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 should be placed stably on the vortex mixer while slowly adding the BC prodrug solution. This way, the BC prodrug solution (in DMSO) can be dispersed uniformly in the IR-783 solution. In the purification step, a differential centrifugation method is used. The first centrifugation at 2,000 x g is used to remove the unloaded BC prodrug solids, while the second centrifugation at 30,000 x g removes DMSO and IR783 in the supernatant that are not incorporated in the nanoparticles. IR783/BC NPs can then be collected as the precipitate and resuspended in filtered deionized water.
Self-assembly is a simple and efficient method for the fabrication of nanoparticles. However, the self-assembly method needs to be optimized according to factors like the hydrophobicity of building blocks. In this protocol, one of the components, IR783, is a water-soluble dye that is dissolved in water. However, in some cases, all the components may be hydrophobic. Under such a circumstance, they should be dissolved in DMSO like the BC prodrug in this protocol. A stabilizer such as 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG) can be used to help form and stabilize self-assembled nanoparticles14,26,27.
Light has limited tissue penetration, which limits the application of photoactivatable nanoparticles in clinical use. One solution is to develop long-wavelength light-activatable systems, such as red or near-infrared light28. Using optic fibers to deliver light is another way to solve the limited tissue penetration issue of light for some disease lesions29. Moreover, the co-assembly of components mainly relies on intermolecular forces such as hydrophobic interaction, &#960;-&#960; stacking, and hydrogen bonding, which suggests that this method may not be applicable to all prodrug and dye molecules30. The feasibility of co-assembling functional molecules like drugs, prodrugs, dyes, photosensitizers, and photothermal agents needs to be evaluated through theoretical calculation and experimental characterization.
The photoactivatable prodrug-dye nanoscale drug delivery system has several advantages. Firstly, dyes like IR783 can be decomposed under light irradiation, subsequently enabling local and specific disassembly of the nanoparticles25. Furthermore, the incorporated dye can function as an imaging agent for monitoring the drug delivery system, with its fluorescence used to track the accumulation and disassembly of the nanoparticles. In addition, indocyanine dyes with sulfonate groups have been reported to be able to target caveolae in some cancers19, which enables efficient cellular uptake of the drug delivery systems25. Thus, indocyanine dyes with similar structures have great potential for incorporation in such drug delivery systems.
There have been only a few publications thus far on standardizing operations on the photoactivation of nano drug delivery systems31. Thus, the protocol described here can serve as a useful reference for developing photoresponsive drug delivery systems.

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><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&amp;amp;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&amp;amp;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&amp;amp;K Scientific</td><td>192001</td><td></td></tr><tr><td>Boron trifluoride diethyl etherate</td><td>J&amp;amp;K Scientific</td><td>921076</td><td></td></tr><tr><td>B&amp;uuml;chner funnel</td><td>AS ONE</td><td>3-6466-01</td><td></td></tr><tr><td>Chlorambucil</td><td>J&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;amp;K Scientific</td><td>203402</td><td></td></tr><tr><td>N,N&#039;-Dicyclohexylcarbodiimide (DCC)</td><td>J&amp;amp;K Scientific</td><td>275928</td><td></td></tr><tr><td>penicillin&amp;ndash;streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Phosphate-buffered saline (10&amp;times;)</td><td>&amp;nbsp;Sigma-Aldrich</td><td>P5493</td><td></td></tr><tr><td>&amp;nbsp;Power and energy meter&amp;nbsp;</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&amp;amp;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&amp;nbsp;</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 (Figure 4C), which could be attributed to the negatively charged sulfonate groups of IR783. Figure 4D shows the stability of the IR783/BC NPs. The size of the nanoparticles was maintained at around 85 nm for at least 48 h (in PBS at 37 &#176;C) after fabrication, while its PDI also remained less than 0.2. No significant change was observed in the size distribution of IR783/BC NPs at 0 h, 24 h, and 48 h after fabrication (Figure 4E).
Figure 5A,B displays the morphology of IR783/BC NPs before and after light irradiation, respectively. Both aggregates and fragments could be observed after light irradiation, which indicates the dissociation and aggregation of the nanoparticles, respectively. Figure 5C presents the change in nanoparticle size and its distribution after 3 min and 5 min of light irradiation. An increased size and broader distribution were observed for the irradiated nanoparticles. The photorelease profiles of the IR783/BC NPs were also measured by HPLC. As shown in Figure 5D,E, prodrug BC was photocleaved in 10 min. Meanwhile, Cb was released with a recovery efficiency of around 22% within the same period.
The IR783/BC NPs displayed significant cytotoxicity on human colorectal tumor cells (HCT116) under light irradiation at 530 nm compared with the non-irradiation group (Figure 6). The IC50 of IR783/BC NPs with light irradiation (6.62 &#181;M) was lower than that of free BC with light irradiation (9.24 &#181;M), which demonstrates a higher in vitro antitumor efficacy of IR783/BC NPs with light irradiation than that of free BC with light irradiation25. The presented cytotoxicity resulted from both released Cb and generated reactive oxygen species (ROS) (Supplemental Figure 1 and Supplemental Figure 2). The higher cytotoxicity of the IR783/BC NPs under light irradiation was mainly caused by the efficient cellular uptake of the IR783/BC NPs25.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64677/64677fig01.jpg" /> 
Figure 1: Formation of IR783/BC NPs. (A) Self-assembly and light-triggered dissociation of IR783/BC NPs. (B) Fabrication scheme of IR783/BC NPs by flash precipitation. Reproduced with permission25. Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64677/64677fig02.jpg" /> 
Figure 2: Synthesis route of prodrug BODIPY-Cb (BC). BODIPY derivates were first synthesized from acetoxyacetyl chloride and 2,4-dimethyl pyrrole, and then conjugated with Cb. 1H-NMR spectrum of the BC prodrug is shown in Supplemental Figure 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64677/64677fig03.jpg" /> 
Figure 3: LED irradiation setup. (A,B) LED lamp setting for irradiance measurement. (C,D) LED lamp setting for the irradiation of IR783/BC NPs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64677/64677fig04.jpg" /> 
Figure 4: Characterization of IR783/BC NPs. (A) Appearance of free IR783, free BC, and IR783/BC NP solutions. (B) Size distribution of IR783/BC NPs. (C) Surface charge of IR783/BC NPs. (D) Change of size of IR783/BC NPs in PBS at 37 &#176;C during 48 h after fabrication. (E) Size distribution of IR783/BC NPs at 0 h, 24 h, and 48 h after fabrication. Reproduced with permission25. Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64677/64677fig05.jpg" /> 
Figure 5: Photorelease of IR783/BC NPs. TEM images of IR783/BC NPs (A) before and (B) after light irradiation. Scale bar = 100 &#181;m. (C) Size distribution of IR783/NPs after 0 min, 3 min, and 5 min of light irradiation. (D) HPLC analysis of IR783/BC NPs subjected to increasing duration of light irradiation until 10 min. (E) Quantification of BC and IR783 degradation and Cb release (n = 3). Error bars represent standard deviation. Light irradiation: 530 nm LED lamp, 50 mW/cm2. Reproduced with permission25. Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64677/64677fig06.jpg" /> 
Figure 6: Cytotoxicity of IR783/BC NPs against HCT116 cells. Significant cytotoxicity appeared at BC concentrations &#62;1 &#181;M (n = 4). Error bars represent standard deviation. An independent sample t-test was adopted to determine the statistical significance of differences. *p &#60; 0.05, **p &#60; 0.01. Light irradiation: 530 nm LED lamp, 50 mW/cm2. Reproduced with permission25.Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/64677fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Photochemistry mechanism. (A) Photocleavage of BC prodrug. (B) Decomposition of IR783. Adapted with permission25. Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/Supplementary Figure 1.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure 2: ROS generation profile. ROS generation of different samples under light irradiation quantified with a singlet oxygen sensor green (SOSG) probe. Light irradiation: 530 nm LED lamp, 50 mW/cm2. Adapted with permission25. Copyright 2022, Wiley. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/Supplementary Figure 2.jpg" target="_blank">Please click here to download this File.</a>
Supplemental Figure 3: 1H-NMR spectrum of BODIPY-Cb. Reprinted with permission22. Copyright 2022, American Chemical Society. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64677/Supplementary Figure 3.jpg" target="_blank">Please click here to download this File.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

970 Views.  The University of Hong Kong. Dit protocol biedt een referentie over de constructie en karakterisering van fotoresponsieve medicijnkleurstofsystemen, met name de opstelling van lichtstraling. Deze techniek toont het voordeel van eenvoudige fabricage, hoge laadcapaciteit van geneesmiddelen en fotocontroleerbaarheid. Deze techniek kan worden gebruikt om colorectale tumoren te behandelen met behulp van optische vezels om lichten te leveren voor het activeren van de medicijnafgifte op de tumorplaats.Begin met het wegen...

Video: Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Chemistry

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biology

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Bioengineering

Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood or interstitial fluid); 2) the object is illuminated with light of appropriate wavelength in order to excite the probe into its triplet state; 3) the emitted phosphorescence is collected, and its time course is analyzed to yield the phosphorescence lifetime, which is converted into the oxygen concentration (or partial pressure, pO2). The probe must not interact with the biological environment and in some cases to be 4) excreted from the medium upon the measurement completion. Each of these steps imposes requirements on the molecular design of the phosphorescent probes, which constitute the only invasive component of the measurement protocol. Here we review the design of dendritic phosphorescent nanosensors for oxygen measurements in biological systems. The probes consist of Pt or Pd porphyrin-based polyarylglycine (AG) dendrimers, modified peripherally with polyethylene glycol (PEG's) residues. For effective two-photon excitation, termini of the dendrimers may be modified with two-photon antenna chromophores, which capture the excitation energy and channel it to the triplet cores of the probes via intramolecular FRET (F&ouml;rster Resonance Energy Transfer). We describe the key photophysical properties of the probes and present detailed calibration protocols.

We present principles of oxygen measurements by phosphorescence quenching and review design of porphyrin-based dendritic nanosensors for oxygen imaging in biological systems.

Oxygen measurement by phosphorescence quenching [1, 2] consists of the following steps: 1) the probe is delivered into the medium of interest (e.g. blood ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Synthesis of Near-Infrared Emitting Gold Nanoclusters for Biological Applications

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been devoted to their development. In this protocol, a recently developed, facile method for preparation of water soluble, biocompatible, and colloidally stable near-infrared emitting AuNCs have been described in detail. This room-temperature, bottom-up chemical synthesis provides easily functionalizable AuNCs capped with thioctic acid and thiol-modified polyethylene glycol in aqueous solution. The synthetic approach requires neither organic solvents or additional ligand exchange nor extensive knowledge of synthetic chemistry to reproduce. The resulting AuNCs offer free surface carboxylic acids, which can be functionalized with various biological molecules bearing a free amine group without adversely affecting the photoluminescent properties of the AuNCs. A quick, reliable procedure for flow cytometric quantification and confocal microscopic imaging of AuNC uptake by HeLa cells also been described. Due to the large Stokes shift, proper setting of filters in flow cytometry and confocal microscopy is necessary for efficient detection of near-infrared photoluminescence of AuNCs.

A reliable and easily reproducible method for preparation of functionalizable, near-infrared emitting photoluminescent gold nanoclusters and their direct detection inside HeLa cells by flow cytometry and confocal laser scanning microscopy is described.

Over the past decade, fluorescent gold nanoclusters (AuNCs) have witnessed growing popularity in biological applications and enormous efforts have been ...

Facile Preparation and Photoactivation of Prodrug-Dye Nanoassemblies

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