chitosan and 5 mg of EDTA were dissolved in 4 mL of water via ultrasonication and stirring. The two oppositely charged components would bind together through electrostatic interactions, and subsequently form chitosan-EDTA nanoparticles upon the dropwise addition of 8 mL of ethanol. After the clear solution gradually became milky (NPs formed), 250 µL glutaraldehyde solution (1 wt %) was added into the colloid system to solidify the formed NPs. The resultant solution was dialyzed against distilled water to remove excess EDTA, ethanol and unassembled chitosan. The stirring rate, crosslinking time and degree of crosslinking for CNPs fabrication were optimized using orthogonal analysis, and the final concentration of the CNPs was determined by centrifugation and lyophilization. In order to ensure the formation of glutaraldehyde-free CNPs, the residual glutaraldehyde was analyzed by hydroxylamine hydrochloride method (which would react with


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glutaraldehyde to yield a new absorption perk at 235 nm). A linear curve of glutaraldehyde was first prepared by using this method. Briefly, various concentrations of glutaraldehyde were added with fixed concentration of hydroxylamine hydrochloride. And UV-vis spectra were recorded after 30 min reaction. The concentration of glutaraldehyde in supernatant was monitored and calculated by the absorbance at 235 nm based on the standard linear curve. Photosensitizer Ce6 was encapsulated into CNPs by physical absorption.36 Briefly, different concentrations of Ce6 (DMSO as solvent) were added dropwise to a CNP solution and stirred overnight in the dark at room temperature. Subsequently, the photosensitizer Ce6-loaded NPs (CNPs-Ce6) were separated by centrifugation (10,000 rpm, 10 min) and re-dispersed in water. The NPs were washed with water and ethanol for three times to avoid the adhesion of free Ce6 onto the surfaces of the NPs. The supernatant was collected and the total amount of un-trapped Ce6 was determined by UV-vis spectroscopy, based on a linear curve of Ce6 established by measuring the UV-Vis absorbance (410 nm) of increased concentration of Ce6 in 1% DMSO aqueous solution. The loading and encapsulation efficiency of Ce6 by the NPs were calculated by using the following equations: Ce6 loading efficiency (%) = (Ce6 in CS NPs) / (loaded CS NPs) × 100%

(1)

Ce6 encapsulation efficiency (%) = (Ce6 in total - Ce6 in supernatant) / (Ce6 in total) × 100% (2) 2.3. Characterization of the CNPs and CNPs-Ce6. UV-vis absorption and fluorescence properties of the CNPs and CNPs-Ce6 were monitored using a UV-3600 spectrophotometer (Shimadzu, Japan) and RF-5301PC spectrofluorophotometer (Shimadzu, Japan), respectively. Infrared spectra were acquired using a Bruker Vertex 70 FT-IR spectrometer. The morphologies of the NPs were examined with a transmission electron microscope (TEM, Hitachi, Japan). The


6

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fluid properties of NPs including the hydraulic diameter, polymer dispersion index, and zeta potential were measured on Zeta-PALS instrument (Brookhaven, USA). 2.4. Detection of singlet oxygen between free Ce6 and CNPs-Ce6. Singlet oxygen was detected by a 1O2 sensor ADPA using a classical method.26 Briefly, 100 uL of ADPA (DMSO, 3 mM) was added into 3 mL CNPs, free Ce6 and CNPs-Ce6, respectively. Subsequently, UV-vis spectra of each solution were recorded after they had been irradiated by a laser beam at under 635 nm laser beam (50 mW/cm2) at different time points. The decrease in the absorbance of ADPA corresponded to the generation of 1O2. 2.5. Cell Culture and cytotoxicity evaluation of CNPs. A549 cell line (human lung adenocarcinoma) and L02 cell line (human liver cell) were cultured in 25 cm2 flasks using 1640medium and DMEM supplemented with 10% FBS at 37 °C with 5% CO2, respectively. The cytotoxicity of CNPs to these cells was evaluated with MTT assays.37 Typically, A549 cells and L02 cells (3×104 cells per well) were cultured in 96-well plates with 150 µL of culture medium for 24 h to allow attachment. Subsequently, the cells were treated with various concentrations of CNPs. After 12 and 24 h of incubation, 50 µL of MTT was added into each well, followed by the addition of DMSO (200 µL) after 4 h of incubation. The optical density was recorded on an ELIASA at wavelengths of 490 and 570 nm, respectively.

The relative cell viability was

calculated as follows: Relative Cell Viability (%) = (ODSample - ODBlank) / (ODControl - ODBlank)×100%

(3)

2.6. Photodynamic therapy and singlet oxygen evaluation in vitro. The therapeutic efficacy of free Ce6 and CNPs-Ce6 was studied by MTT assays. Briefly, A549 cells treated with various concentrations of free Ce6 and CNPs-Ce6 were incubated for 24 h in the absence and presence of


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10 min laser (635 nm, 50 mW/cm2) irradiation. Subsequently, the cell viability was calculated to evaluate the therapeutic efficacy. DCHFDA, a fluorescence singlet oxygen sensor, can directly react with 1O2, resulting in a dramatic increase in fluorescence intensity.38 To visualize and quantify 1O2 generation in cytoplasm, 1 µL of DCHFDA was added into internalized cells prior to irradiation. Subsequently, confocal images were taken before and immediately after irradiation. Additionally, the changes in the fluorescence intensity were collected at the beginning and the end of the photoreaction. 2.7. Confocal imaging. A549 cells were first incubated in 35 mm confocal dishes for 24 h, and subsequently treated with free Ce6 and CNPs-Ce6 containing the same concentration of Ce6. Confocal images were recorded by an inverted confocal laser scanning microscope after the cells were incubated for 1, 2, 4, 12 and 24 h, respectively. All treated cells were washed with PBS prior to confocal imaging in order to avoid accumulation of the non-internalized Ce6. 2.8. Flow cytometry analysis. In order to quantify the cell uptake, A549 cells were treated with fresh culture media containing free Ce6 and CNPs-Ce6, respectively. The same with confocal imaging, the cells were cultured for different periods (1 h, 2 h, 4 h, 12 h and 24 h). After internalization, cells were harvested and analyzed using a flow cytometer. The FL3-A channel was selected to detect the fluorescence signal of Ce6. 2.9. Statistical analysis. The data presented as the average value ± the standard deviation of independent experiments which were performed at least 3 times. The p-value of statistical differences between pairs between groups were statistically analyzed by one-way ANOVA followed by Tukey's range test using the SPSS package, (*P < 0.05, **P < 0.01, or ***P < 0.001).


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3. RESULTS AND DISCUSSION The details for preparing highly biocompatible chitosan nanoparticles (CNPs) were described in the experimental section. As depicted in Fig. 2a, non-solvent-aided counterion complexation method was employed to fabricate the drug nanocarrier. The preparation parameters were optimized by using orthogonal analysis (Table S1). The diameter of CNPs containing no drugs was at an average of 165 nm (±8.0 nm) in an aqueous solution, as determined by DLS (Fig. 2b). TEM measurements characterization (Fig. 2b inset) revealed uniform spherical morphology of these blank CNPs, with an average diameter of 130 nm (± 12.5 nm) (Fig. S1a) which is consistent with the DLS result. The slight decrease of the diameter determined by TEM in comparison to that by DLS was due to the shrinking of the NPs in the dry state that incurred during TEM measurements.39 To prepare photosensitizer-loaded chitosan nanoparticles, Ce6 solution (in DMSO) was added dropwise into the CNPs solution with moderate stirring (400 rpm) overnight. After purification, Ce6-loaded CNPs (CNPs-Ce6) were obtained with an average diameter of 180 nm (± 11.4 nm), as determined by DLS (Fig. 2c). TEM characterization (Fig. 2c inset) confirmed the formation of uniform spherical CNPs-Ce6, similar to that of the blank CNPs, and the diameter was found to be 135 nm (± 15.6 nm) (Fig. S1b). In aqueous solutions, the zeta potentials of the CNPs and CNPs-Ce6 were 28.62 and 24.77 mV, respectively (Fig. S1c). The very moderate decrease of the zeta potential of CNPs was likely due to the successful loading of electronegative Ce6, which counteracted a fraction of the positive charges of CNPs. Spectroscopic experiments were employed to further evaluate the successful loading of Ce6. In the FT-IR spectra (Fig. 2d), the intensity of the peak at 1631cm-1 corresponding to the amino groups in the chitosan chain, was dramatically decreased and the peak shape became sharper and exhibited a slight blue shift to 1625 cm−1, upon loading of Ce6, likely attributed to the reactions


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between the carboxyl groups of Ce6 and the amino groups of chitosan to form a Schiff base.40 Subsequently, UV-vis experiments were conducted to determine the encapsulation efficiency (EE) and loading efficiency (LE) of the CNPs-Ce6 (Fig. 2e, Fig. S1d). The LE was found to increase with an increasing feeding concentration of Ce6, whereas the EE was maintained at approximately 80% or above, indicating that CNPs could be an efficient drug carrier for Ce6. Additionally, after encapsulation in CNPs, the fluorescence intensity of Ce6 still remained at a high level (approximately 73% retained), suggesting that Ce6 could still serve as an efficient imaging agent that could be potentially used for tracking its delivery (Fig. S1e). 25-26

Figure 2. Representative scheme depicting the preparation CNPs-Ce6 (GA = glutaraldehyde) (a); DLS and TEM (inset) images of CNPs (b), and CNPs-Ce6 (c); UV-vis spectra of CNPs, and CNPs-Ce6 (d) FT-IR spectra of chitosan chains, CNPs and CNPs-Ce6; EE and LE results


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determined by UV-vis (e); physiological stability of CNPs-Ce6 in water; PBS; 1640-medium and FBS (f). After confirmation that there was no residual glutaraldehyde in the blank CNPs after dialysis (Fig. S1f), the physiological stability of the CNPs-Ce6 was investigated subsequently. The lyophilized CNP-Ce6 was firstly dispersed in various biologically relevant solutions including water, PBS buffer, 1640 medium and FBS, and the particle size was determined by DLS. As illustrated in Fig. 2f, the particle sizes of CNP-Ce6 in water, PBS buffer, 1640 medium, and FBS, were rather similar, with an average diameter of around 160~200 nm. The slight increase of the particle size in FBS and 1640 medium might be attributed to moderate adhesion of protein and polysaccharides on the surface of the CNPs-Ce6. Additionally, the CNPs were found to be stable under neutral conditions (PBS buffer, pH = 7.4) after 5 days (Fig. S2a), whereas the particle size of the CNPs increased along with time when under weak acidic environment (pH = 5.5) (Fig. S2a), suggesting that the prepared CNPs could also be employed as pH responsive drug delivery platform. Finally, the CNPs aqueous solution was kept under room temperature for examination of its storage stability, and the DLS results showed no obvious aggregations observed after storing the sample for 7 days (Fig. S2b). Singlet oxygen (1O2) generation is a crucial indicator when evaluating PDT efficiency. 9,10anthracenedipropionic acid (ADPA) and 2,7-duchlorodihydrofluorscein diacetate (DCHFDA) were employed to measure the singlet oxygen generation in aqueous solutions and in cells, respectively, as the generated singlet oxygen upon irradiation of a PDT agent would decompose ADPA causing an absorbance decrease in aqueous solution, and would interact with DCHFDA in the cells causing a green fluorescence increase, respectively. In an aqueous solution, the absorbance of ADPA at 375 nm in all three groups (CNPs, free Ce6, and CNPs-Ce6) decreased,


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suggesting the generation of singlet oxygens in all three samples upon laser irradiation (635 nm, 50 mW/cm2, Fig. S3).25 The singlet oxygen generation efficiencies of the three groups, are linearly correlated to the reaction rates constants (k) between ADPA and singlet oxygen, which were 0.05×10-3 (negligible), 0.6×10-3, and 1.0×10-3, respectively, as calculated via the classic equations proposed by Raoul Kopelman research group (Fig. 3a).41 In comparison with that of the free Ce6, the k value for CNPs-Ce6 had remarkably increased, indicating that the singlet oxygen generation efficiency of Ce6 was dramatically enhanced after encapsulation in the CNPs. This was likely due to the inhibition of the aggregation of Ce6, and the associated inhibition of the quenching of 1O2 upon irradiation.26 The singlet oxygen generation was further evaluated in human lung cancer cells (A549). After incubating the cells with free Ce6 (5 µM) and CNPs-Ce6 (Ce6 5 µM), respectively, for 2 h, 1 µL of DCHFDA (10 mM) was added to the medium, which was followed by irradiation for 0, 3, 6 and 10 min at room temperature, and the fluorescence images were recorded on a confocal fluorescence microscope after each irradiation treatment. As shown in Fig. S4, the intracellular green fluorescence of DCHFDA increased with increased irradiation time for both free Ce6 and CNPs-Ce6 treated groups, suggesting an irradiation-time dependent generation of singlet oxygen in both cases. Meanwhile, the CNPs-Ce6 treated cells exhibited much stronger fluorescence inside cells in comparison with that of free Ce6. Furthermore, the average green fluorescence intensity inside cells quantified by randomly choosing 10 cells in the view by confocal microscopy (Fig. 3b), demonstrated that the singlet oxygen generated inside cells by Ce6 was also significantly enhanced when it is encapsulated in CNPs, likely due to the higher singlet oxygen generation efficiency of CNPs-Ce6, as well as the increased cellular uptake (will be discussed in the following section of “cellular imaging and uptake), at least in part, of CNPs-Ce6


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as a nanomedicine. Taken together, the much improved single oxygen generation by CNPs-Ce6, in comparison with that of free Ce6, will likely contribute to better PDT therapeutic efficacy.

Figure 3. The reaction rate constants comparison between CNPs, free Ce6 and CNPs-Ce6 (a), green fluorescence intensity of DCHFDA in living cells incubated with PBS, free Ce6, and CNPs-Ce6 upon radiation with a 635 nm laser (50 mW/cm2) for 0, 3, 6, and 10 min, respectively (b). Data were expressed as the mean ± SD (n = 3). The cytotoxicity (including phototoxicity) of both free Ce6 and CNPs-Ce6 was evaluated by using a human lung cancer cell line (A549). After incubation with Ce6 and CNP-Ce6, respectively, for 24 h, the cells were irradiated (635 nm laser, 50 mW/cm2) for 5 min (light dose = 30 J/cm2), and the relative viability of cells was determined by MTT assay. Without laser irradiation, the relative viability of the cells treated with free Ce6 showed a dose-dependent manner, with an approximate IC50 of 5 µM (Fig. 4a), suggesting its significant cytotoxicity even in the absence of laser irradiation. After encapsulation by the CNPs, Ce6 at a concentration of up to 5 µM, showed negligible cytotoxicity, and is comparable to the control group, suggesting that the biocompatibility of Ce6 was significantly improved upon its loading into the CNPs


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nanocarrier. Meanwhile, as expected, free CNPs exhibited an excellent in vitro biocompatible profile on both A549 cell line (Fig. S5a) and L02 cell line (Fig. S5b). In addition, a human liver cell line, L02, was also employed for evaluation of the nonspecific toxicity of free Ce6. The data also suggested that the biocompatibility of Ce6 was dramatically improved upon its loading into the CNPs (Fig. S6). Furthermore, in vitro phototoxicity of Ce6 in the free form and the CNPs form was evaluated as a direct assessment of its therapeutic efficacy as a PDT agent. Upon laser irradiation (Fig. 4b), the cell viability of both free Ce6 and CNPs-Ce6 treated groups decreased in comparison with the non-irradiated groups (Fig. 4a), suggesting a much increased cytotoxicity induced by the generated singlet oxygen upon irradiation. Of note, the cytotoxicity of the CNPsCe6 upon laser irradiation was significantly improved, when compared with that of the free Ce6, suggesting that CNPs significantly enhanced the phototoxicity of Ce6, consistent with our observations on their singlet oxygen generation rates in vitro (Fig. 3).

Figure 4. Cytotoxicity of free Ce6 and CNPs-Ce6 in A549 cell line in the dark (a) and irradiated with a laser (635 nm laser, 50 mW/cm2) for 5 min (b). Data were expressed as the mean ± SD (n = 6).


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As determined by fluorescence experiment (Fig. S1e), the encapsulation of Ce6 by CNPs well maintained its fluorescent properties, with a very modest fluorescence decrease observed. Therefore, the intracellular distribution and cellular uptake of Ce6 and CNPs-Ce6 in A549 cell line was investigated via confocal microscopy and flow cytometry (FCM), respectively. Cells incubated with free Ce6 and CNPs-Ce6 for different incubation time was analysed by FCM (Fig. 5a and b). The cancer cell line exhibited increased uptake of both Ce6 and CNPs-Ce6 on a timedependent manner. In comparison with those incubated with free Ce6, cells incubated with CNPs-Ce6 showed significantly stronger red fluorescence (from Ce6) within the cells, suggesting that encapsulation of Ce6 by CNPs significantly improved its cellular uptake, likely attributed to the endocytosis of the nano-scale particles by the cells.42

Figure 5. Flow cytometry analysis of CNPs-Ce6 in A549 cell line (a) and quantitative analysis of cellular uptake of free Ce6 and CNPs-Ce6 upon incubation (b) for 1 h, 2 h, 4 h, 12 h and 24 h, respectively. Data were expressed as the mean ± SD (n = 6). In addition, confocal microscopy was utilized to visually show the cellular internalization Ce6 and for the purpose of molecular imaging (Fig. 6). The observation of bright, red fluorescence in cells demonstrated that Ce6 may serve as an efficient imaging agent for possible tracking of PDT


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delivery and therapy. In addition, both free Ce6 and CNPs-Ce6 treated cells exhibited increased red fluorescence as time elapsed, suggesting a time-dependent uptake manner of both free and CNPs-encapsulated Ce6 by the cancer cells, consistent with FCM analysis. After all, both FCM analysis and confocal microscopic analysis have confirm that the encapsulation of Ce6 into CNPs significantly improved its cellular uptake and facilitated cellular imaging.

Figure 6. Confocal images of A549 cells incubated with CNPs (top row), free Ce6 (middle row), and CNPs-Ce6 (bottom row) after different incubation times (1, 2, 4, 12 and 24 h). 4. CONCLUSION In this study, a natural polysaccharide, chitosan, was directly employed to fabricate nanocarriers for the delivery of a photodynamic therapy photosensitizer Chlorin e6 (Ce6) towards improved cancer treatment. The as-prepared chitosan nanoparticles (CNPs) were stable in various biologically-relevant media. Very importantly, the inherent non-specific toxicity of Ce6 was significantly decreased after it had been loaded in the CNPs, whereas its phototoxicity was dramatically improved, due to increased cellular uptake of the nanocarriers as well as the


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increased singlet oxygen generation of Ce6. Meanwhile, the fluorescent properties of Ce6 were not affected upon its encapsulation by the CNPs, thus fluorescent tracking of its delivery and cellular uptake became possible. We provide a facile approach to prepare a nano-PDT medicine using a natural, biocompatible polysaccharide for improved PDT agent delivery and cancer therapy with improved toxicity and therapeutic efficacy profile, which may be of much practical value in the field of PDT delivery and therapy and also provides new insights for design and preparation of novel, biocompatible nanomedicine platform. ASSOCIATED CONTENT Supporting Information Orthogonal experiments for preparing chitosan nanoparticles; TEM images (DLS inset) and zeta potential data of blank CNPs and CNPs-Ce6; UV-vis and fluorescence spectra of Ce6 and CNPsCe6, stability study of CNPs, glutaraldehyde residue determination; UV-vis spectra of CNPs, free Ce6, CNPs-Ce6 after irradiation by laser; confocal images for in vitro singlet oxygen evaluation, in vitro biocompatibility study of CNPs; and cytotoxicity study of Ce6 and CNPsCe6 under dark in L02 cell line are provided in the supporting information. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT The work was financially supported by the National Key Research and Development Program of China (2017YFA0205302), Macau Science and Technology Development Fund, Macao SAR (FDCT/030/2017/A1), and Research Committee at University of Macau (MYRG2016-00165ICMS-QRCM). REFERENCES (1) Moorthi, C.; Manavalan, R.; Kathiresan, K., Nanotherapeutics to Overcome Conventional Cancer Chemotherapy Limitations. J. Pharm. Pharmaceut. Sci. 2011, 14, 67-77. (2) Hu, B.; Leow, W. R.; Cai, P.; Li, Y. Q.; Wu, Y. L.; Chen, X., Nanomechanical Force Mapping of Restricted Cell-To-Cell Collisions Oscillating between Contraction and Relaxation. ACS Nano 2017, 11, 12302-12310. (3) Wu, Y. L.; Engl, W.; Hu, B.; Cai, P.; Leow, W. R.; Tan, N. S.; Lim, C. T.; Chen, X., Nanomechanically Visualizing Drug-Cell Interaction at the Early Stage of Chemotherapy. ACS Nano 2017, 11, 6996-7005. (4) Hu, B.; Leow, W. R.; Amini, S.; Nai, B.; Zhang, X.; Liu, Z.; Cai, P.; Li, Z.; Wu, Y. L.; Miserez, A.; Lim, C. T.; Chen, X., Orientational Coupling Locally Orchestrates a Cell Migration Pattern for Re-Epithelialization. Adv. Mater. 2017, 29, DOI: 10.1002/adma.201700145. (5) Allison, R. R.; Sibata, C. H., Oncologic photodynamic therapy photosensitizers: a clinical review. Photodiagnosis Photodyn. Ther. 2010, 7, 61-75. (6) Kim, J.; Kim, J.; Jeong, C.; Kim, W. J., Synergistic nanomedicine by combined gene and photothermal therapy. Adv. Drug Deliv. Rev. 2016, 98, 99-112. (7) Chow, E. K.; Ho, D., Cancer nanomedicine: from drug delivery to imaging. Sci. Transl. Med. 2013, 5, 216rv4.


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Highly Biocompatible Chlorin e6-Loaded Chitosan Nanoparticles for

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