Pluronic F-68

The magnolol-loaded mixed micelles (MMs) and magnolol nanosuspensions (MNs) were prepared to use film hydration and antisolvent methods, respectively. The optimal MMs and MNs formulations were prepared to use magnolol, Soluplus®, and Poloxamer 188 in ratios of 1:12:5 and 2:1:1, respectively. The average particle size of MMs was 111.8 ± 14.6, and MNs was 78.53 ± 5.4 nm. The entrapment and drug loading efficiency for MMs were 89.58 ± 2.54% and 5.46 ± 0.65%, correspondingly. The drug loading efficiency of MNs was 42.50 ± 1.57%. In the in vitro release study, MMs showed a slow drug release while that of MNs was fast. The results of the Caco-2 transcellular transport study indicated that both MMs and MNs increased the permeation of magnolol. MMs and MNs markedly promoted gastrointestinal drug absorption by 2.85 and 2.27-fold, respectively, as shown in the pharmacokinetics study [1].

Traumatic Brain Injury (TBI), the main contributor to morbidity and mortality worldwide, can disrupt the cell membrane integrity of the vascular endothelial system, endangering blood–brain barrier function and threatening cellular subsistence. Protection of the vascular endothelial system might enhance clinical outcomes after TBI. Poloxamer 188 (P188) has been shown to improve neuronal function after ischemia/reperfusion (I/R) injury as well as after TBI. We aimed to establish an in vitro compression-type TBI model, comparing mild-to-moderate and severe injury, to observe the direct effects of P188 on Mouse Brain Microvascular Endothelial Cells (MBEC). Confluent MBEC were exposed to normoxic or hypoxic conditions for either 5 or 15 h (hours). 1 h compression was added, and P188 was administered during 2 h reoxygenation. A direct effect of P188 on MBEC was tested by assessing cell number/viability, cytotoxicity/membrane damage, metabolic activity, and total nitric oxide production (tNOp). While P188 enhanced cell number/viability, metabolic activity, and tNOp, an increase in cytotoxicity/membrane damage after mild-to-moderate injury was prevented. In severely injured MBEC, P188 improved metabolic activity only. P188, present during reoxygenation, influenced MBEC function directly in simulated I/R and compression-type TBI [2].

Metabolic Activity[2]
The metabolic activity was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay. The tetrazolium compound (3- (4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) used in this assay is bio-reduced by metabolically active cells forming a colored formazan product, soluble in culture medium. After thawing for 90 min at 20–25 °C, 20 µL of CellTiter 96 AQueous One Solution Reagent was added to each well containing 100 µL of media (achieving a concentration of approximately 16.67%). The plates were incubated in the cell culture incubator at 37 °C for 1–4 h in humified air with 5% CO2. The abs were read in the plate reader at 490 nm. Lastly, the blank abs were subtracted from the total abs.[2]

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Total NO Production[2]
The tNOp was assayed using the Cayman’s Nitrate/Nitrite Colorimetric Assay Kit. Nitric Oxide (NO), a highly reactive, short-lived free radical, is synthesized in biological systems by enzymes of the NO synthase (NOS) family, including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Different cell types can produce NO, including endothelial cells. The main effect of NO is paracrine activation of guanylate cyclase, which leads to an intracellular increase in cyclic guanosine monophosphate causing smooth muscle cells to relax. This assay measures the total production of NO by adding the amount of nitrite (NO2−) and the amount of nitrate (NO3−), which both represent the final products of NO. First the NO3− is exposed to NO3− reductase, converting it to NO2−. In a second step, the Griess Reagents are used to convert NO2− to an azo chromophore. Measurement of the resulting abs can determine the NO2− concentration. Reagents and samples were prepared and the assay was performed as instructed in the assay protocol. In short, 10 µL of enzyme cofactor mixture and nitrate reductase mixture were added to up to 80 µL samples (resulting concentrations: 10% enzyme cofactor mixture, 10% nitrate reductase mixture, 80% sample). After the required incubation time, 50 µL Griess Reagent R1 followed by 50 µL Griess Reagent R2 were supplemented (a concentration of 25% of each result). The abs were measured at 540 nm using a plate reader. A standard curve was prepared, following the instructions in the assay protocol, whenever the assay was performed.

Pierce Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA) was used to determine cytotoxicity/membrane damage. In healthy cells, LDH is a cytosolic enzyme. Following injury of the plasma membrane, LDH is released into the medium. We used a colorimetric method quantifying cellular cytotoxicity by measuring extracellular LDH, using a coupled enzymatic reaction. LDH catalyzes the reaction from lactate to pyruvate leading to a reduction of nicotinamide adenine dinucleotide from its oxidized (NAD+) to its reduced (NADH) form. The tetrazolium salt iodonitrotetrazolium was reduced to a red formazan product by diaphorase using NADH. The formazan formation is directly proportional to the amount of LDH release. This can be used to indicate the level of cytotoxicity/membrane disruption. The reagent was prepared and stored following the manufacturer’s instructions. Briefly, the vial containing the substrate mix (lyophilizate) was diluted with 11.4 mL ultrapure water. The assay buffer was thawed, while shielded from light. The reaction mix consists of 0.6 mL assay buffer (5%) and 11.4 mL substrate mix (95%). Firstly, 50 µL of the media of each well were transferred to a not pre-coated, clear 96-well plate. Afterward, 50 µL of the reaction mix (achieving a concentration of 50% reaction mix) was added, the plates were tapped gently and protected from light using aluminum foil. The plates were incubated at room temperature. After 30 min, 50 µL stop solution (concentration of approximately 33%) were added. Then, 10 min later, the absorbance within the media was measured at 490 nm using a plate reader (Synergy H1, BioTek Instruments Inc.). In a second step, 5 µL of lysis buffer (10X) were added to the original plate, containing the cells and 50 µL of residual media. The plates were incubated at 37 °C for 60 min and the assay was performed as described above. The absorbance (abs) was calculated: abs(media)/(abs[media] + abs[lysed cells]) [2].

Animal/Disease Models: TourniquetInduced Ischemia-Reperfusion Injury in Rats [2]
Doses: 150 mg/kg
Route of Administration: intravenous/i.v.
Experimental Results: dramatically decreased the elevated TBARS but not to control levels and SOD activity was at control levels.

[1]. Enhanced Oral Bioavailability of Magnolol via Mixed Micelles and Nanosuspensions Based on Soluplus ®-Poloxamer 188. Drug Deliv. 2020 Dec;27(1):1010-1017.

[2]. Lotze FP, et al. Poloxamer 188 Exerts Direct Protective Effects on Mouse Brain Microvascular Endothelial Cells in an In Vitro Traumatic Brain Injury Model. Biomedicines. 2021 Aug 19;9(8):1043.
[3]. Walters TJ, et al. Poloxamer-188 reduces muscular edema after tourniquet-induced ischemia-reperfusion injury in rats. J Trauma. 2011 May;70(5):1192-7.
[4]. Yan F, et al. The effect of poloxamer 188 on nanoparticle morphology, size, cancer cell uptake, and cytotoxicity. Nanomedicine. 2010 Feb;6(1):170-8.

8800 (Average)

9003-11-6

White to yellow solid

25.0600

OQNWUUGFAWNUME-UHFFFAOYSA-N

InChI=1S/C7H16O4/c1-7(11-5-3-9)6-10-4-2-8/h7-9H,2-6H2,1H3

2-[2-(2-hydroxyethoxy)propoxy]ethanol

Pluronic F-68

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)

DMSO: ~100 mg/mL
Water: ~100 mg/mL
Ethanol: ~100 mg/mL

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.)