C12-200 is a formulation excipient that facilitates intracellular delivery of nucleic acids. It does not have a protein or enzyme target. No IC50, Ki, EC50, or binding affinity values are provided. [1][2][3]
C12-200 does not have a specific biological target; rather, it serves as a delivery vehicle that facilitates cellular uptake and endosomal escape of nucleic acid cargo (siRNA, mRNA, saRNA). The ionizable nature of C12-200 allows it to bind negatively charged nucleic acid molecules under acidic conditions, forming stable complexes that are internalized by cells via endocytosis. Once inside the cell, the acidic endosomal environment triggers protonation of the lipidoid, which promotes membrane destabilization and release of the nucleic acid cargo into the cytosol. This mechanism of action is characteristic of ionizable cationic lipids and is critical for efficient nucleic acid delivery. C12-200 does not interact with specific cell surface receptors, and its cellular uptake is primarily mediated by non-specific endocytic pathways, although the specific formulation and particle size can influence the route of internalization.

- Cytocompatibility in breast cancer cell lines (MCF-7, MDA-MB-231, BT-549): Cells were incubated with C12-200/siGAPDH LNPs (50 nM siRNA, 4 h transfection). Cell viability measured 24 h post-transfection using ATP assay showed >95% viability compared to untreated controls. Positive control PEI (50 μg/mL) reduced viability to 20%. [1]
- Cytocompatibility in primary rat dorsal root ganglion (DRG) neurons: Cells were incubated with C12-200/siTRPV1 LNPs (50 nM siRNA, 4 h). Cell viability was approximately 100% 24 h post-transfection. [1]
- Dose escalation cytocompatibility in MDA-MB-231 cells: Cells were incubated with increasing concentrations of siGFP-loaded C12-200 LNPs (10, 25, 50, 75, and 100 nM siRNA). Cell viabilities remained >85% 24 h post-transfection. [1]
- Cellular uptake of Cy5 siRNA in primary rat cortical neurons (flow cytometry): Cy5 siRNA-loaded C12-200 LNPs (with PEG-DMG, 50 nM siRNA, 24 h incubation) resulted in 94-96% Cy5-positive cells. LNPs without PEG-DMG resulted in 81-83% Cy5-positive cells. Naked Cy5 siRNA resulted in 44-46% uptake. Lipofectamine RNAiMAX (positive control) resulted in 94-96% uptake but with visible toxicity. [1]
- Cellular uptake in human cortical neuron cell line (HCN-2, fluorescence microscopy): HCN-2 cells were incubated with C12-200 LNPs (50 or 100 nM Cy5 siRNA) for 2, 4, or 24 h. LNPs with PEG-DMG showed time-dependent increase in Cy5 siRNA uptake, with nearly all cells showing fluorescence at 24 h. LNPs without PEG-DMG showed lower uptake. Lipofectamine RNAiMAX caused cell toxicity within 2 h. [1]
C12-200 demonstrates high transfection efficiency in vitro, forming stable complexes with negatively charged nucleic acids via electrostatic interactions. It has been shown to effectively deliver siRNA and mRNA to various cell lines, including HepG2, HeLa, and primary neural cells, with low cytotoxicity. In cell culture, C12-200 LNPs facilitate efficient gene knockdown (for siRNA) or protein expression (for mRNA) at low nanomolar concentrations. The transfection efficiency of C12-200 is comparable to or exceeds that of other benchmark ionizable lipids such as DLin-MC3-DMA. C12-200 demonstrates good biocompatibility in vitro, with minimal effects on cell viability at concentrations used for effective transfection. The lipidoid's ability to mediate endosomal escape is a key factor contributing to its high transfection efficiency.

- hEPO protein production in mice (CD-1): Intravenous injection of C12-200 LNPs containing hEPO mRNA (1.0 mg/kg, based on encapsulated mRNA) resulted in serum hEPO levels of ~11 μg/mL at 6 h post-administration (>125,000-fold over normal physiological levels). Detectable levels (~47 ng/mL) remained at 72 h, and slightly above normal (~120 pg/mL) at 1 week. Rapid onset: ~190 ng/mL at 30 min. [2]
- Hematocrit (Hct) increase in mice: Wild-type mice treated with C12-200 LNPs containing hEPO mRNA (single 30 μg dose or three 10 μg doses on days 1, 3, 5) showed a ~20% increase in Hct over 15 days, demonstrating that MRT-derived EPO protein was physiologically active. [2]
- hEPO protein production in cynomolgus monkeys: Male cynomolgus monkeys (~4 kg) received single intravenous injection of C12-200 LNPs containing hEPO mRNA at 0.025 or 0.050 mg/kg. Serum hEPO levels peaked at 6 h (~9000 pg/mL at 0.050 mg/kg) and remained detectable at 72 h. No adverse clinical signs were observed; ALT/AST levels were within normal range. [2]
- hFIX protein production in wild-type mice (CD-1): Intravenous injection of C12-200 LNPs containing hFIX mRNA (1.0 mg/kg) resulted in plasma hFIX levels reaching ~4.4 μg/mL at 12 h post-administration, sustained for 72 h. [2]
- hFIX protein production in FIX knockout (hemophilia B) mice: FIX KO mice received single intravenous dose of C12-200 LNPs containing hFIX mRNA at 0.25 or 0.50 mg/kg. Plasma hFIX levels at 12 h: ~400 ng/mL (0.25 mg/kg) and ~1.77 μg/mL (0.50 mg/kg). FIX activity reached 40.9% (0.25 mg/kg) and 87% (0.50 mg/kg) of wild-type levels. [2]
- Efficacy in hemophilia B mouse model (incision model): FIX KO mice treated with hFIX mRNA-loaded C12-200 LNPs (0.25 or 0.50 mg/kg) underwent a 1 cm dorsal incision at 12 h post-dose. Hct levels 12 h post-incision (24 h post-dose) were 36.1% (0.25 mg/kg) and 39.0% (0.50 mg/kg), compared to 17.9% in saline-treated KO mice (pre-treatment Hct ~47.7%). This demonstrated reduced bleeding due to active FIX protein. [2]
- Luciferase mRNA delivery in mice: C12-200 LNPs containing firefly luciferase mRNA (15 μg total mRNA, intravenous) resulted in ~3-fold higher liver luminescence with the optimized formulation (C-35) compared to the original formulation. Expression was predominantly in the liver. [3]
- siRNA delivery (Factor VII silencing) in mice: C12-200 LNPs containing FVII siRNA were administered intravenously at doses from 0.01 to 0.1 mg/kg. ED50 for both original and optimized (C-35) formulations was approximately 0.03 mg/kg total siRNA. No significant difference in silencing efficacy was observed between formulations. [3]
C12-200 displays favorable biodistribution in vivo, with LNPs containing C12-200 selectively accumulating in the liver upon intravenous administration. In mouse models, C12-200-containing LNPs encapsulating human erythropoietin (EPO) mRNA have been shown to increase serum EPO levels, while LNPs encapsulating factor VII siRNA reduce serum factor VII levels. These findings demonstrate the functional delivery of both mRNA and siRNA cargo in vivo. C12-200 LNPs have also been used to deliver therapeutic nucleic acids in preclinical models, including siRNA for KRAS-mutant tumors. The lipidoid's ionizable properties contribute to efficient endosomal escape and cytosolic delivery of the nucleic acid cargo, which is essential for achieving pharmacological activity. The in vivo performance of C12-200 is formulation-dependent, and optimization of particle size, lipid composition, and nucleic acid-to-lipid ratio is critical for achieving maximal efficacy.

The following in vivo activity assays were performed on expressed proteins: - FIX activity assay: Plasma samples from FIX KO mice were analyzed using a chromogenic Factor IX Activity Assay. Activity was normalized to wild-type (C57Bl/6) saline-treated mice (100% activity). FIX KO saline-treated mice showed ~1.15% activity. Treatment with hFIX mRNA-loaded C12-200 LNPs (0.50 mg/kg) restored activity to 87% of wild-type levels. [2]
C12-200 is evaluated using dynamic light scattering (DLS) to measure particle size and polydispersity index (PDI), and zeta potential analysis to assess surface charge. Encapsulation efficiency is determined by the RiboGreen assay, which measures fluorescence before and after detergent disruption of LNPs; the difference in fluorescence correlates with the amount of encapsulated nucleic acid. The stability of C12-200 formulations under various storage conditions (temperature, pH, freeze-thaw cycles) is assessed to determine shelf-life and suitability for clinical use. The pKa of C12-200 is determined by titration, which is a critical parameter for predicting endosomal escape efficiency. These physicochemical characterization methods are essential for quality control and batch-to-batch consistency in LNP formulation.

- Cell viability (ATP assay): Cells were seeded in 96-well plates. After 4 h transfection with LNPs (50 nM siRNA unless otherwise specified) and 24 h incubation, CellTiter-Glo 2.0 reagent (60 μL) was added to each well with 60 μL complete medium. After 15 min incubation in the dark at room temperature on a shaker, 60 μL of mixture was transferred to a white opaque plate, and luminescence was measured. Relative ATP levels (%) were calculated by normalizing to untreated cells. [1]
- Flow cytometry for Cy5 siRNA uptake: Primary rat cortical neurons (50,000 cells/well in 24-well plate) were incubated with Cy5 siRNA-loaded LNPs (50 nM, 24 h). Cells were collected, centrifuged (300 g, 5 min), resuspended in PBS, and analyzed on an acoustic focusing cytometer (excitation 638 nm, emission 720/30 nm). 30,000 events recorded. Untreated cells were used to set the gate for Cy5-negative population. [1]
- Fluorescence microscopy for Cy5 siRNA uptake: Human cortical neurons (HCN-2, 7,000 cells/well in 48-well plate) were cultured for 1-2 weeks. Cells were incubated with Cy5 siRNA-loaded LNPs (50 or 100 nM) for 2, 4, or 24 h. Cells were washed with PBS and imaged using an epifluorescent inverted microscope (excitation 651 nm, emission 670 nm). [1]
- In situ hybridization (ISH) for mRNA detection: Liver tissues from mice treated with hEPO or hFIX mRNA-loaded LNPs were analyzed using proprietary probe design technology that allows single-molecule visualization. Probes were designed to not cross-react with mouse, rat, pig, and rhesus monkey. Positive staining was observed in hepatocytes and sinusoidal cells from 30 min to 72 h post-administration. [2]
- Luciferase assay: Mice were injected intravenously with Luc mRNA-loaded LNPs (15 μg total mRNA). Six hours post-injection, organs were harvested and luciferase expression was quantified using an IVIS imaging system (total flux from luminescence). [3]
C12-200 LNPs are tested on cultured cells (e.g., HepG2, HeLa, primary neural cells) by incubating with fluorescently labeled nucleic acids or functional nucleic acids (siRNA, mRNA). Transfection efficiency is measured by flow cytometry or fluorescence microscopy for fluorescently labeled cargo; gene knockdown is assessed by qPCR or Western blot for target protein expression; protein expression from mRNA is measured by ELISA or other immunoassays. Cytotoxicity is assessed using MTT, CellTiter-Glo, or LDH release assays. The optimal nucleic acid-to-lipid ratio and formulation conditions are determined through systematic screening in cell-based assays. These experiments help establish the relationship between formulation parameters and biological activity.

- Mice (CD-1, C57BL/6, FIX KO): Male mice aged 6-8 weeks (CD-1) or 10-12 weeks (C57BL/6, FIX KO) were used. All formulations were administered via tail vein injection as a single bolus. Doses: hEPO mRNA at 1.0 mg/kg; hFIX mRNA at 0.25, 0.50, or 1.0 mg/kg; siRNA at 0.01-0.1 mg/kg; luciferase mRNA at 15 μg total mRNA per mouse. Blood samples were collected at designated time points via facial vein puncture, tail snip, or terminal cardiac puncture. For serum, blood was clotted at room temperature for ≥30 min, centrifuged at 9300 g for 10 min. For plasma, blood was collected into lithium heparin or citrate-coated tubes. [2][3]
- Cynomolgus monkeys: Male monkeys (~4 kg body weight) received a single intravenous injection via ear vein at doses of 0.025 or 0.050 mg/kg (hEPO mRNA-loaded LNPs) or 0.025 mg/kg (control untranslatable mRNA). Total dose volume was 1 mL. Blood samples were collected over 72 h. ALT/AST levels were measured to assess tolerability. [2]
- Hemophilia B mouse model incision procedure: FIX KO mice were anesthetized with isoflurane at 12 h post-dose. The dorsal thoracic region (pre-shaved) was aseptically prepared with betadine and alcohol. A ~1.0 cm surgical skin incision was made and closed with surgical sutures. Blood samples were collected 12 h post-incision (24 h post-dose) for Hct measurement. [2]
- Hematocrit measurement: Whole blood samples were collected in heparinized micro-capillary tubes and centrifuged at 10,000-15,000 g for 5 min at ambient temperature. Packed cell volume was calculated vs total volume. [2]
- LNP formulation (general): C12-200, helper lipids (DSPC, DOPE, or DSPE), cholesterol, and PEG-lipid (DMG-PEG-2K) were dissolved in ethanol at specific molar ratios. An aqueous solution of mRNA (or siRNA) in citrate buffer (10 mM, pH 4.0-4.5) was prepared. The lipid solution was rapidly injected into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1× PBS (pH 7.4), concentrated, and stored at 2-8°C. Typical encapsulation efficiency: 75-78%; particle size: 86-102 nm; polydispersity: 0.14-0.16. [2][3]
Animal studies involve intravenous administration of C12-200 LNPs encapsulating therapeutic nucleic acids in mice or rats. Tissue distribution is evaluated by measuring nucleic acid levels (e.g., by qPCR or radiolabeling) in liver, spleen, lung, kidney, and other organs. Efficacy is assessed by measuring target gene knockdown (for siRNA) or protein expression (for mRNA) in relevant tissues. For example, in factor VII siRNA studies, serum factor VII levels are measured at various time points post-administration. Pharmacodynamic studies may also evaluate the duration of effect and dose-response relationships. Formulation parameters such as particle size, lipid composition, and nucleic acid-to-lipid ratio are optimized based on in vivo performance.

C12-200 has favorable pharmacokinetic properties as an LNP component. It is rapidly cleared from the circulation following intravenous administration, with peak accumulation in the liver occurring within minutes post-injection. The ionizable nature of C12-200 allows endosomal escape and cytosolic delivery of the nucleic acid cargo, which is essential for achieving pharmacological activity. The pharmacokinetic profile of C12-200 is formulation-dependent, and factors such as particle size, lipid composition, and PEGylation can influence circulation time and tissue distribution. LNPs containing C12-200 typically exhibit a biphasic clearance pattern, with an initial rapid distribution phase followed by a slower elimination phase. The majority of the administered lipidoid is cleared by the liver and metabolized, with minimal accumulation in non-target tissues.

- In vitro cytocompatibility: C12-200 LNPs showed >85-95% cell viability in MCF-7, MDA-MB-231, BT-549 breast cancer cells, and primary rat DRG neurons at 50 nM siRNA dose. Dose escalation up to 100 nM siRNA (and correspondingly higher lipid dose) maintained >85% viability in MDA-MB-231 cells. [1]
- In vivo tolerability in mice: All C12-200 LNP formulations were well tolerated in mice at the tested doses (hEPO mRNA up to 1.0 mg/kg; hFIX mRNA up to 1.0 mg/kg; siRNA up to 0.1 mg/kg) with no observable adverse events. [2][3]
- In vivo tolerability in cynomolgus monkeys: Monkeys treated with C12-200 LNPs at 0.025 or 0.050 mg/kg showed no clinical signs and normal physiological ALT/AST levels. Control monkey (untranslatable mRNA at 0.025 mg/kg) also showed no adverse effects. [2]
Preclinical toxicity studies indicate that C12-200 is well-tolerated at therapeutic doses. Common adverse effects include transient elevation of liver enzymes and mild inflammatory responses associated with LNP administration. Cytotoxicity is minimal in vitro at concentrations used for transfection. The neutral charge of C12-200 at physiological pH contributes to its favorable safety profile by reducing non-specific interactions with serum proteins and cell membranes. High-dose studies may reveal hepatotoxicity or immunogenicity, which are typical for cationic lipid-based delivery systems. The safety profile of C12-200 is formulation-dependent, and the inclusion of helper lipids and PEG-lipids can modulate the toxicological profile. In general, C12-200 is considered a relatively safe and well-tolerated ionizable lipidoid for research applications.

[1]. Development of Lipidoid Nanoparticles for siRNA Delivery to Neural Cells. AAPS J. 2021;24(1):8. Published 2021 Dec 6.

[2]. Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system. Gene Ther. 2016;23(10):699-707.

[3]. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015 Nov 11;15(11):7300-6.

- Chemical structure: C12-200 is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol). It is an ionizable cationic lipidoid with a pKa of approximately 6.96-7.25. [1][2][3]
- Role in LNPs: C12-200 is the ionizable cationic lipidoid component that electrostatically complexes with negatively charged nucleic acids (siRNA/mRNA), facilitates cellular uptake, and promotes endosomal escape due to its pH-dependent charge (positive at acidic pH, neutral at physiological pH). [1][2][3]
- Formulation optimization: Using Design of Experiment (DOE) methodologies (Fractional Factorial and Definitive Screening Designs), an optimized mRNA delivery formulation (C-35) was developed with parameters: C12-200:mRNA weight ratio 10:1; molar composition: 35% C12-200, 16% DOPE, 46.5% cholesterol, 2.5% PEG-DMG; particle diameter 102 nm; encapsulation efficiency 43%; pKa 6.96; zeta potential -5.0 mV. This formulation increased hEPO production 7-fold compared to the original siRNA-optimized formulation. [3]
- Comparison of siRNA vs mRNA delivery: The optimized mRNA formulation (C-35) did not improve siRNA delivery (FVII silencing) compared to the original siRNA-optimized formulation (ED50 ~0.03 mg/kg for both). This indicates different optimized formulation parameter spaces for siRNA and mRNA delivery. [3]
C12-200 is a research-use lipidoid that has not yet been approved as a therapeutic agent by regulatory authorities. It is widely used in the field of RNA therapeutics, gene delivery, and vaccine development. C12-200 is employed in combination with helper lipids (DSPC, cholesterol, PEG-lipid) to formulate LNPs for mRNA and siRNA delivery. The lipidoid has been extensively characterized in both academic and industrial research settings, and serves as a benchmark for the development of novel ionizable lipids. Its favorable properties, including efficient nucleic acid encapsulation, high transfection efficiency, and acceptable safety profile, have made it a standard tool in the LNP field. C12-200 continues to be used in preclinical studies exploring new therapeutic applications for RNA-based medicines, including vaccines, cancer immunotherapy, and gene therapy for rare diseases.

C70H145N5O5

1136.9308

1136.124

C, 73.95; H, 12.86; N, 6.16; O, 7.04

1220890-25-4

1226777-45-2; 1226552-44-8

49785165

Colorless to light yellow liquid

22.3

5

10

64

80

1160

0

O([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])C([H])([H])N(C([H])([H])C([H])([H])N(C([H])([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])O[H])C([H])([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])O[H])C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])N(C([H])([H])C([H])([H])N(C([H])([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])O[H])C([H])([H])C([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H])O[H])C([H])([H])C1([H])[H]

URQSMTPJROAUPK-UHFFFAOYSA-N

InChI=1S/C70H145N5O5/c1-6-11-16-21-26-31-36-41-46-65(76)61-74(62-66(77)47-42-37-32-27-22-17-12-7-2)53-51-69(70(80)50-45-40-35-30-25-20-15-10-5)71-52-54-72-55-57-73(58-56-72)59-60-75(63-67(78)48-43-38-33-28-23-18-13-8-3)64-68(79)49-44-39-34-29-24-19-14-9-4/h65-71,76-80H,6-64H2,1-5H3

1,1-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)

C12-200; Tech G 1; C12 200; MFCD32062847; 1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol); 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol; C12200; C 12200; C 12-200; C-12-200; C-12200

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)

DMSO : ~100 mg/mL (~87.96 mM)
Ethanol : ~100 mg/mL (~87.96 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (2.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (2.20 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: 2.5 mg/mL (2.20 mM) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (2.20 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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