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Abstract
Background:
Nonspecific tumor targeting, potential relapse and metastasis of tumor after treatment are the main barriers in clinical photodynamic therapy (PDT) for cancer, hence, inhibiting relapse and metastasis of tumor is significant issues in clinic.
Purpose:
In this work, chidamide as a histone deacetylases inhibitor (HADCi) was bound onto a pH-responsive block polymer folate polyethylene glycol-b-poly(aspartic acid) (PEG-b-PAsp) grafted folate (FA-PEG-b-PAsp) to obtain the block polymer folate polyethylene glycol-b-poly(asparaginyl-chidamide) (FA-PEG-b-PAsp-chidamide, FPPC) as multimodal tumor-targeting drug-delivery carrier to inhibiting tumor cell proliferation and tumor metastasis in mice.
Methods:
Model photosensitizer pyropheophorbide-a (Pha) was encapsulated by FPPC in PBS to form the polymer micelles Pha@FPPC [folate polyethylene glycol-b-poly(asparaginyl-chidamide) micelles encapsulating Pha]. Pha@FPPC was characterized by transmission electron microscope and dynamic light scattering; also, antitumor activity in vivo and in vitro were investigated by determination of cellular ROS level, detection of cell apoptosis and cell cycle arrest, PDT antitumor activity in vivo and histological analysis.
Results:
With favorable and stable sphere morphology under transmission electron microscope (TEM) (~93.0 nm), Pha@FPPC greatly enhanced the cellular uptake due to its folate-mediated effective endocytosis by mouse melanoma B16-F10 cells and the yield of ROS in tumor cells induced by PDT, and mainly caused necrocytosis and blocked cell growth cycle not only in G2 phase but also in G1/G0 phase after PDT. Pha@FPPC exhibited lower dark cytotoxicity in vitro and a better therapeutic index because of its higher dark cytotoxicity/photocytotoxicity ratio. Moreover, Pha@FPPC not only significantly inhibited the growth of implanted tumor and prolonged the survival time of melanoma-bearing mice due to both its folate-mediated tumor-targeting and selectively accumulation at tumor site by EPR (enhanced permeability and retention)effect as micelle nanoparticles but also remarkably prevented pulmonary metastasis of mice melanoma after PDT compared to free Pha, demonstrating its dual antitumor characteristics of PDT and HDACi.
Conclusion:
As a folate-mediated and acid-activated chidamide-grafted drug-delivery carrier, FPPC may have great potential to inhibit tumor metastasis in clinical photodynamic treatment for cancer because of its effective and multimodal tumor-targeting performance as photosensitizer vehicle.
Acknowledgments
This work was financially supported by grants from Shanghai Municipal Planning Commission of Science Research Fund for Youth Project (2016Y0039), Shanghai Natural Science Foundation (16ZR1444200), and National Natural Science Foundation of General Program (21778022) and Youth Program (81603124).
Disclosure
The authors report no conflicts of interest in this work.
Supplementary materials
Synthesis of folate polyethylene glycol (PEG)-b-poly(asparaginyl-chidamide) (FA-PEG-b-PAsp-chidamide, FPPC)
Synthesis of L-aspartate benzyl ester-N-carboxylic acid anhydride (BLA-NCA)
L-Aspartic acid-4-benzyl ester (BLA, 4.7 g, 0.02 mol) was dissolved in 60 mL of anhydrous THF and triphosgene (6.0 g, 0.02 mol) was then added and allowed to stir at 60°C until the mixture solution turned clear. After evaporation of the solvent, the residue was re-crystallized with chloroform and n-hexane at 0°C to provide BLA-NCA as white crystal solid.
Synthesis of folate polyethylene glycol-b-poly(aspartic acid benzyl ester) (FA-PEG-b-PBLA)
FA-PEG-NH2 (0.1 g) was dissolved in 10 mL anhydrous DCM and BLA-NCA (1.0 g) in 30 mL of DCM was then added. The reaction mixture was stirred at room temperature under N2 for 72 hrs. The mixture solution washed successively by H2O and saturated brine, dried over anhydrous Na2SO4, and filtrated. After evaporation of the solvent, the residue was then re-crystallized with DCM and ether (Et2O) at 0°C to provide FA-PEG-b-PBLA as a light yellow powder.
Synthesis of folate polyethylene glycol-b-poly(aspartic acid) (FA-PEG-b-PAsp)
FA-PEG-b-PBLA (1.0 g) was dissolved in a mixed solution (25 mL THF and 25 mL 1M NaOH aqueous solution) and stirred at 35°C for 10 hrs. Then, the concentrated mixture was dialyzed in purified water to provide FA-PEG-b-PAsp as brown fluffy powder.
Conjugation of chidamide and FA-PEG-b-PAsp
FA-PEG-b-PAsp (0.2 g) was dissolved in 10 mL anhydrous DCM and chidamide (0.243 g), HATU (0.238), and DIPEA (0.1 mL) was then added and stirred at room temperature. Then, the concentrated mixture was dialyzed in purified water for 48 hrs to provide FPPC as brown fluffy powder(1HNMR in ).
Meanwhile, polymer polyethylene glycol-b- poly(asparaginyl-chidamide) (PEG-b-PAsp-chidamide, PPC) was synthesized in a similar process only by replacement FA-PEG-NH2 with PEG-NH2 in step two(1HNMR in ).
Figure S1 1HNMR of (A:FPPC and B: PPC).
Establishment of standard curves
1 mg Pha was dissolved in 1 mL DMSO, doubling diluted. 100 μL solution of each sample was then transferred to 96-well plates, and triple duplicate wells for each concentration, and the absorbance of each concentration was measured via ELIASA at 405 nm. Finally, the standard curve was established according to the absorbances (Abs) ().
Figure S2 Standard curve of Pha.
Analysis of maximum fluorescence intensity on Cellular uptake images
Maximum fluorescence intensity of three individual cells from each group in was detected and analyzed by Image J. (National Institutes of Health, US, version: 1.4.3.67). Due to the high cytotoxicity of free Pha and cell images overlaid, cells in free Pha group were chosen from mono-cell area.
Figure S3 Maximum fluorescence intensity of Pha (Turkey’s Multiple comparison Test, Pha@FPPC vs control ***P<0.001, Pha@FPPC vs Pha@PPC ##P<0.01).
