A Bright NIR-II Fluorescence Probe for Vascular and Tumor Imaging

Summary

The present protocol describes a detailed, real-time NIR-II fluorescence imaging operation of a mouse using a NIR-II optics imaging device.

Abstract

As an emerging imaging technology, near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging has significant potential in the biomedical field, owing to its high sensitivity, deep tissue penetration, and superior imaging with spatial and temporal resolution. However, the method to facilitate the implementation of NIR-II fluorescence imaging for some urgently needed fields, such as medical science and pharmacy, has puzzled relevant researchers. This protocol describes in detail the construction and bioimaging applications of a NIR-II fluorescence molecular probe, HLY1, with a D-A-D (donor-acceptor-donor) skeleton. HLY1 showed good optical properties and biocompatibility. Furthermore, NIR-II vascular and tumor imaging in mice was performed using a NIR-II optics imaging device. Real-time high-resolution NIR-II fluorescence images were acquired to guide the detection of tumors and vascular diseases. From probe preparation to data acquisition, the imaging quality is greatly improved, and the authenticity of the NIR-II molecular probes for data recording in intravital imaging is ensured.

Introduction

Fluorescence imaging is the commonly used molecular imaging tool in basic research, and is also often used to guide surgical tumor resection in clinics¹. The essential principle of fluorescence imaging is to employ a camera to receive fluorescence emitted by a laser after the irradiation of samples (tissues, organs, etc.)². The process is completed within a few milliseconds³. The fluorescence imaging wavelengths can be divided into ultraviolet (200-400 nm), visible region (400-700 nm), near-infrared I (NIR-I, 700-900 nm), and near-infrared II (NIR-II, 1000-1700 nm)^4,5,6. Because the endogenous molecules such as hemoglobin, melanin, deoxyhemoglobin, and bilirubin in biological tissues have strong absorption and a scattering effect on the light in visible regions, the penetration and sensitivity of light are greatly reduced, and the fluorescence imaging in visible light wavelengths is adversely affected^7,8,9.

NIR-II fluorescence imaging has low photon absorption and scattering, high imaging speed, and high image contrast (or sensitivity)^10,11. As the fluorescence wavelength increases, the absorption and scattering of fluorescence in biological tissues decrease gradually, and the auto-fluorescence in the NIR-II region is extremely low¹². Thus, the NIR-II window significantly increases the penetration depth of tissues and obtains a higher resolution and signal-to-noise ratio^13,14,15. The NIR-II window can be further subdivided into the NIR-IIa (1300-1400 nm) and NIR-lIb (1500-1700 nm) windows¹⁶. To date, several milestone NIR-II materials have been reported, including inorganic material single-walled carbon nanotubes, rare earth nanoparticles, quantum dots, and organic material semiconductor polymer nanoparticles, small-molecule dyes, aggregation-induced luminescent materials, etc.^{1,17,18,19,20,21,22}. Inorganic nanomaterials are easily accumulated in the liver, spleen, etc., and have potential long-term biotoxicity²³. Organic small-molecule fluorophore has the advantages of rapid metabolism, low toxicity, easy modification, and a clear structure, which is the most promising probe for clinical use²⁴.

The NIR-II optics imaging system is also a critical component of fluorescence bioimaging because it can efficaciously collect NIR-II fluorescence signals from the NIR-II probe, thus rendering precise functional, anatomical, and molecular images^25,26. The NIR-II imaging system mainly comprises shortwave infrared cameras, long-pass (LP) filters, lasers, and computer processors. In vivo NIR-II fluorescent imaging is considered one of the most feasible imaging approaches for elucidating the mechanisms of diseases and the nature of life^27,28,29. NIR-II imaging technology has been widely used in biomedical fields such as cancer cell detection, dynamic imaging, in vivo targeted tracing, and targeted therapy, especially in oncology research^30,31. However, considering the high technical requirements of NIR-II imaging technology on imaging probes and instruments, it also puzzles and restricts the practical use of researchers in different fields. Therefore, the preparation of NIR-II imaging probes and the applications of NIR-II imaging are introduced in detail in this article.

Protocol

Animal experiments for NIR-II imaging studies were conducted at the Animal Experiment Center of Wuhan University, which has been awarded the International Association for Experimental Animal Care (AALAC). All animal studies were conducted following the China Animal Welfare Commission Guidelines for the Care and Use of Experimental Animals and approved by the Animal Care and Use Committee (IACUC) of the Animal Experimental Center of Wuhan University.

Female BALB/c nude mice (~20 g) at 6 weeks of age were used for the present study.

1. NIR-II imaging preparation

1. Place commercially available black cardboard (see Table of Materials) in the center of the carrier. Then, place the sample on top of the black cardboard, so that the sample is in the center of the carrier (a stage located in the imaging device).
   NOTE: Compared with white cardboard, black cardboard has less background interference during NIR-II imaging.
2. Select a suitable filter based on the wavelength of the NIR-II probe. Press long (>2 s) to control the box area (such as 900 LP) corresponding to the filter model in the screen interface when the system moves the filter into the optical imaging path.
3. Long press platform up on the touch screen interface of the carrier console control area so that the carrier consoles up; long press platform down so the carrier consoles down.
4. Adjust the platform height to "0 mm" (height adjustment) and use automatic focusing to make the NIR-II image clear.

2. Synthesis of NIR-II dye (HLY1)

1. Weigh the raw materials required for the synthesis experiment. Ensure they do not deteriorate.
2. Add Compound 1 (200 mg, 0.18 mmol), PdCl₂(dppf)₂CH₂Cl₂ (28 mg, 0.04 mmol), N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine (170 mg, 0.4 mmol), and K₂CO₃ (46 mg, 0.34 mmol) to tetrahydrofuran (THF) solution in a 25 mLround-bottom flask. Stir the mixture for 4 h at 75 °C under N₂ atmosphere (Figure 1A).
   NOTE: For the synthesis procedure of Compound 1 and N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-2-amine, refer to Li et al²¹. The chemical structures are shown in Figure 1A.
3. After cooling to ambient temperature, quench the reaction with distilled (DI) water (80 mL), and extract the mixture with DCM (dichloromethane)/H₂O (30 mL) (three times). Purify the crude product by column chromatography¹⁶ (petroleum ether:DCM = 10:1) to make HLY1 a green solid (78 mg, 30% yield).
4. Place the dye HLY1 under the protection of nitrogen in the refrigerator for later use. This can be stored for up to 6 months.

3. Preparation of water-suspensible nanoprobe

1. Weigh HLY1 (1 mg) and amphipathic encapsulation materials, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2k (DSPE-PEG2k, 10 mg; see Table of Materials).
2. Prepare HLY1 dots²⁰ by employing DSPE-PEG2k as an encapsulation matrix (nanoprecipitation method¹²) (Figure 1C). Dissolve HLY1 in THF (1 mL) and slowly add into a beaker containing DSPE-PEG2k aqueous solution (9 mL) with sonication at 25 °C. Subsequently, remove THF from the mixture by dialysis²⁰.
3. Concentrate the above solution centrifugally with ultrafiltration¹⁸ (7100 x g for 10 min) and then place it in a 4 °C refrigerator for future use. This can be stored for up to 1 month.
   NOTE: The nanoprobe aqueous solution loaded by DSPE-PEG_2k should be stored above 0 °C and used as soon as possible.

4. Construction of tumor-bearing mice

1. Culture 4T1 mouse breast cancer cells (4T1) in Dulbecco's Modified Eagle Medium (DMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (see Table of Materials), and maintain in a humidified incubator with 5% CO₂ at 37 °C.
2. For the NIR-II fluorescent imaging experiment, culture 4T1 cells (5 x 10⁷) for 24 h, digest with trypsin (1 mL), and wash twice with serum-free DMEM (4 mL).
3. Anesthetize the mice by treating with isoflurane (2%). Confirm adequate anesthetization by stimulating the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient³².
4. Then, using an insulin injection needle, inject the 4T1 cell mixture into the mice through subcutaneous injection (100 µL).
   ​NOTE: NIR-II imaging studies were performed ~2 weeks after inoculation, when the tumor had grown to a volume of ~100 mm³. Before NIR-II tumor imaging, please confirm the tumor size. The tumor size was estimated by an electronic vernier caliper for the present study¹¹.

5. In vivo NIR-II fluorescence imaging

1. Anesthetize the mice by treating with isoflurane (2%) and perform NIR-II imaging of the whole body of the mice using an optical NIR-II imaging system (see Table of Materials).
   NOTE: Pay attention to the dosage of anesthetic to avoid mice death. Generally, anesthesia lasts for 5-10 min. Stimulate the toes or the soles of the feet of the mice, and observe whether the mice respond. If there is no response, it means that the anesthesia is sufficient.
2. Take a solution of HLY1 dots (0.8 mg/mL, 200 µL). Inject the HLY1 dots intravenously into the anesthetized mice, and 3 min later, perform NIR-II fluorescence imaging of the blood vessels of the whole body of mice using an NIR-II imaging system. Focus further on the mouse's head to collect brain vascular imaging.
   NOTE: Use clean experimental gloves during imaging, which will help to obtain clean NIR-II images.
3. Collect the images 5 min after the injection of HLY1 dots in mice, and process the data using ImageJ software. The instrument parameters of the optical NIR-II imaging system are 90 mW/cm² (808 nm laser).
4. On completion of the experiment, euthanize the animals following institutionally approved protocols.
   NOTE: For the present study, the animals were euthanized by exposing them to excess isoflurane³².

Results

The fluorescent intensity and brightness of water-suspensible HLY1 dots were determined by an NIR-II imaging instrument. The fluorescent intensity of HLY1 in the 90% f_wTHF/H₂O mixture was five times that in the THF solution, which indicated a prominent AIE feature of HLY1 (Figure 1B). Moreover, HLY1 dots emitted strong fluorescent signals under a 1,500 nm LP filter, showing that HLY1 dots can be used for NIR-IIb imaging (Figure 1D)...

Discussion

NIR-I fluorescent imaging can be used to some extent for tumor and vascular imaging, but due to the limited maximum emission wavelength of NIR-I fluorophores (<900 nm), it results in poor tissue penetration and tumor signal background ratio^33,34. Poor and low imaging resolution may cause a deviation between the outcome of the imaging feedback treatment and the actual therapeutic effect. In addition, most NIR-I fluorophores have poor optical stability and extr...

Materials

Name	Company	Catalog Number	Comments
Anhydrous pyridine	Perimed	110-86-1
Anhydrous sodium sulfate	China national medicines Co.,Ltd	SY006376
Black cardboard	Suzhou Yingrui Optical Technology Co., Ltd	AO00158
Column chromatography	Energy Chemical	E080498
Diphenylphosphine palladium dichloride	Sigma-Aldrich	B2161-1g
DSPE-PEG2000	Ponsure	PS-E1
Dulbecco's modified eagle medium	Gibco	8121587
EGTA	Biofroxx	EZ6789D115
Fetal bovine serum	Gibco	2166090RP
Isoflurane	GLPBIO	GC45487-1
K2CO3	Macklin	P816305-5g
N. N '- dimethylformamide	China national medicines Co.,Ltd	02-12-1968
NIR-II imaging instrument	Suzhou Yingrui Optical Technology Co., Ltd	16011109
N-sulfenanilide	Enerry chemical	1250030-5g
PdCl2(dppf)2CH2Cl2	TCI	B2064-1g
penicillin-streptomycin	Gibco	15140-122
Tetrahydrofuran	China national medicines Co.,Ltd	M005197
Tetratriphenylphosphine palladium	Immochem	1021232-5g
Tetratriphenylphosphine palladium	Sigma-Aldrich	1021232-5g
Tributyltin chloride	Immochem	QH004335
Trimethylchlorosilane	China national medicines Co.,Ltd	40060560