Fluorescent dye

In study pertaining to life sciences, rhodamine B isothiocyananate is a biochemical reagent that can be utilized as an organic substance or biological material.

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To understand the mechanism of tumor-specific fluorescence of RBITC-PEG, we first investigated the in vitro fluorescence of the PEGylated dyes in the presence of fetal bovine serum (FBS) and mouse whole blood. As shown in Fig. 3a, the fluorescence of RBITC-PEG in water increased after the addition of FBS but was quenched by whole blood. In contrast, the Cy5.5 (Cy7)-PEG fluorescence was not quenched but enhanced by the blood and FBS because the better dispersion reduces the self-quenching of the dye. Surprisingly, the ground tissues of the liver, spleen, kidney, and lung could also quench RBITC-PEG fluorescence but the tumor tissue did not; however, these ground tissues did not affect the fluorescence of Cy5.5-PEG (Fig. 3b&c). Another important finding is that perfusion of the mice with phosphate buffer solution (PBS) before their sacrifice to remove the blood, the fluorescence in the organs was recovered (Fig. 3d&e). These results indicate that red blood cells in the organs might account for the fluorescence quenching of RBITC-PEG. [1]

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Then, the iv injected 4 T1-Luc-GFP cells were used to mimic circulating cancer cells to further probe their effects on the main organs and early metastasis (Fig. 6a). After 2 days of the cell injection, there were no bioluminescent signals detected in the whole mouse body, suggesting no tumor formation yet. After 7 days, only one big bioluminescent spot in the lung of the mouse appeared and became even stronger after 14 days (Fig. 6b&c). However, the fluorescence imaging of the injected RBITC-PEG showed that even at 2 days after the cancer cell injection, intense fluorescent signals were already observed in the liver, little in the lung and kidneys. At 7 days, the liver had massive and spread fluorescence, and the fluorescence in the lung and kidneys also became stronger. At 14 days, the liver, lung, and kidneys all had strong fluorescence (Fig. 6d&e). The confocal microscopy and histological analysis of the main organ slices found that, very surprisingly, viable GFP fluorescent cancer cells were only found in the lung, not in the liver, kidneys, and spleen, given their extensive fluorescence (Fig. 6f). Thus, the iv injected cancer cells were easily trapped in the lung and formed tumors due to their large sizes. Another important finding is that the circulating tumor cells did modulate the microenvironments of the liver and other organs well before forming metastatic tumors (Fig. 6g). By hematological analysis of mice with orthotopic and intravenous 4 T1 tumors (Fig. S3), we found a significant drop in blood HgB concentration in intravenous administration model, indicating metastases do influence the HgB and this change could be reflected by the fluorescence of RBITC derivatives. Further, we established an intracardiac injection metastases model (Fig. S4), the fluorescent signal increased along with the severe degree of metastasis (from No.1 to No.3). So, RBITC-PEG may be useful for detecting microenvironment change and pre-metastatic niches [1].

Fluorescence is routinely used for in vivo tracking and imaging of molecules and nanostructures with assuming that the fluorescence intensity is proportional to the dye concentration. Herein, we report the unique tumor-specific fluorescence character of rhodamine B isothiocyanate derivatives (RBITCs), which emits fluorescence selectively in cancerous tissues, including small metastatic tumors, but is quenched in blood and healthy tissues. A preliminary mechanism study shows that binding of the thiourea group in the RBITCs on hemoglobin quenches their fluorescence, but the oxidation of the thiourea by the elevated reactive oxygen species in tumor activates the fluorescence. Thus, the fluorescent intensity of RBITCs is associated with the microenvironment of tissues and positively correlates with the cancer stages. These findings suggest that the RBITCs are not suitable for tracking of cargos in the presence of red blood cells but may be useful for cancer imaging and early diagnosis, and probing the tumor microenvironment. [1]

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In vivo tumor-specific fluorescence imaging [1]
The RBITC derivatives were prepared by the reaction of RBITC's isothiocyanate group with the terminal amine group of PEG (500 Da), obtaining the RBITC conjugate with PEG (RBITC-PEG) (Fig. 1). The widely used dyes, Cy7 and Cy5.5, were also PEGylated to obtain Cy7-PEG and Cy5.5-PEG for comparison.
We first evaluated the in vivo fluorescence characters of RBITC-PEG by ex vivo fluorescence imaging. The mice bearing subcutaneous (sc) BCap37 xenografted tumors were iv injected with dyes. After 12 h, they were sacrificed and their blood, tumors, and main organs, including heart, liver, spleen, lung, and kidneys, were dissected and imaged (Fig. 2a). The tumors of the mice injected with RBITC-PEG showed strong fluorescence, while their blood and healthy organs had very weak fluorescence. In contrast, Cy5.5-PEG or Cy7-PEG gave very weak fluorescence in tumors but very strong fluorescence in normal organs, especially in the liver, lung, and kidneys. The fluorescence intensity ratios of tumor to liver (T/Li) and tumor to lung (T/Lu) were calculated to quantitatively analyze the fluorescence characters of these dyes (Fig. 2b and c). Both the T/Li and T/Lu ratios of the RBITC-PEG-treated mice were more than 5-fold of those treated with Cy5 or Cy7-PEG. We also verified the tumor-specific fluorescence of RBITC-PEG in the mice bearing sc cervical HeLa tumors, pancreatic BxPC3 tumors, and breast MCF-7/ADR drug-resistant tumors, and observed the same phenomenon that only tumors had strong fluorescence (Fig. 2d). The time-dependence of the fluorescence in the organs and tumors after the iv injection of RBITC-PEG was tracked using ex vivo imaging (Fig. 2e). The fluorescence of RBITC-PEG in tumors was already strong with half an hour and remained the same after that, while the fluorescence in the liver, lung, and kidneys was initially strong but decayed over time and became very weak after 12 h.

The strong fluorescence in tumor seemed that RBITC-PEG had an exciting tumor-specific targeting. However, analysis of the real RBITC-PEG concentrations in the organs and tumors by high-performance liquid chromatography (HPLC) revealed that only 1.15% dose of the injected RBITC-PEG accumulated in the tumors at 12 h post-injection, but the liver, spleen, and lung had concentrations of about 7–9 fold of that in the tumor (Fig. 2f). So, the RBITC fluorescence was activated in the tumor but quenched in healthy organs, including liver and lung, indicating that the RBITC fluorescence was not correlated with its concentration. Thus, an important indication is that the RBITCs' fluorescence behaviors must be carefully calibrated before usage for in vivo studies such as labeling carriers for biodistribution analysis. [1]

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To further confirm the assumption, we collected the whole blood of the mice after injection of RBITC-PEG for 12 h. The blood had no fluorescence. However, after centrifugation to separate the serum and red blood cells, the upper serum layer had strong fluorescence, but the red blood cells had no fluorescence (Fig. 3f). Interestingly, adding salt increased the fluorescence slightly at the salt concentrations from 0.5% to 5%, and became very strong when the salt concentration reached 7.5% or higher.
To further test if hemoglobin (HgB) of the red blood cells was responsible for RBC quenching the RBITC-PEG fluorescence, its fluorescence with cells before and after generating HgB was compared. K562 human erythroid progenitor cells, an in vitro experimental model mimicking erythroid differentiation, can be induced to be hemoglobin-generating cells by hemin. RBITC-PEG mixed with K562 cells emitted strong fluorescence, but once with the K562 cells induced to produce HgB, its fluorescence sharply reduced (Fig. 3g). Thus, it was HgB that quenched the fluorescence of RBITC-PEG.
It can be concluded that in blood and healthy tissues, RBCs' HgB quenches the RBITC-PEG fluorescence, but why RBCs in tumors could not quench it was unknown. Tumors are known to suffer persistent systemic oxidative stress with elevated levels of reactive oxygen speices (ROS). Thus, the role of ROS in activation of the HgB-quenched RBITC-PEG was further probed. RBCs' membrane and HgB were separated and separately mixed with RBITC-PEG. Within expected, HgB quenched the RBITC-PEG immediately while the RBC membrane didn't affect the fluorescence (Fig. 3h&i). Once H2O2 (1 mM), the most common type of ROS, was added, the RBITC-PEG with HgB exhibited strong fluorescence, indicating the de-quenching the fluorescence, while the fluorescence of RBITC-PEG with RBC membrane was unchanged by H2O2. This result suggests that the tumor oxidation environment reversed the HgB quenching of RBITC-PEG.
Phenylhydrazine (PHZ) induces the oxidation of RBCs, and the PHZ- induced toxicity is the drug-induced oxidative stress in the erythrocytes. So, the PHZ-induced-anemia mouse model was established by multiple peritoneal injections of PHZ to further study the in vivo oxidative environment effect on the RBITC-PEG fluorescence (Fig. 4a). After multiple treatments of PHZ, the RBCs in PHZ treated mice shrank and deformed (Fig. 4b); the paws of the mice turned to a pale colour, indicating a significant reduction of the blood RBCs and HgB, and the heme and bilirubin levels also increased (Fig. 4c), suggesting the successful construction of the anemia mice. The anemia mice were iv injected with RBITC-PEG, and their blood was sampled and imaged at 12 h post-injection. The blood from the anemia mice had much stronger fluorescence than that from the control mice (Fig. 4d&e). The PHZ-induced anemia mouse model had a reduced number of HgB, which accordingly caused less fluorescence quenching. Enhanced level of heme and bilirubin, as a consequence of anemia, had no effect on the fluorescence of RBITC-PEG. Adding 7.5% NaCl to the blood from the control mice destroyed the binding between RBITC-PEG and HgB, and recovered the fluorescence, confirming that RBITC-PEG was on the RBCs but quenched. Thus, in vivo oxidation microenvironment of RBCs inhibited the quenching effect of HgB. [1]

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We further tested the effect of the oxidative microenvironment in normal tissues on the RBITC-PEG fluorescence. Sodium arsenite (NaAsO2) is known to induce liver oxidation stress, which can further progress to liver cirrhosis and carcinogenesis. The NaAsO2-treated mice were iv injected with RBITC-PEG, and their major organs were collected for ex vivo imaging. In contrast to normal mice whose livers were not fluorescent, the livers from the NaAsO2-treated mice emitted strong fluorescence, while no noticeable fluorescence was found in other organs (Fig. 4f&g). This result confirmed that the oxidation stress in the liver did prevent fluorescence quenching of RBITC-PEG.
The next question is which group in RBITC-PEG rendered its HgB-quenchable but oxidation-de-quenchable fluorescence. In the molecular level, the thiourea group easily complex with the iron ion center and is very susceptible to oxidation. So, the thiourea group was reacted with benzyl bromide to convert it to S-benzyl-substituted RBITC-PEG (M-RBITC-PEG, Fig. 5a). M-RBITC-PEG showed the same absorption and fluorescence spectra with a slight decrease in fluorescent intensity as RBITC-PEG (Fig. 5b). RBITC-PEG and M-RBITC-PEG were then iv injected into the mice bearing BCap37 tumors, and their major organs and tumors were dissected at 12 h post-injection for fluorescent imaging. As shown in Fig. 5c&d, M-RBITC-PEG's fluorescence lost tumor-specificity and exhibited strong fluorescence in the liver and kidneys, and low fluorescence in the tumor compared to RBITC-PEG. The total fluorescence signal of M-RBITC-PEG was much higher than RBITC-PEG, which means that M-RBITC-PEG fluorescence was unaffected by RBCs or HgB. Thus, it was the thiourea/HgB complexation that quenches the fluorescence of RBITC-PEG [1].

Cell lines [1]
Human pancreatic cancer cell line BxPC3, human cervical adenocarcinoma cell line Hela and human chronic myelogenous leukemia K562 cells were purchased from the American Type Culture Collection. Human adriamycin resistant mammary carcinoma cell line MCF7/ADR, Human breast carcinoma cell line BCap37, mouse mammary adenocarcinoma stably expressing luciferase (Luc) and green fluorescent protein (GFP) cell line (4 T1-Luc-GFP) were used. Cells were maintained in RPMI-1640 medium. All culture media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. MCF7/ADR cells were maintained continuously with 0.5 μM of doxorubicin. K562 was suspension-cultured cells. After pre-culturing of for 24 h, 50 μM of hemin was added for 3 days for observation of erythroid differentiation.

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Assay of erythroid differentiation [1]
Successful erythroid differentiation of K562 cells was confirmed by detection of hemoglobin-expressing cells with benzidine‑hydrogen peroxide staining. Briefly, cells were collected and washed twice with PBS. Fresh cells were stained with benzidine solution containing 3% H2O2 for 3 min and checked under the light microscope. Blue indicates the produce of HgB.

Mice Model [1]
(1) Subcutaneous tumor models: Experiments were carried out on nude mice bearing subcutaneous grown xenografts of breast cancer cells. Each experimental group consisted of 3 animals, the tumor was measured by a caliper every day and calculated as a2 × b/2 where a and b are the shortest and longest diameter of the tumor in mm, respectively. When the tumor reached a mean size of 100 mm3, mice were treated intravenously with different dyes-PEG conjugates (200 μL, 1 mM in PBS). [1]
(2) Models of orthotopic metastases carcinoma by intra-mammary gland injection, lung metastases tumor model by tail intravenous injection, and general metastases tumor model by intracardiac injection of 4 T1-Luc-GFP cells: 4 T1-Luc-GFP cells at a dose of 2 × 105 cells were injected into 6- to 8-week-old BALB/c mice. Tumor metastasis was measured by quantitative bioluminescence imaging.

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In vivo and ex vivo fluorescent imaging [1]
Whole-body optical imaging was performed at 12 h post-injection of dyes on a Kodak In-Vivo FX Professional Imaging Syste equipped with fluorescent filter sets. For in vivo imaging, prior to imaging, the mice were anesthetized by intra-abdominal injection of 1% pentobarbital sodium (45 mg/kg). For ex vivo imaging, major organs and tumors were excised and washed with 0.9% saline.

[1]. Tumor-specific fluorescence activation of rhodamine isothiocyanate derivatives. J Control Release. 2021 Feb 10:330:842-850.

In summary, RBITC derivatives can complex with HgB via the thiourea group and became nonfluorescent, while the oxidation microenvironment can oxidize the thiourea group and reactivate their fluorescence. Therefore, the fluorescence of RBITCs is sensitive to the RBCs or HgB and the microenvironment, and thus uses of the dyes, for instance, biodistribution study, must be carefully calibrated first. On the other hand, this unique property makes the dyes useful fluorescent probes for cancer imaging and diagnosis. [1]

C29H30CLN3O3S

536.08

535.169

36877-69-7

44134928

Green to dark green solid powder

1.23g/cm3

717.2ºC at 760 mmHg

387.6ºC

1.639

3.299

1

7

8

37

965

0

CCN(CC)C1=CC2=C(C=C1)C(=C3C=CC(=[N+](CC)CC)C=C3O2)C4=C(C=CC=C4N=C=S)C(=O)O.[Cl-]

ASPXTKVHMNAXGJ-UHFFFAOYSA-N

InChI=1S/C29H29N3O3S.ClH/c1-5-31(6-2)19-12-14-21-25(16-19)35-26-17-20(32(7-3)8-4)13-15-22(26)27(21)28-23(29(33)34)10-9-11-24(28)30-18-36;/h9-17H,5-8H2,1-4H3;1H

[9-(2-carboxy-6-isothiocyanatophenyl)-6-(diethylamino)xanthen-3-ylidene]-diethylazanium;chloride

Rhodamine B isothiocyanate; RBITC; [9-(2-carboxy-6-isothiocyanatophenyl)-6-(diethylamino)xanthen-3-ylidene]-diethylazanium;chloride; 1879951-72-0; N-(9-(2-carboxy-6-isothiocyanatophenyl)-6-(diethylamino)-3H-xanthen-3-ylidene)-N-ethylethanaminium chloride; Isothiocyanatorhodamine B; MFCD00136007;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: 12.5 mg/mL (23.32 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.)