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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes the fabrication and characterization of a photoresponsive prodrug-dye nanoassembly. The methodology for drug release from the nanoparticles by light-triggered disassembly, including the light irradiation setup, is explicitly described. The drugs released from the nanoparticles following light irradiation exhibited excellent anti-proliferation effects on human colorectal tumor cells.

Abstract

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release from nanocarriers at target sites modulated by light irradiation. In this protocol, a facile method for fabricating photoactivatable prodrug-dye nanoparticles via molecular self-assembly is presented. The procedures for prodrug synthesis, nanoparticle fabrication, physical characterization of the nanoassembly, photocleavage demonstration, and in vitro cytotoxicity verification are described in detail. A photocleavable boron-dipyrromethene-chlorambucil (BC) prodrug was first synthesized. BC and a near-infrared dye, IR-783, at an optimized ratio, could self-assemble into nanoparticles (IR783/BC NPs). The synthesized nanoparticles had an average size of 87.22 nm and a surface charge of -29.8 mV. The nanoparticles disassembled upon light irradiation, which could be observed by transmission electronic microscopy. The photocleavage of BC was completed within 10 min, with a 22% recovery efficiency for chlorambucil. The nanoparticles displayed enhanced cytotoxicity under light irradiation at 530 nm compared with the non-irradiated nanoparticles and irradiated free BC prodrug. This protocol provides a reference for the construction and evaluation of photoresponsive drug delivery systems.

Introduction

Chemotherapy is a common cancer treatment that employs cytotoxic agents to kill cancer cells and thus inhibits tumor growth1. However, patients may suffer from side effects such as cardiotoxicity and hepatotoxicity due to the off-target absorption of the chemotherapy drugs2,3,4. Therefore, localized drug delivery through the spatiotemporal control of drug release/activation in tumors is essential to minimize drug exposure in normal tissues.

Prodrugs are chemically modified drugs that exhibit reduced tox....

Protocol

1. Synthesis of boron-dipyrromethene-chlorambucil (BC) prodrug (Figure 2)22

  1. Synthesis of BODIPY-OAc
    1. Weigh 1.903 g of 2,4-dimethyl pyrrole and dissolve it in 20 mL of anhydrous dichloromethane (DCM) in a round-bottom flask under a nitrogen atmosphere. Weigh 1.638 g of acetoxy acetyl chloride and add it dropwise into the solution. Keep stirring for 10 min at room temperature and then reflux the solution for 1 h a.......

Representative Results

IR783/BC NPs were successfully fabricated in this study using a flash precipitation method. The synthesized IR783/BC NPs presented as a purple solution, while the aqueous solution of IR783 was blue (Figure 4A). As shown in Figure 4B, the IR783/BC NPs exhibited an average size of approximately 87.22 nm with a polydispersity index (PDI) of 0.089, demonstrating a narrow size distribution. The surface charge of the IR783/NPs was approximately -29.8 mV (

Discussion

This protocol outlines a facile flash precipitation method for the fabrication of prodrug-dye nanoparticles, which offers a simple and convenient approach for nanoparticle formation. There are several critical steps in this method. Firstly, for all steps of synthesis, fabrication, and characterization, containers like microtubes should be covered with foil to avoid unnecessary photocleavage of the BC prodrug by environmental light. Moreover, in the flash precipitation step, the microtube containing the IR-783 solution sh.......

Acknowledgements

We acknowledge assistance from the University of Hong Kong Li Ka Shing Faculty of Medicine Faculty Core Facility. We thank Professor Chi-Ming Che at the University of Hong Kong for providing the human HCT116 cell line. This work was supported by Ming Wai Lau Centre for Reparative Medicine Associate Member Program and the Research Grants Council of Hong Kong (Early Career Scheme, No. 27115220).

....

Materials

NameCompanyCatalog NumberComments
1260 Infinity II HPLCAgilent Technologies
2,4-Dimethyl pyrroleJ&K Scientific315305
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)GibcoM6494
4-Dimethylaminopyridine (4-DMAP)J&K Scientific212279
90 mm Petri Dish Clear Treated SterileSPL11090
96-well Tissue Culture Plate Clear Treated SterileSPL30096
Acetoxyacetyl chlorideJ&K Scientific192001
Boron trifluoride diethyl etherateJ&K Scientific921076
Büchner funnelAS ONE3-6466-01
ChlorambucilJ&K Scientific321407-1G
CM100 Transmission Electron MicroscopePhilips
CombiFlash RF chromatography system Teledyne ISCO
DichloromethaneDUKSAN Pure ChemicalsJT9315-88
Dimethyl sulfoxideDUKSAN Pure Chemicals2762
Disposable cuvetteMalvern PanalyticalDTS1070Zeta potential measurement
Disposable cuvetteMalvern PanalyticalZEN0040
Empty Disposable Sample Load CartridgesTeledyne ISCO693873225can hold up to 65 g
Fetal bovine serumGibco10270106
Filtering flaskAS ONE3-7089-03
HexaneDUKSAN Pure Chemicals4198
Holey carbon film on copper gridBeijing Zhongjingkeyi Technology Co.,LtdBZ10023a
HPLC column (InfinityLab Poroshell 120)Agilent Technologies695975-902T
Integrating sphere photodiode power sensorThorlabsS142C
IR783Tokyo Chemical Industry (TCI) Co., LtdI1031
LED MightexLCS-0530-15-11
LED Driver Control Panel V3.2.0 (Software)Mightex
Lithium Hydroxide AnhydrousTCIL0225
Methylmagnesium iodide, 3M solution in diethyl etherAladdinM140783
N,N-Diisopropyl ethyl amine (DIPEA)J&K Scientific203402
N,N'-Dicyclohexylcarbodiimide (DCC)J&K Scientific275928
penicillin–streptomycinGibco15140122
Phosphate-buffered saline (10×) Sigma-AldrichP5493
 Power and energy meter ThorlabsPM100 USB
RotavaporBUCHI Rotavapor R300
RMPI 1640Gibco21870076
Separatory funnel (125 mL)SynthwareF474125L
Silver Silica Gel Disposable Flash Columns, 40 gTeledyne ISCO692203340
Sodium sulfate, anhydrousAlfa AesarA19890
SpectraMax M4Molecular Devices LLC
Tetrahydrofuran (THF), anhydrousJ&K Scientific943616
Trypsin-EDTA (0.25%), phenol redGibco25200056
VortexDLAB Scientific Co., LtdMX-S
Zetasizer Nano ZS90 Malvern Instrument

References

  1. Chabner, B. A., Roberts, T. G. Chemotherapy and the war on cancer. Nature Reviews Cancer. 5 (1), 65-72 (2005).
  2. Monsuez, J. -. J., Charniot, J. -. C., Vignat, N., Artigou, J. -. Y. Cardiac side-effects of cancer chemotherapy. International ....

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Prodrug dye NanoassembliesPhotoactivationDrug DeliveryColorectal TumorBoron Dipyrromethene ChlorambucilIR783Nanoparticle FabricationEncapsulation EfficiencyLoading CapacityDynamic Light ScatteringSurface Charge

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