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 from nanocarriers at target sites modulated by light irradiation. In this protocol, a facile method for fabricating photoactivatable prodrug-dye nanoparticles via molecular self-assembly is presented. The procedures for prodrug synthesis, nanoparticle fabrication, physical characterization of the nanoassembly, photocleavage demonstration, and in vitro cytotoxicity verification are described in detail. A photocleavable boron-dipyrromethene-chlorambucil (BC) prodrug was first synthesized. BC and a near-infrared dye, IR-783, at an optimized ratio, could self-assemble into nanoparticles (IR783/BC NPs). The synthesized nanoparticles had an average size of 87.22 nm and a surface charge of -29.8 mV. The nanoparticles disassembled upon light irradiation, which could be observed by transmission electronic microscopy. The photocleavage of BC was completed within 10 min, with a 22% recovery efficiency for chlorambucil. The nanoparticles displayed enhanced cytotoxicity under light irradiation at 530 nm compared with the non-irradiated nanoparticles and irradiated free BC prodrug. This protocol provides a reference for the construction and evaluation of photoresponsive drug delivery systems.
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).
1. Synthesis of boron-dipyrromethene-chlorambucil (BC) prodrug (Figure 2)22
2. Preparation of IR783/BC NPs by the flash precipitation method
Time (min) | Acetonitrile (%) | Water (%) |
0 | 20 | 80 |
5 | 20 | 80 |
30 | 95 | 5 |
35 | 95 | 5 |
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
4. Photoactivation of IR783/BC NPs
5. Testing cytotoxicity of IR783/BC NPs with and without light irradiation
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 °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 µM) was lower than that of free BC with light irradiation (9.24 µM), 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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 °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. Please click here to view a larger version of this figure.
Figure 5: Photorelease of IR783/BC NPs. TEM images of IR783/BC NPs (A) before and (B) after light irradiation. Scale bar = 100 µm. (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. Please click here to view a larger version of this figure.
Figure 6: Cytotoxicity of IR783/BC NPs against HCT116 cells. Significant cytotoxicity appeared at BC concentrations >1 µM (n = 4). Error bars represent standard deviation. An independent sample t-test was adopted to determine the statistical significance of differences. *p < 0.05, **p < 0.01. Light irradiation: 530 nm LED lamp, 50 mW/cm2. Reproduced with permission25.Copyright 2022, Wiley. Please click here to view a larger version of this figure.
Supplemental Figure 1: Photochemistry mechanism. (A) Photocleavage of BC prodrug. (B) Decomposition of IR783. Adapted with permission25. Copyright 2022, Wiley. Please click here to download this File.
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. Please click here to download this File.
Supplemental Figure 3: 1H-NMR spectrum of BODIPY-Cb. Reprinted with permission22. Copyright 2022, American Chemical Society. Please click here to download this File.
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, π-π 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.
The authors have nothing to disclose.
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).
1260 Infinity II HPLC | Agilent Technologies | ||
2,4-Dimethyl pyrrole | J&K Scientific | 315305 | |
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) | Gibco | M6494 | |
4-Dimethylaminopyridine (4-DMAP) | J&K Scientific | 212279 | |
90 mm Petri Dish Clear Treated Sterile | SPL | 11090 | |
96-well Tissue Culture Plate Clear Treated Sterile | SPL | 30096 | |
Acetoxyacetyl chloride | J&K Scientific | 192001 | |
Boron trifluoride diethyl etherate | J&K Scientific | 921076 | |
Büchner funnel | AS ONE | 3-6466-01 | |
Chlorambucil | J&K Scientific | 321407-1G | |
CM100 Transmission Electron Microscope | Philips | ||
CombiFlash RF chromatography system | Teledyne ISCO | ||
Dichloromethane | DUKSAN Pure Chemicals | JT9315-88 | |
Dimethyl sulfoxide | DUKSAN Pure Chemicals | 2762 | |
Disposable cuvette | Malvern Panalytical | DTS1070 | Zeta potential measurement |
Disposable cuvette | Malvern Panalytical | ZEN0040 | |
Empty Disposable Sample Load Cartridges | Teledyne ISCO | 693873225 | can hold up to 65 g |
Fetal bovine serum | Gibco | 10270106 | |
Filtering flask | AS ONE | 3-7089-03 | |
Hexane | DUKSAN Pure Chemicals | 4198 | |
Holey carbon film on copper grid | Beijing Zhongjingkeyi Technology Co.,Ltd | BZ10023a | |
HPLC column (InfinityLab Poroshell 120) | Agilent Technologies | 695975-902T | |
Integrating sphere photodiode power sensor | Thorlabs | S142C | |
IR783 | Tokyo Chemical Industry (TCI) Co., Ltd | I1031 | |
LED | Mightex | LCS-0530-15-11 | |
LED Driver Control Panel V3.2.0 (Software) | Mightex | ||
Lithium Hydroxide Anhydrous | TCI | L0225 | |
Methylmagnesium iodide, 3M solution in diethyl ether | Aladdin | M140783 | |
N,N-Diisopropyl ethyl amine (DIPEA) | J&K Scientific | 203402 | |
N,N'-Dicyclohexylcarbodiimide (DCC) | J&K Scientific | 275928 | |
penicillin–streptomycin | Gibco | 15140122 | |
Phosphate-buffered saline (10×) | Sigma-Aldrich | P5493 | |
Power and energy meter | Thorlabs | PM100 USB | |
Rotavapor | BUCHI Rotavapor R300 | ||
RMPI 1640 | Gibco | 21870076 | |
Separatory funnel (125 mL) | Synthware | F474125L | |
Silver Silica Gel Disposable Flash Columns, 40 g | Teledyne ISCO | 692203340 | |
Sodium sulfate, anhydrous | Alfa Aesar | A19890 | |
SpectraMax M4 | Molecular Devices LLC | ||
Tetrahydrofuran (THF), anhydrous | J&K Scientific | 943616 | |
Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
Vortex | DLAB Scientific Co., Ltd | MX-S | |
Zetasizer Nano ZS90 | Malvern Instrument |