Fluorescent Dye

Cyanine dyes, as used in super-resolution fluorescence microscopy, undergo light-induced “blinking”, enabling localization of fluorophores with spatial resolution beyond the optical diffraction limit. Despite a plethora of studies, the molecular origins of this blinking are not well understood. Here, we examine the photophysical properties of a bio-conjugate cyanine dye (AF-647), used extensively in dSTORM imaging. In the absence of a potent sacrificial reductant, light-induced electron transfer and intermediates formed via the metastable, triplet excited state are considered unlikely to play a significant role in the blinking events. Instead, it is found that, under conditions appropriate to dSTORM microscopy, AF-647 undergoes reversible photo-induced isomerization to at least two long-lived dark species. These photo-isomers are characterized spectroscopically and their interconversion probed by computational means. The first-formed isomer is light sensitive and transforms to a longer-lived species in modest yield that could be involved in dSTORM related blinking. Permanent photobleaching of AF-647 occurs with very low quantum yield and is partially suppressed by the anaerobic redox buffer [2].

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8-35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8-35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10-20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8-35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8-35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs [1].

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Biodistribution of FAPolAF647 [1]
Following IV Injection [1]
IV injection is one of the most common routes of administration and is considered to give the best bioavailability. Indeed, following injection of FAPolAF647 in the retro-orbital sinus, the fluorescent signal propagated rapidly throughout the whole body of the animals (Fig. 3). Strikingly, the signal was extremely stable, profiles obtained 10 min post-injection being similar to those obtained at 72 h. A significant decrease was first seen at 10 days post-injection. Ventral views of the animals revealed, as expected from IV injection, a rapid concentration in the liver. This hepatic signal was clearly detectable up to 72 h, with a maximal intensity seen at 4 h. The concentration of the signal in the liver was confirmed when collecting organs at different time-points in some of the animals. As seen in Fig. 3, 94 % of the signal was measured in the liver, the rest being distributed among the lungs (3.3 %), the heart (0.6 %), and the kidneys (2.1 %), the latter signal being consistent with an excretion process. Interestingly, there was no detectable signal in the spleen or in the brain. Since all organs (except liver) were negative at 24 h when removed from the body, the observed fluorescent signal in the whole body at this time-point is mainly due to the persistence of APols in the circulation.

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Biodistribution of FAPolAF647 Following IP Injection [1]
IP injections are widely used in animal studies because of the ease of the procedure. Following injection of FAPolAF647, the fluorescent signal remained concentrated in the peritoneal cavity for ~1 h (Fig. 4). It then reached the circulation to produce a uniform signal in the body, concomitant with a persistent and intense staining of the liver for up to 72 h. As for the IV route, the signal progressively disappeared at 10 and 20 days post-injection, confirming the slow release of APols from the body. The collection of organs at different time-points showed a large amount of signal in abdominal fat pads (45 %) and in the liver (55 %) up to 72 h. With the exception of a faint signal in the liver, signals were barely undetectable at 10 and 20 days, suggesting complete elimination. In all cases, no signal was observed in the lung, heart, spleen, and brain, implying no accumulation of APols in these tissues. A very modest signal in kidneys presumably reflected renal elimination. It was difficult to monitor due to the slow release from fat and liver, generating very low doses of APol to eliminate at any given time point. Hence, as for the IV route, a majority of APols remains circulating once diffusing from fat and is progressively eliminated, presumably via the hepatic route.

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Biodistribution of FAPolAF647 Following SC Injection [1]
The SC route is known to lead to slow but complete absorption of drugs. Indeed, following injection of the FAPolAF647 bolus, a compact and restricted signal was observed at the injection site for the entire period of analysis (Fig. 5). The intensity of the spot decreased very slowly, but was still detectable after 20 days. This high site-specific concentration effect impeded detection of APols in the rest of the body, including at the ventral view. Consistently, fluorescence was found associated with the dorsal fat pads, which appear to be a very efficient natural reservoir for APols. With the exception of the liver, which showed significant staining, suggesting hepatic elimination of APols, and hence, diffusion from the injection site, APols could not be detected in any of the collected organs, due to their low circulating concentration.

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Biodistribution of a Rhodamine-Labeled APol [1]
To validate the biodistribution profiles, experiments were repeated using a rhodamine-labeled version of A8–35 (FAPolrhod; see Giusti et al. 2012). As seen in Fig. 6, distribution profiles fully matched those obtained for FAPolAF647. The profiles are presented for each adminis tration route with the corresponding distribution of free rhodamine, which served as a control experiment. This confirmed that the observed persistent signals do correspond to FAPolrhod, given that the signal of the free dye (although ~100× concentrated compared to the label carried by FAPolrhod) reaches a peak around 1 h and is almost gone at 72 h. Free rhodamine was detectable in urine 1–4 h post-injection using a plate reader (Tristar II, Berthold; not shown). In all cases, FAPolrhod remained undetectable in spite of a detection sensitivity compatible with the measure of 1,000× less FAPolrhod-containing solution than the one being injected (data not shown). This is consistent with very slow release of FAPolrhod, generating fluorescent signal intensity below the detection threshold.

Whole-Body In Vivo Biodistribution [1]
18 female NMRI Nude 10-week-old mice (two per injection mode) were subjected to IP, SC, or IV retro-orbital sinus injections of 100 μL of FAPolAF647, FAPolrhod, or free rhodamine solutions, corresponding to 10 μg of products. Fluorescence was detected on gas-anesthetized animals, thanks to a Bioimager, as previously described (Destouches et al. 2011; Page et al. 2011). The fluorescence was measured for each animal from the ventral and the dorsal sides at t0 (before injection), 10 min, 1 h, 4 h, 24 h, 48 h, 72 h, 10 days, and 20 days. Acquisitions were performed during 30 s for IP and IV injections and 1 s for SC injections.

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Ex Vivo Biodistribution [1]
30 female Balb/C 10-week-old mice were subjected to IP, SC, or IV retro-orbital sinus injections (two animals per injection mode) with 100 μL of FAPolAF647 solution. Two mice of each group were sacrificed at 4 h, 24 h, 72 h, 10 days, and 20 days after injection and dissected in order to collect organs (liver, kidney, spleen, fat pads, heart, lungs, and brain). Acquisitions were performed during 1 s.

[1]. In vivo characterization of the biodistribution profile of amphipol A8-35. J Membr Biol. 2014;247(9-10):1043-1051.

[2]. Photo‐isomerization of the Cyanine Dye Alexa‐Fluor 647 (AF‐647) in the Context of dSTORM Super‐Resolution Microscopy[J]. Chemistry–A European Journal, 2019, 25(65): 14983-14998.

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8-35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8-35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10-20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8-35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8-35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs [1]

C40H46N3NA3O16S4

1022

1021.14539

AF647-NHS ester triTEA;407627-61-6;AF647-NHS ester;407627-60-5;AF647-NHS ester tripotassium;1453856-34-2

169450060

Typically exists as solid at room temperature

0

17

20

66

2310

0

CC1(C2=C(C=CC(=C2)S(=O)(=O)[O-])[N+](=C1/C=C/C=C/C=C\3/C(C4=C(N3CCCS(=O)(=O)[O-])C=CC(=C4)S(=O)(=O)[O-])(C)CCCCCC(=O)ON5C(=O)CCC5=O)CCCS(=O)(=O)[O-])C.[Na+].[Na+].[Na+]

BMJDGMFKBUDRTQ-UHFFFAOYSA-K

InChI=1S/C40H49N3O16S4.3Na/c1-39(2)30-26-28(62(53,54)55)15-17-32(30)41(22-10-24-60(47,48)49)34(39)12-6-4-7-13-35-40(3,21-9-5-8-14-38(46)59-43-36(44)19-20-37(43)45)31-27-29(63(56,57)58)16-18-33(31)42(35)23-11-25-61(50,51)52;;;/h4,6-7,12-13,15-18,26-27H,5,8-11,14,19-25H2,1-3H3,(H3-,47,48,49,50,51,52,53,54,55,56,57,58);;;/q;3*+1/p-3

trisodium;(2Z)-2-[(2E,4E)-5-[3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indol-1-ium-2-yl]penta-2,4-dienylidene]-3-[6-(2,5-dioxopyrrolidin-1-yl)oxy-6-oxohexyl]-3-methyl-1-(3-sulfonatopropyl)indole-5-sulfonate

AF647-NHS ester (trisodium);

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)