An intelligent dual stimuli-responsive photosensitizer delivery system with O2-supplying for efficient photodynamic therapy
Graphical abstract
Introduction
Photodynamic therapy (PDT) involves photosensitizers and light to produce reactive oxygen species (ROS), which damage cells and induce apoptosis [1]. However, the application of PDT is limited by tumor hypoxia (pO2 ≤2.5 mmHg) due to the oxygen (O2)-dependent nature of PDT [2]. More serious hypoxia caused by PDT-induced O2 consumption may lead to irreversible drug resistance and tumor metastasis [3]. Therefore, optimizing the efficacy with O2-supplying is of great importance for PDT.
Graphitic-phase carbon nitride nanosheets (g-C3N4, CNs) are a novel type of nanocarrier that can be used in PDT due to their photosensitivity. Unlike traditional metal oxides, such as TiO2 and ZnO, which are only activated upon ultraviolet (UV) light irradiation, g-C3N4 is a visible light-driven photosensitizer [4,5]. Moreover, the high degree of condensation of the tri-s-triazine ring structure in g-C3N4 makes it highly photoluminescent and suitable for bioimaging [5,6]. g-C3N4 can be synthesized as nanosheets [7,8], quantum dots [9], hollow structures [10], and mesoporous structures [11]. Graphitic-phase mesoporous carbon nitride nanosheets (mpg-C3N4, MCNs) possess a uniform mesoporous sheet structure, which not only enables storage of drugs but also enhances ROS generation under visible light illumination due to the higher surface area [4,5,10]. Nevertheless, its low selectivity and tumor targeting function hamper use of mpg-C3N4 in PDT [12].
Malignant cancer cells generate excess H2O2 (50–100 μM) in the tumor microenvironment because of the aberrant metabolism of cancer cells [13,14]. Catalase (CAT) mediates decomposition of H2O2 to oxygen and water inside the tumor [15]. CAT, which can convert about 5 million H2O2 molecules per minute, has been explored in recent years to overcome tumor relieve [13,16]. Therefore, a CAT-based strategy to relieve tumor hypoxia and concentrate ROS in the tumor area seems feasible [17].
In this study, we developed a HAase/H2O2 dual stimulation photosensitizer-loading system to promote photosensitizer release, achieving O2-enriched PDT to relieve tumor hypoxia and improve PDT effect. HMME and CAT were wrapped in the pores of mpg-C3N4, and HA was coated onto the surface to facilitate photosensitizer delivery to tumors (HA@mpg-C3N4-HMME/CAT). HMME was used due to its high quantum yield of ROS in the therapeutic excitation window (350–550 nm). CAT encapsulated in mpg-C3N4 with largely retained enzyme activity and increased stability protect from protease is able to degrade endogenous H2O2 inside tumor to enhance in situ O2 concentration. HA targets tumors via CD44 receptors, which are overexpressed on tumor cells [18,19]. It also enhances the intracellular uptake of MCNs and improves the therapeutic efficacy. After being taken up by cancer cells, HA@mpg-C3N4-HMME/CAT intracellular HAase degraded the HA coating, resulting in release of HMME and CAT. Intracellular H2O2 penetrated the pores and was degraded by CAT to generate O2; this also promoted release of HMME due to efflux of O2. Finally, HMME and mpg-C3N4 generated ROS in the presence of O2 to kill cancer cells under light-emitting diode (LED) light irradiation. The mechanism of the dual stimulation (H2O2-activatable and hyaluronidase-stimulation), dual response (photosensitizer mpg-C3N4 and HMME), and O2-supplying photosensitizer-delivery system (HA@mpg-C3N4-HMME/CAT) is shown in Fig. 1.
Section snippets
Materials and reagents
Hematoporphyrin monomethyl ether (HMME, purity >98%) was sourced from Beijing Yi-He Biotech Co. Ltd (Beijing, China). All solvents and reagents were analytical grade. Sodium hyaluronate (purity >98%, MW = 7.7 kDa) was bought from Bloomage Freda Biopharm Co. Ltd (Jinan, Shandong). The dialysis bags (molecular weight cutoff = 8–14 kDa) were got from Spectrum Laboratories (Rancho Dominguez, CA, USA). Cyanamide and 12 nm SiO2 particles (Ludox HS40), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
Cell line
The B16-F10 murine melanoma cell line was originally obtained from the Chinese Academy of Sciences Cell Bank (TCM36). Cells were cultured in the normal DMEM with 10% heat-inactivated fetal bovine serum (FBS) and 10 IU/mL of antibiotics (penicillin/streptomycin) in 5% CO2 at 37 °C in a humidified incubator (BPN-80CRW, Shanghai).
Cellular uptake
B16-F10 cells were allowed to adhere on the 6-well plates for 24 h, and then incubated cells with HMME and HA@mpg-C3N4-HMME/CAT (HMME concentration: 30 μg/mL) for 1 h,
Xenograft tumor mouse model
Female C57 mice were purchased from Henan Laboratory Animal Center and all animal experiments were performed under a protocol approved by the Henan Laboratory Animal Center. To develop the tumor model, 2 × 106 B16-F10 cells suspended in 0.1 mL PBS were subcutaneously into the right shoulder of the female C57 mice (18–22 g, 4–6 weeks old). The mice can be used when tumor volume reached 80–120 mm3.
Biodistribution of HA@mpg-C3N4-HMME/CAT
The tumor-bearing mice were intravenous injection with HMME or HA@mpg-C3N4-HMME/CAT (HMME
Characterization of the preparations
The use of g-C3N4 (CNs) as nanocarriers has attracted much attention for PDT due to their photosensitivity [4,5]. Enhancement of the photosensitivity and drug-loading capacity of bulk g-C3N4 by introducing a mesoporous structure was reported by Ho et al. [10] However, the use of MCNs in photosensitizer delivery systems has not been extensively investigated. In this study, a multifunctional nanocomposite was produced (Fig. 1). As shown in Fig. 1, first, the MCN nanosheets were synthesized. Then
Conclusions
In summary, a tumor-targeting HAase/CAT dual activatable and MCNs/HMME dual response photosensitizer-delivery system was developed. MCNs, which were used as the carrier loaded with HMME and CAT, and HA was grafted onto the surface of MCNs. HA@mpg-C3N4-HMME/CAT had a relatively high photosensitizer loading efficiency up to 59.82%. In vitro, HMME release was limited in the absence of H2O2 and HAase, but increased upon addition of H2O2/HAase. Uptake of HA@mpg-C3N4-HMME/CAT via CD44
Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (Nos. 81673021, 81573364), the China Postdoctoral Science Foundation (no. 2014M562002 and 2015T80783), the scientific and technological project of Henan Provience (182102310117). Thanks the supported of modern analysis and computering center of Zhengzhou university.
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