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 at 40 &#176;C.</li>
		<li>Cool the mixture to room temperature. Weigh 5.170 g of N,N-diisopropylethylamine (DIPEA) and add it dropwise into the mixture under stirring. After 30 min, weigh 5.677 g of boron trifluoride diethyl etherate (BF3&#183;OEt2), add it dropwise into the solution, and keep stirring for an additional 30 min.</li>
		<li>Add 10 g of silica gel (200-400 mesh) into the mixture and remove the solvent by rotary evaporation at 45 &#176;C. Stop the evaporation when the silica gel returns to dry powder.</li>
		<li>Add a frit into the bottom of a cartridge (see Table of Materials). Fill the silica gel (from step 1.1.3) into the cartridge and then add another frit into the cartridge on the top of the filled gel.</li>
		<li>Fix the cartridge into the collar connected with the flash chromatography system (see Table of Materials) and turn it to lock it. Install the cartridge on top of the six-way valve in the flash chromatography system and install a flash column (see Table of Materials) under the valve.</li>
		<li>Start the chromatography instrument and set 515 nm and 365 nm as the detection wavelengths. Perform elution with 4/3 (v/v) hexane/DCM. Collect the eluent fractions as the 515 nm signal appears.</li>
		<li>Remove the solvent from the collected fractions by rotary evaporation at 40 &#176;C until no more solvent is collected in the solvent collection flask. Place the solid product in a vacuum drying chamber overnight to remove the rest of solvent.</li>
	</ol>
	</li>
	<li>Synthesis of BODIPY-OH
	<ol>
		<li>Weigh 1.120 g of BODIPY-OAc (synthesized in step 1.1) and dissolve it in 70 mL of tetrahydrofuran (THF) at room temperature, fully covered with foil. Add 70 mL of 0.1 M LiOH aqueous solution dropwise into the BODIPY-OAc solution.</li>
		<li>Stir the mixture for 30 min and remove the solvent at 40&#160;&#176;C by rotary evaporation until no more solvent is collected in the solvent collection flask. Place the residue in a vacuum drying chamber overnight to remove water.</li>
		<li>Dissolve the dry residue in 30 mL of DCM and add 10 g of silica gel into the solution. Remove the solvent by rotary evaporation at 40 &#176;C. Stop the evaporation when the silica gel returns to dry powder.</li>
		<li>Purify the product BODIPY-OH by column chromatography, following steps 1.1.4 to 1.1.6, with DCM alone as the eluent.</li>
		<li>Compare fractions collected at different elution time points with a THF solution of BODIPY-OAc on thin layer chromatography (TLC) and identify the product23.
		<ol>
			<li>Spot 3-4 &#181;L of the eluted fraction and the BODIPY-OAc solution separately on one edge of a TLC plate at the same height. Place the TLC plate in a glass chamber containing 1 mL of DCM, submerging the spotted edge into the DCM solvent but with the two spots out of the solvent.</li>
			<li>Take out the TLC plate when the DCM solvent reaches over half the height of the plate. Select the eluted fraction with a TLC spot at a different height from the BODIPY-OAc spot.</li>
		</ol>
		</li>
		<li>Remove the solvent by rotary evaporation at 40 &#176;C (step 1.1.7) to obtain the product BODIPY-OH.</li>
	</ol>
	</li>
	<li>Synthesis of BODIPY-(Me)2-OH
	<ol>
		<li>Weigh 313 mg of BODIPY-OH and dissolve it in 35 mL of anhydrous diethyl ether in the dark under a nitrogen atmosphere. Add 3.75 mL of methyl magnesium iodide (3 M in diethyl ether) dropwise into the solution and keep stirring for 3 h at room temperature.</li>
		<li>Quench the reaction by adding 3.5 mL of water dropwise.</li>
		<li>Extract the mixture with DCM and water.
		<ol>
			<li>Transfer the mixture into a 125 mL separatory funnel. Add 20 mL of DCM into the mixture.</li>
			<li>Close the cap of the separatory funnel. Tilt the funnel at around 45&#176;&#160;and slightly shake the funnel. Open the cap to deflate. Repeat this step 3 times, and allow to stand for 3 min.</li>
			<li>Open the bottom valve and collect the lower organic phase in a beaker.</li>
			<li>Add 30 mL of DCM to the aqueous phase. Repeat the extraction (steps 1.3.3.2 and 1.3.3.3) 3 times with 30 mL of DCM each time.</li>
		</ol>
		</li>
		<li>Add 10 g of solid Na2SO4 into the collected organic phase to dry the organic phase overnight.</li>
		<li>Link a filtering flask to a vacuum pump with a rubber tube. Lay a piece of filter paper on the B&#252;chner funnel and insert the funnel into the top of the flask. Wet the filter paper with 1 mL of DCM, transfer the mixture into the funnel, and turn on the vacuum pump. Collect the organic solution in the flask.</li>
		<li>Add 10 g of silica gel into the organic solution. Remove the organic solvent by rotary evaporation at 40&#160;&#176;C until the silica gel returns to&#160;dry powder. Purify the product BODIPY-(Me)2-OH by column chromatography (steps 1.1.4 to 1.1.6) with hexane/DCM = 1/1 (v/v) as the eluent.</li>
		<li>Select the eluted fractions containing the product using TLC analysis as described in step 1.2.5 (TLC spot at a different height from BODIPY-OH). Remove the solvent by rotary evaporation at 40 &#176;C to obtain the product as described in step 1.1.7.</li>
	</ol>
	</li>
	<li>Synthesis of BODIPY-(Me)2-I2-OH
	<ol>
		<li>Weigh 41 mg of BODIPY-(Me)2-OH and dissolve it in 2.5 mL of anhydrous THF in the dark under a nitrogen atmosphere. Weigh 74 mg of N-iodosuccinimide and dissolve it in 1 mL of anhydrous THF.</li>
		<li>Add the N-iodosuccinimide solution dropwise into the BODIPY-(Me)2-OH solution. After stirring for 3.5 h at room temperature, remove the solvent by rotary evaporation at 40 &#176;C until no more solvent is collected in the solvent collection flask on the rotary evaporator.</li>
		<li>Dissolve the residue in 10 mL of DCM and wash it 3 times with 30 mL of water each time, as described in step 1.3.3. Dry the organic phase with Na2SO4 (steps 1.3.4 and 1.3.5). Remove the solvent by rotary evaporation at 40 &#176;C (step 1.1.7)&#160;to obtain the product BODIPY-(Me)2-I2-OH.</li>
	</ol>
	</li>
	<li>Synthesis of BODIPY-Cb
	<ol>
		<li>Weigh 85 mg of chlorambucil and dissolve it in 2 mL of anhydrous DCM in the dark under a nitrogen atmosphere. Weigh 69 mg of N,N&#39;-dicyclohexylcarbodiimide and dissolve it in 1 mL of anhydrous DCM. Add it dropwise into the chlorambucil solution with stirring for 10 min.</li>
		<li>Dissolve 1.7 mg of 4-dimethylaminopyridine in 0.5 mL of anhydrous DCM. Add this solution into the mixture and keep stirring for 10 min at room temperature. Then add 73 mg of BODIPY-(Me)2-I2-OH dissolved in 2 mL of anhydrous DCM and keep stirring for 2 h.</li>
		<li>Add 10 g of silica gel into the mixture and remove the solvent by rotary evaporation at 40 &#176;C. Stop the evaporation when the silica gel returns to dry powder. Purify the product BODIPY-Cb by column chromatography (steps 1.1.4 to 1.1.6, 540 nm and 365 nm signal wavelength) with hexane/DCM = 7/3 (v/v) as the eluent.</li>
		<li>Select the eluted fractions containing the product different from BODIPY-(Me)2-I2-OH using TLC analysis (step 1.2.5). Remove the solvent by rotary evaporation at 40 &#176;C (step 1.1.7) to obtain the product BODIPY-Cb.</li>
	</ol>
	</li>
</ol>
2. Preparation of IR783/BC NPs by the flash precipitation method
<ol>
	<li>Weigh 10 mg of the BC prodrug (BODIPY-Cb) and dissolve it in 1 mL of DMSO in a 1.5 mL microtube&#160;to obtain a 10 mg/mL stock solution. Cover the BC solution with foil.</li>
	<li>Prepare 300 &#181;L of 0.4 mg/mL IR-783 in filtered deionized water in a 1.5 mL microtube. Place this microtube on a vortex mixer at 1,500 rpm.</li>
	<li>Add 20 &#181;L of the BC solution in DMSO to the IR-783 solution over 10 s at a constant rate using a 20 &#181;L pipette. The end of the pipette tip should touch the inner wall of the microtube (Figure 1B).</li>
	<li>Keep the microtube on the vortex mixer for an additional 30 s to obtain the IR783/BC NP solution. Then, place the nanoparticle solution on a rack fully covered with foil.</li>
	<li>Centrifuge the resulting IR783/BC NP solution for 10 min at 2,000 x g and 4 &#176;C to remove aggregates. Collect the supernatant, leaving ~20 &#181;L in the tube to avoid disturbing the pellet. Discard the pellet.</li>
	<li>Centrifuge the supernatant twice for 30 min at 30,000 x g and 4 &#176;C and collect the nanoparticle precipitate from both the centrifugations. Resuspend the nanoparticles in 300 &#181;L of 1x PBS. 
	NOTE: When the hydrophobic BC prodrug in DMSO is dispersed in water with vortexing, the DMSO is dissolved by water, and the prodrug molecules tend to form nanoscale assemblies to keep themselves stable under the local supersaturation situation24.</li>
	<li>Quantify the content of IR-783 and BC by high performance liquid chromatography (HPLC), using the elution method shown in Table 1. 
	NOTE: The HPLC sample is prepared by mixing equal volumes of nanoparticle solution and acetonitrile. The injection volume is 20 &#181;L. The detection wavelength for chlorambucil and BC prodrug is 260 nm, and the detection wavelength for IR783 is 783 nm. The HPLC column is an analytical 4.6 mm (inner diameter) x 100 mm (length) C18 column, with a 2.7 &#181;m particle size and 120 &#197; pore size.</li>
	<li>Calculate the prodrug encapsulation efficiency (EE%) and loading capacity (LC%) according to the following equations: 
	<img alt="Equation 1" src="/files/ftp_upload/64677/64677eq01v4.jpg" style="margin: auto;" /></li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>Acetonitrile (%)</td>
			<td>Water (%)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>20</td>
			<td>80</td>
		</tr>
		<tr>
			<td>5</td>
			<td>20</td>
			<td>80</td>
		</tr>
		<tr>
			<td>30</td>
			<td>95</td>
			<td>5</td>
		</tr>
		<tr>
			<td>35</td>
			<td>95</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: HPLC method for qualitative and quantitative analysis of BC prodrug and its photocleavage. Reproduced with permission25. Copyright 2022, Wiley.
3. Characterization of IR783/BC NPs
<ol>
	<li>Measure the average size of the IR783/BC NPs with a dynamic light scattering (DLS) instrument (see Table of Materials). Add 200 &#181;L of IR783/BC NP solution in a cuvette and insert the cuvette in the holder for measurement. Set the measurement type as &#39;size&#39; and measurement temperature as 25 &#176;C. Perform three measurements with a duration of 20 s for each measurement.</li>
	<li>Measure the surface charge of the IR783/BC NPs with the DLS instrument using a zeta-potential test cuvette.
	<ol>
		<li>Dilute 25 &#181;L of IR783/BC NP&#160;solution with 725 &#181;L of deionized water in a 1.5 mL microtube and add the solution into a zeta-potential test cuvette. Place the cuvette in the sample groove. Cap the sample groove.</li>
		<li>Set the measurement type as &#39;zeta-potential&#39; and measurement temperature as 25 &#176;C. Perform 10 measurements.</li>
	</ol>
	</li>
	<li>Prepare the samples for transmission electron microscopy (TEM) imaging. Add 10 &#181;L of IR783/BC NP solution on a piece of holey carbon film on a copper grid (300 mesh) and remove 7 &#181;L. Leave 3 &#181;L of solution on the film overnight for auto-evaporation. 
	NOTE: Adding 10 &#181;L of the NP solution followed by the removal of 7 &#181;L allows the droplet to cover a broader area on the film.</li>
</ol>
4. Photoactivation of IR783/BC NPs
<ol>
	<li>Set up an LED lamp (530 nm; see Table of Materials) with an iron stand so that the light directly faces the operating floor. Place an integrating sphere photodiode photometer (see Table of Materials) directly under the LED lamp. 
	NOTE: To prevent the influence of environmental light, all light irradiation experiments are&#160;conducted in a darkroom.</li>
	<li>Turn on the LED lamp and open the cap of the photometer. Record the irradiance and set the lamp parameters using the associated software (see Table of Materials). Adjust the input current (mA) to set the irradiance as 50 mW/cm2. 
	NOTE: The irradiance is also affected by the distance between the LED lamp and photometer. In the setup used here (Figure 3A,B), the distance is fixed at 5 cm.</li>
	<li>Dilute the IR783/BC NP&#160;solution with deionized water to 50 &#181;M based on BC concentration. Add 200 &#181;L of the IR783/BC NP solution into a 1.5 mL microtube. Place the tube on a foam block with a groove fitting size of the microtube and the same height as the photometer in step 4.1 (Figure 3C,D).</li>
	<li>Open the cap of the tube. Turn on the LED lamp and irradiate the nanoparticle solution for 1, 2, 3, 5, 7, and 10 min.</li>
	<li>Quantify BC consumption and Cb release by HPLC after light irradiation. Calculate the percentage of remaining BC and Cb release using the following equations: 
	<img alt="Equation 3" src="/files/ftp_upload/64677/64677eq3v2.jpg" style="margin: auto;" /></li>
</ol>
5. Testing cytotoxicity of IR783/BC NPs with and without light irradiation
<ol>
	<li>Culture HCT116 cells (human colorectal tumor cell line) in RPMI 1640 medium containing 10% fetal bovine serum and 1% Penicillin-Streptomycin (complete medium) in a 5% CO2 atmosphere at 37 &#176;C (~2 x 106 cells per dish in a 90 mm cell culture dish). Routinely subculture the cells every 2-3 days. 
	NOTE: HCT116 is a human colon cell line. Compared to other cancer cells such as HeLa, MCF7, and A549 cells, HCT-116 cells express a higher level of caveolin-125, which can be targeted by IR783 and enhance the cellular uptake of IR783/BC NPs.</li>
	<li>Plate the HCT116 cells in 96-well plates with RPMI 1640 complete medium at a density of 104 cells per well.
	<ol>
		<li>Aspirate the medium from the culture dish when the cell confluency exceeds 50%. Wash the cells with 1x PBS and remove the PBS. Add 1 mL of 0.25% trypsin solution and incubate at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>After 3 min, add 2 mL of complete medium to quench trypsin digestion. Resuspend the cells, transfer the cell suspension into a 15 mL centrifugation tube, and centrifuge at 300 x g for 3 min. Discard the supernatant and resuspend the cell pellet in 1 mL of complete medium.</li>
		<li>Dilute 10 &#181;L of the cell suspension to 200 &#181;L with complete medium. Place 10 &#181;L on a hemocytometer and cap it with a coverslip.</li>
		<li>Observe the hemocytometer under a microscope (eyepiece: 10x; objective lens: 4x). Count and record the cell numbers at the four corner squares and the center. Calculate the cell concentration using the formula: 
		<img alt="Equation 5" src="/files/ftp_upload/64677/64677eq05.jpg" style="margin: auto;" /> 
		Where n = the average of the cell numbers of the five squares.</li>
		<li>Dilute the cell suspension to 1 x 105 cells/mL. Add 100 &#181;L of the cell suspension per well in a 96-well plate to seed the cells. Add 100 &#181;L of PBS per well in the unseeded wells.</li>
	</ol>
	</li>
	<li>Treat the cells with (1) 0.1-150 &#181;M free BC, (2) 0.1-150 &#181;M IR783/BC NPs (based on BC concentration), (3) 0.1-150 &#181;M free BC with light irradiation, or (4) 0.1-150 &#181;M IR783/BC NPs (based on BC concentration) with light irradiation. Incubate the cells at 37 &#176;C in a 5% CO2 incubator for 6 h. 
	NOTE: The free BC and IR783/BC NP solutions are diluted from their respective stock solutions with complete medium.</li>
	<li>After 6 h of incubation, replace the prodrug/nanoparticles-containing medium with fresh complete medium. Incubate the non-irradiation Groups 1 and 2 in the dark for 24 h. For Groups 3 and 4, irradiate the cells with a 530 nm LED lamp (50 mW/cm2) for 5 min, and incubate for 24 h. 
	NOTE: Cell plates are placed on a foam block to ensure the same height as the photometer in step 4.1.</li>
	<li>Determine the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.
	<ol>
		<li>After the BC or nanoparticle treatment, add 10 &#181;L of MTT (10 mg/mL in PBS) to each well and incubate the plates at 37 &#176;C for 3 h. Then, remove the medium and add 100 &#181;L of DMSO to each well. Read the absorbance with a microplate reader at 490 nm, 570 nm, and 630 nm.</li>
		<li>Calculate the cell viability using the following equation: 
		<img alt="Equation 6" src="/files/ftp_upload/64677/64677eq06v2.jpg" style="margin: auto;" /> 
		NOTE: Four independent experiments (n = 4) of each group are conducted for analysis. OD490 can be replaced by OD570-OD630 in calculation of cell viability.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+mechanism+of+chlorambucil+hydrolysis.">Kinetics and mechanism of chlorambucil hydrolysis.</a> Journal of Pharmaceutical Sciences. 68 (8), 992-996 (1979).</li><li>Lv, 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=Upconversion-like+photolysis+of+BODIPY-based+prodrugs+via+a+one-photon+process.">Upconversion-like photolysis of BODIPY-based prodrugs via a one-photon process.</a> Journal of the American Chemical Society. 141 (44), 17482-17486 (2019).</li><li>Silver, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let+us+teach+proper+thin+layer+chromatography+technique.">Let us teach proper thin layer chromatography technique.</a> Journal of Chemical Education. 97 (12), 4217-4219 (2020).</li><li>Saad, W. S., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+nanoparticle+formation+by+flash+nanoprecipitation.">Principles of nanoparticle formation by flash nanoprecipitation.</a> Nano Today. 11 (2), 212-227 (2016).</li><li>Long, K., 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=Photoresponsive+prodrug-dye+nanoassembly+for+in-situ+monitorable+cancer+therapy.">Photoresponsive prodrug-dye nanoassembly for in-situ monitorable cancer therapy.</a> Bioengineering &amp; Translational Medicine. 7 (3), 10311 (2022).</li><li>Zhong, T., 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=A+self-assembling+nanomedicine+of+conjugated+linoleic+acid-paclitaxel+conjugate+(CLA-PTX)+with+higher+drug+loading+and+carrier-free+characteristic.">A self-assembling nanomedicine of conjugated linoleic acid-paclitaxel conjugate (CLA-PTX) with higher drug loading and carrier-free characteristic.</a> Scientific Reports. 6 (1), 36614 (2016).</li><li>Long, K., 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=Green+light-triggered+intraocular+drug+release+for+intravenous+chemotherapy+of+retinoblastoma.">Green light-triggered intraocular drug release for intravenous chemotherapy of retinoblastoma.</a> Advanced Science. 8 (20), 2101754 (2021).</li><li>Lv, W., Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-photon+upconversion-like+photolysis:+a+new+strategy+to+achieve+long-wavelength+light-excitable+photolysis.">One-photon upconversion-like photolysis: a new strategy to achieve long-wavelength light-excitable photolysis.</a> Synlett. 31 (12), 1129-1134 (2020).</li><li>Rwei, A. Y., Wang, W., Kohane, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoresponsive+nanoparticles+for+drug+delivery.">Photoresponsive nanoparticles for drug delivery.</a> Nano Today. 10 (4), 451-467 (2015).</li><li>Grzelczak, M., Vermant, J., Furst, E. M., Liz-Marz&#225;n, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+self-assembly+of+nanoparticles.">Directed self-assembly of nanoparticles.</a> ACS Nano. 4 (7), 3591-3605 (2010).</li><li>Gnanasammandhan, M. K., Idris, N. M., Bansal, A., Huang, K., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-IR+photoactivation+using+mesoporous+silica-coated+NaYF4:Yb,Er/Tm+upconversion+nanoparticles.">Near-IR photoactivation using mesoporous silica-coated NaYF4:Yb,Er/Tm upconversion nanoparticles.</a> Nature Protocols. 11 (4), 688-713 (2016).</li></ol>

A PCT application has been filed with No. PCT/CN2021/081262.

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...

Watch this Scientific Journal Video about Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies at JoVE.com

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 ...

facile-preparation-and-photoactivation-of-prodrug-dye-nanoassemblies

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JoVE Journal

Medicine

859 Views.  The University of Hong Kong. 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.

Video: Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...