Name: facile preparation and photoactivation of prodrug-dye nanoassemblies
Uploaded: 2023-02-17T14:05:00
Description: Watch this Scientific Journal Video about Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies at JoVE.com

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).

1. Synthesis of boron-dipyrromethene-chlorambucil (BC) prodrug (Figure 2)22
<ol>
	<li>Synthesis of BODIPY-OAc
	<ol>
		<li>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...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+enzyme/prodrug+cancer+therapy.">Strategies for enzyme/prodrug cancer therapy.</a> Clinical Cancer Research. 7 (11), 3314-3324 (2001).</li><li>Luo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-targeted+and+pH-sensitive+doxorubicin+prodrug-microbubble+complex+with+ultrasound+for+tumor+treatment.">Dual-targeted and pH-sensitive doxorubicin prodrug-microbubble complex with ultrasound for tumor treatment.</a> Theranostics. 7 (2), 452 (2017).</li><li>Gao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+triggered+phase-change+nanodroplets+for+doxorubicin+prodrug+delivery+and+ultrasound+diagnosis:+An+in+vitro+study.">Ultrasound triggered phase-change nanodroplets for doxorubicin prodrug delivery and ultrasound diagnosis: An in vitro study.</a> Colloids and Surfaces B: Biointerfaces. 174, 416-425 (2019).</li><li>Brade, A. M., Szmitko, P., Ngo, D., Liu, F. -. F., Klamut, H. 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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...

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 toxicity in normal tissues while retaining their action in diseased lesions upon activation5,6. Prodrugs can be responsive to a variety of stimuli, such as pH7,8, enzymes9,10, ultrasound11,12, heat13, and light14,15,16, and release their parent drugs specifically in the lesions. Nevertheless, many prodrugs exhibit inherent drawbacks, such as poor solubility, incorrect absorption rate, and early metabolic destruction, which may limit their development17. In this context, the formation of prodrug nanoassemblies offers advantages like decreased side effects, in situ drug release, better retention, and the combination of treatment and imaging, indicating great application potential for these nanoassemblies. Many prodrug nanoassemblies have been developed for disease treatment, including doxorubicin prodrug nanospheres, curcumin prodrug micelles, and camptothecin prodrug nanofibers18.
In this protocol, we present a simple method for the preparation of prodrug-dye nanoassemblies that exhibit high prodrug content, good water dispersibility, long-term stability, and sensitive responding ability. IR783 is a water soluble near-infrared dye that can serve as stabilizer of the nanoassemblies19. The other component of the nanoassembly is boron-dipyrromethene-chlorambucil (BODIPY-Cb, BC), a prodrug that was designed for two main reasons. As chlorambucil (Cb) displays systemic toxicity in vivo, the prodrug form can decrease its toxicity20. The BC prodrug can be photocleaved using 530 nm light irradiation directed at disease lesions, enabling the local release of Cb. On the other hand, Cb is prone to hydrolysis in aqueous environments, and can be protected by transforming it into a prodrug form21. Thus, the co-assembly of the BC prodrug and IR-783 dye was expected to form a stable and effective drug delivery nanosystem (Figure 1A). This prodrug-dye nanoassembly improves the dispersibility and stability of the prodrug molecules, suggesting its potential for application in light-controllable drug delivery. The photocleavage of the BC prodrug enables the disassembly of nanoparticles and the light-controlled release of Cb in the lesions (Supplemental Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1260 Infinity II HPLC</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2,4-Dimethyl pyrrole</td>
			<td>J&#38;K Scientific</td>
			<td>315305</td>
			<td></td>
		</tr>
		<tr>
			<td>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT)</td>
			<td>Gibco</td>
			<td>M6494</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine (4-DMAP)</td>
			<td>J&#38;K Scientific</td>
			<td>212279</td>
			<td></td>
		</tr>
		<tr>
			<td>90 mm Petri Dish Clear Treated Sterile</td>
			<td>SPL</td>
			<td>11090</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Tissue Culture Plate Clear Treated Sterile</td>
			<td>SPL</td>
			<td>30096</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetoxyacetyl chloride</td>
			<td>J&#38;K Scientific</td>
			<td>192001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boron trifluoride diethyl etherate</td>
			<td>J&#38;K Scientific</td>
			<td>921076</td>
			<td></td>
		</tr>
		<tr>
			<td>B&#252;chner funnel</td>
			<td>AS ONE</td>
			<td>3-6466-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorambucil</td>
			<td>J&#38;K Scientific</td>
			<td>321407-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>CM100 Transmission Electron Microscope</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CombiFlash RF chromatography system&#160;</td>
			<td>Teledyne ISCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>DUKSAN Pure Chemicals</td>
			<td>JT9315-88</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>DUKSAN Pure Chemicals</td>
			<td>2762</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable cuvette</td>
			<td>Malvern Panalytical</td>
			<td>DTS1070</td>
			<td>Zeta potential measurement</td>
		</tr>
		<tr>
			<td>Disposable cuvette</td>
			<td>Malvern Panalytical</td>
			<td>ZEN0040</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty Disposable Sample Load Cartridges</td>
			<td>Teledyne ISCO</td>
			<td>693873225</td>
			<td>can hold up to 65 g</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtering flask</td>
			<td>AS ONE</td>
			<td>3-7089-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexane</td>
			<td>DUKSAN Pure Chemicals</td>
			<td>4198</td>
			<td></td>
		</tr>
		<tr>
			<td>Holey carbon film on copper grid</td>
			<td>Beijing Zhongjingkeyi Technology Co.,Ltd</td>
			<td>BZ10023a</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC column (InfinityLab Poroshell 120)</td>
			<td>Agilent Technologies</td>
			<td>695975-902T</td>
			<td></td>
		</tr>
		<tr>
			<td>Integrating sphere photodiode power sensor</td>
			<td>Thorlabs</td>
			<td>S142C</td>
			<td></td>
		</tr>
		<tr>
			<td>IR783</td>
			<td>Tokyo Chemical Industry (TCI) Co., Ltd</td>
			<td>I1031</td>
			<td></td>
		</tr>
		<tr>
			<td>LED&#160;</td>
			<td>Mightex</td>
			<td>LCS-0530-15-11</td>
			<td></td>
		</tr>
		<tr>
			<td>LED Driver Control Panel V3.2.0 (Software)</td>
			<td>Mightex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Hydroxide Anhydrous</td>
			<td>TCI</td>
			<td>L0225</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylmagnesium iodide, 3M solution in diethyl ether</td>
			<td>Aladdin</td>
			<td>M140783</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropyl ethyl amine (DIPEA)</td>
			<td>J&#38;K Scientific</td>
			<td>203402</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#39;-Dicyclohexylcarbodiimide (DCC)</td>
			<td>J&#38;K Scientific</td>
			<td>275928</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin&#8211;streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (10&#215;)</td>
			<td>&#160;Sigma-Aldrich</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Power and energy meter&#160;</td>
			<td>Thorlabs</td>
			<td>PM100 USB</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotavapor</td>
			<td>BUCHI Rotavapor R300</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RMPI 1640</td>
			<td>Gibco</td>
			<td>21870076</td>
			<td></td>
		</tr>
		<tr>
			<td>Separatory funnel (125 mL)</td>
			<td>Synthware</td>
			<td>F474125L</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver Silica Gel Disposable Flash Columns, 40 g</td>
			<td>Teledyne ISCO</td>
			<td>692203340</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium sulfate, anhydrous</td>
			<td>Alfa Aesar</td>
			<td>A19890</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M4</td>
			<td>Molecular Devices LLC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF), anhydrous</td>
			<td>J&#38;K Scientific</td>
			<td>943616</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>DLAB Scientific Co., Ltd</td>
			<td>MX-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Zetasizer Nano ZS90&#160;</td>
			<td>Malvern Instrument</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

facile preparation and photoactivation of prodrug-dye nanoassemblies

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&#160;for the construction and evaluation of photoresponsive drug delivery systems.

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 (<strong class...

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Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

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.

Self-assembly is a simple yet reliable method for constructing nanoscale drug delivery systems. Photoactivatable prodrugs enable controllable drug release ...

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