Near-infrared fluorescent dye

Protocol
I. Protein Sample Pretreatment
1) To optimize labeling efficiency, it is recommended to prepare the target protein (e.g., antibody) at a concentration of 2 mg/mL.
2) Ensure the pH of the protein solution is 8.5±0.5. If the pH is below 8.0, adjust it using 1 M sodium bicarbonate solution.
3) If the protein concentration is lower than 2 mg/mL, labeling efficiency will significantly decrease. It is recommended to maintain the final protein concentration within the range of 2–10 mg/mL for optimal labeling results.
4) The protein solution should be in a buffer system free of primary amines (such as Tris or glycine) and ammonium ions to avoid interference with the labeling reaction.

II. Dye Working Solution Preparation (Using ICG-OSu as an Example)
Add anhydrous DMSO to the ICG-OSu vial to prepare a 10 mM stock solution, and ensure thorough dissolution and mixing via pipetting or vortexing.

III. Dye Dosage Calculation
The amount of ICG-OSu required depends on the quantity of protein to be labeled. It is recommended to maintain a molar ratio of dye to protein at approximately 10:1.
Example:
Assuming 500 μL of IgG at a concentration of 2 mg/mL (molecular weight 150,000) needs to be labeled, and 1 mg of ICG-OSu is dissolved in 100 μL DMSO, the required volume of ICG-OSu is calculated as follows:
1) Micromoles of IgG = concentration (mg/mL) × volume (mL) / molecular weight (mg/mmol)
= 2 × 0.5 / 150,000 = 6.7×10⁻⁶ mmol
2) Required micromoles of ICG-OSu = micromoles of IgG × 10
= 6.7×10⁻⁶ × 10 = 6.7×10⁻⁵ mmol
3) Volume of ICG-OSu (μL) = micromoles of ICG-OSu × molecular weight (mg/mmol) / concentration (mg/μL)
= 6.7×10⁻⁵ × 753.88 / 0.01 = 5.05 μL

IV. Labeling Reaction Steps
1) Slowly add an appropriate amount of freshly prepared 10 mg/mL ICG-OSu to 0.5 mL of the protein sample. Mix gently and then centrifuge briefly to collect the liquid at the bottom of the tube. Avoid vigorous shaking to prevent protein denaturation.
2) Place the reaction tube in a light-protected environment and incubate at room temperature for 60 minutes. During incubation, gently invert the tube every 10–15 minutes for mixing.

V. Purification of the Conjugate
The following is an example procedure for purifying the dye-protein conjugate using a Sephadex G-25 column:
1) Pretreat the Sephadex G-25 column according to the manufacturer’s instructions.
2) Load the reaction mixture onto the top of the Sephadex G-25 column.
3) Once the sample enters the column bed, immediately add PBS (pH 7.2–7.4) for elution.
4) Continue washing the column with PBS (pH 7.2–7.4), collect the eluted fractions containing the target conjugate, and combine them for further use.

The study did not perform independent in vivo activity assays specifically for ICG-OSu. All in vivo imaging and biodistribution studies were conducted using the final conjugated probe TMTP1-PEG4-ICG or free ICG dye as a control [1].

The cyclic structure of TMTP1 (cTMTP1) was designed to be consistent with the previous research. We designed the fluorescent probe as follows: tumor targeted peptide TMTP1 equipped with PEG4 was covalently conjugated to ICG derivative ICG-OSu, as demonstrated in Figure1a. A solid carrier 2-chlorotrityl chloride was used to synthesize solid-phase polypeptide. At first the amino acid is synthesized according to a predetermined sequence, ligated to PEG4 with −NH2 tail, and excised from the resin. Then it was oxidized with a MeOH/I2 solution and the polypeptide sequence looped through the disulfide bond. After purification and lyophilization, the sample was mixed with ICG-OSu (equal molar amount) dissolved in DMF and twice the volume of DIEA was added. LC-MS identified whether the reaction was complete. After completion, the reaction solution was directly separated and purified, then lyophilized and packed. The chemistry purity of TMTP1-PEG4-ICG was analyzed by HPLC and the compound was further evaluated by LC-MS. The optical absorbance spectrum was measured by a UV–vis spectrophotometer and compared with ICG.[1]

No cell assay specifically for ICG-OSu was described in the study. The cell binding assay described was used to compare the targeting ability of the final probe TMTP1-PEG4-ICG against free ICG.
For the probe evaluation, cells (4T1, SiHa, HeLa, S12, HaCaT) were seeded at a density of 1x10^5 per well in 6-well plates and cultured until ~80% confluent. After washing with cold PBS, cells were incubated with 1 µM of either TMTP1-PEG4-ICG or free ICG (as control) on ice for 1 hour. Cells were then washed, harvested, and analyzed by flow cytometry. For blocking studies, cells were pre-incubated with 10 µM cTMTP1 peptide for 30 minutes before adding the TMTP1-PEG4-ICG probe [1].

No animal protocol specifically administering ICG-OSu alone was described. The described protocols were for the evaluation of the final probe TMTP1-PEG4-ICG or free ICG control.
For biodistribution in normal mice, BALB/c mice were intravenously injected with 100 µL of 25 µM TMTP1-PEG4-ICG via the tail vein and imaged using an IVIS system over 48 hours [1].
For tumor targeting studies, 4T1-luc or HeLa-luc tumor-bearing mice (BALB/c or BALB/c nu) were intravenously injected with 100 µL of 25 µM TMTP1-PEG4-ICG or an equivalent amount of free ICG. In blocking studies, mice were pre-injected with 100 µL of 100 µM cTMTP1 peptide before probe administration. Mice were imaged over 48 hours, and organs were excised at specified time points for ex vivo imaging [1].

[1]. A Novel Near-Infrared Fluorescent Probe TMTP1-PEG4-ICG for in Vivo Tumor Imaging. Bioconjug Chem. 2018;29(12):4119-4126.

ICG-OSu, namely indocyanine green N-hydroxysuccinimide ester, is an amine-reactive derivative of the near-infrared dye indocyanine green (ICG)[1].
It is widely used as a building block for designing in vivo imaging probes by coupling its reactive NHS ester group with targeting ligands (e.g., peptides, antibodies)[1].
In this study, ICG-OSu was coupled with the tumor-targeting peptide TMTP1 (via a PEG4 linker) to synthesize the molecular imaging probe TMTP1-PEG4-ICG, aiming to improve its tumor specificity and make it superior to free ICG[1].

C49H53N3O7S

828.026

827.36

1622335-40-3

138376165

Dark purple to black solid powder

8.9

0

8

16

60

1820

0

S(CCCCN1C2C=CC3=CC=CC=C3C=2C(C)(C)/C/1=C/C=C/C=C/C=C/C1C(C)(C)C2C3=CC=CC=C3C=CC=2[N+]=1CCCCCC(=O)ON1C(CCC1=O)=O)(=O)(=O)[O-]

KDJUNNVUQBKNAY-UHFFFAOYSA-N

InChI=1S/C49H53N3O7S/c1-48(2)41(50(39-28-26-35-19-12-14-21-37(35)46(39)48)32-16-8-11-25-45(55)59-52-43(53)30-31-44(52)54)23-9-6-5-7-10-24-42-49(3,4)47-38-22-15-13-20-36(38)27-29-40(47)51(42)33-17-18-34-60(56,57)58/h5-7,9-10,12-15,19-24,26-29H,8,11,16-18,25,30-34H2,1-4H3

4-[(2Z)-2-[(2E,4E,6E)-7-[3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-1,1-dimethylbenzo[e]indol-3-ium-2-yl]hepta-2,4,6-trienylidene]-1,1-dimethylbenzo[e]indol-3-yl]butane-1-sulfonate

ICG-OSu; 1622335-40-3; ICG-NHS; ICG-NHS ester; ICG NHS ester;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 25 mg/mL (30.19 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)