Ionizable cationic lipid; siRNA delivery
D-Lin-MC3-DMA does not act through recognition of specific protein targets but rather through a physicochemical mechanism known as “endosomal escape,” enabled by its unique ionizable lipid properties. With a pKa value of approximately 6.2-6.4, the lipid is essentially neutral at physiological pH (7.4), which helps reduce non-specific interactions in the circulation and systemic toxicity. Upon cellular uptake via endocytosis, the acidic endosomal environment (pH ~5.0) protonates its tertiary amine group, conferring a positive charge. This positively charged lipid then interacts electrostatically with negatively charged endosomal membrane lipids, inducing a transition from a lamellar to a hexagonal phase that disrupts the endosomal membrane and releases the nucleic acid cargo into the cytoplasm.

Preparation of plasmid-containing LNP[1]
LNP-pDNA was prepared as previously described in J Phys Chem B, 119 (28) (2015), pp. 8698-8706. Amino-lipids, helper lipids, cholesterol and PEG-DMG were dissolved in ethanol at a molar ratio of 50/10/39/1. Purified pCI-FLuc (expressing Firefly Luciferase) or pCAX-eGFP (expressing eGFP) was dissolved in 25 mM sodium acetate pH 4 to 0.116 mg/ml. The solutions were mixed using a micromixer. For large-scale formulations, mixing was performed through a T-junction mixer as previously described in Pharm Res, 22 (3) (2005), pp. 362-372. All formulations were produced at 0.029 mg DNA per μmol lipid (corresponding to N/P charge ratio of 6.0), unless otherwise stated.

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Analysis of lipid particles containing DNA[1]
Particle size, lipid concentration, pDNA entrapment, and total pDNA concentration were measured as previously described in J Phys Chem B, 119 (28) (2015), pp. 8698-8706 and J Control Release, 196 (2014), pp. 106-112. Zeta potential was measured as previously described in Mol Ther Nucl Acids, 3 (2014), p. e210.

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Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity[1].

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In vitro, the activity of D-Lin-MC3-DMA is primarily demonstrated through its functional role as a nucleic acid delivery vehicle. Studies have shown that lipid nanoparticles formulated with D-Lin-MC3-DMA can effectively deliver luciferase plasmid DNA (pCI-FLuc) to HeLa cells, resulting in significant luciferase expression at DNA concentrations ranging from 0.75-6.0 μg/mL. In dengue virus research, D-Lin-MC3-DMA liposomes loaded with anti-DENV siRNA significantly reduced viral titers in HepG2 cells (at a working concentration of 40 nM siRNA) without inducing cytotoxicity, hemolysis, or pro-inflammatory cytokine release. Furthermore, covalently conjugating D-Lin-MC3-DMA to siRNA enhanced endosomal escape in cell culture without compromising RNA-induced silencing complex activity. One study reported that CD44-targeting peptide-modified D-Lin-MC3-DMA lipid nanoparticles (AKPC-LNP) achieved 70-80% gene silencing efficiency of YAP/TAZ siRNA in CD44-high breast cancer cells.

Effective internal siRNA delivery vehicles are phosphate nanoparticles (LNP) that contain ionizable phosphate esters, such as dilinoleylmethyl-4-dimethylphosphate (DLin-MC3-DMA), and distearyl acetylphosphatidylcholine (DSPC). With a molar ratio of 50/10/38.5/1.5 (PEG)-Pallet, the LNP-siRNA system procedure is tuned to achieve maximum gene silencing efficacy in hepatocytes following intravenous injection. It contains DLin-MC3-DMA (MC3), DSPC, solution, and polymerization solution. The improved pKa value of DLin-MC3-DMA greatly increases efficacy [1].

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At a dose of 1 mg/kg siRNA in mice, LNPs with ALC-0315 achieved a 2- and 10-fold greater knockdown of FVII and ADAMTS13, respectively, compared to LNPs with MC3. At a high dose (5 mg/kg), ALC-0315 LNPs increased markers of liver toxicity (ALT and bile acids), while the same dose of MC3 LNPs did not. These results demonstrate that ALC-0315 LNPs achieves potent siRNA-mediated knockdown of target proteins in hepatocytes and HSCs, in mice, though markers of liver toxicity can be observed after a high dose. This study provides an initial comparison that may inform the development of ionizable cationic LNP therapeutics with maximal efficacy and limited toxicity.[2]

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D-Lin-MC3-DMA is one of the most potent in vivo-validated lipids for siRNA delivery. In a murine Factor VII silencing model, an optimized LNP-siRNA formulation incorporating D-Lin-MC3-DMA (molar ratio MC3/DSPC/cholesterol/PEG-lipid = 50/10/38.5/1.5) achieved an ED50 as low as 0.005 mg/kg, representing a more than two orders of magnitude improvement in potency over the first-generation lipid DLinDMA. D-Lin-MC3-DMA-conjugated siRNA exhibited a tissue distribution profile in mice similar to cholesterol-conjugated siRNA, with a tendency to accumulate in vascular compartments. CD44-targeting AKPC-LNP (incorporating D-Lin-MC3-DMA) significantly suppressed breast cancer growth in a zebrafish xenograft model, reducing tumor burden by nearly 100% without observable toxicity. Additionally, D-Lin-MC3-DMA is the core lipid component of Onpattro, the first approved siRNA drug, which has been clinically validated to effectively deliver patisiran to hepatocytes for TTR gene silencing.

D-Lin-MC3-DMA is not a conventional enzyme or receptor-binding compound; its function depends on lipid nanoparticle assembly and physicochemical properties. In cell-free systems, its characterization focuses on lipid properties: it can be dissolved in anhydrous ethanol (solubility ≥60 mg/mL) to prepare stock solutions. The size (typically 80-120 nm), polydispersity index (PDI <0.2), and Zeta potential (near neutral at physiological pH) of LNPs are measured by dynamic light scattering (DLS). Encapsulation efficiency of siRNA (typically >90%) is determined using the RiboGreen dye method. Evaluation of ionizable properties can be achieved by monitoring surface charge changes across pH values (from pH 4.0 to pH 7.4) or by measuring pKa (approximately 6.44). Differential scanning calorimetry or fluorescent probes can also be used to assess lipid membrane fluidity and fusion capability with endosomal membranes.

In vitro transfection studies of cultured cells[1]
HeLa, HepG2 and Hep3B cells were cultured in DMEM with 10% FBS. PC12 and MCF7 cells were cultured in RPMI 1640 with 10% FBS. LNPs were diluted into medium at 0.75-6.0 μg/ml pDNA. The luciferase assay was performed 24 h post-treatment. In the case of serum-free treatments, LNP-pDNA systems were diluted into medium alone. To study the role of ApoE, wild-type (C57BL/6) and ApoE−/− (B6.129P2-Apoetm1Unc/J) mouse sera were purchased from The Jackson Laboratory. LNP-pDNA systems were diluted into medium containing 10% of each serum. Measured luminescence was normalized to protein content as measured by the Pierce BCA Protein Assay.

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In vitro LNP uptake measurement[1]
Cellular uptake of LNP formulations was performed as previously described.29 LNP-pDNAs labeled with DiI-C18 (0.2 mol% total lipid) were used. Plates were imaged using a Cellomics Arrayscan VTI HCS reader.

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In vitro cellular assays for D-Lin-MC3-DMA focus on evaluating its efficiency as a nucleic acid delivery vehicle. Typical protocol: 1) Prepare LNPs using microfluidic mixing or ethanol injection: dissolve D-Lin-MC3-DMA, DSPC, cholesterol, and PEG-lipid in ethanol at a specific molar ratio (e.g., 50/10/38.5/1.5) and rapidly mix with nucleic acid-containing citrate buffer (pH 4.0) at a 3:1 volume ratio. 2) Purify by dialysis or ultrafiltration to remove ethanol and exchange buffer to PBS. 3) Cell treatment: seed target cells (e.g., HeLa, HepG2, or breast cancer cells) in culture plates (approximately 1×10⁴-1×10⁵ cells per well), add LNP-containing medium (nucleic acid concentration 0.75-6.0 μg/mL), and incubate for 24-48 hours at 37°C. 4) Evaluate efficacy: assess fluorescent reporter gene expression by flow cytometry, or measure target gene silencing efficiency (e.g., 70-80% YAP/TAZ knockdown) by RT-qPCR or Western blot.

In vivo transfection by injection of LNP-pDNA[1]
Stage ~19-20 white leghorn chicken embryos were stained with neutral red dye, and a small tear was made in the extraembryonic membranes over the forelimb. LNP formulations mixed with ~0.1% Fast Green dye was injected at the distal forelimb at 10 μg/ml pDNA, using a Picospritzer II microinjector (Parker Hannifin Corp). Eggs were incubated overnight. Embryos were rinsed and fixed in 4% paraformaldehyde in PBS, and GFP fluorescence was recorded with a Leica MZFLIII stereofluorescence microscope.

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Transfection of primary embryonic mesenchyme and analysis of reagent toxicity[1]
White leghorn chicken embryos at stage 24 were removed and dissected in Hanks buffered saline solution (HBSS). Forelimbs were pooled and treated for 1 h in dispase to remove epithelial ectoderm. Limb mesenchyme was dissociated, and resuspended in DMEM with 5% FBS and 1% penicillin/streptomycin. Cells were plated onto 96-well plates at a density of 2 × 105 or 2 × 107 cells/ml and treated with 0-40 μg/ml LNP-pDNA. Lipofectamine was used as per manufacturer's protocol. Cultures were incubated overnight at 37 °C and resuspended in PBS buffer for expression analysis using a BD LSRII flow cytometer. Cell survival was assessed in low-density cultures after 24 h of treatment, by counting the adherent cells remaining after PBS washes. Viability was determined based on average counts of all adherent cells within a single field of view (100×) normalized to cell counts of untreated cultures.

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LNP-siRNA injections[2]
siFVII, siADAMTS13, and siLuc were encapsulated in LNPs containing either ALC-0315 or MC3 as the ionizable cationic lipid. We injected mice with 1 mg siRNA per kg body weight (mg/kg) for knockdown studies, and 5 mg/kg dose for toxicity studies. A dose of 1 mg/kg siRNA in mice is standard for inducing knockdown of mRNA for proteins made in hepatocytes using siRNA-LNPs, whereas 5 mg/kg is a higher dose than the one that would normally be used in mice.3 The recommended dose of ONPATTRO (the clinically approved siRNA for hATTR) is 0.3 mg/kg, which corresponds to a human equivalent dose (HED) of 3.69 mg/kg in mice when using body surface area conversion. One week after administration, liver tissue and blood were collected to measure target mRNA and protein levels, respectively, and compared to siLuc-treated mice; half-lives of plasma FVII and ADAMTS13 are 3–6 hours, and 2–3 days, respectively.23,24 mRNA and protein quantification, and toxicity studies are described further below.

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Toxicological analysis[2]
Mice were injected IV with either PBS, or with siLuc encapsulated in LNPs with ALC-0315 (siLuc-ALC-0315) or MC3 (siLuc-MC3) at 5 mg/kg (N = 4). While a dose of any LNP at 10 mg/kg usually causes severe toxicity, such as inflammation and liver necrosis, the toxicity after a 5 mg/kg dose depends on the lipid formulation.26,27 Five hours after the injection, mice were sacrificed, and serum samples were collected as described above. Serum samples were submitted to Idexx BioAnalytics for a toxicology panel. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), bile acids, total bilirubin (TBIL), blood urea nitrogen (BUN), creatine (CREA), gamma-glutamyl transferase (GGT) levels were analyzed. To note, data regarding bile acid levels in mice treated with PBS and with siLuc-ALC-0315 had N = 3 due to the presence of an outlier in each group (data not shown). The presence of the outliers would have not altered the conclusion, siLuc-ALC-0315 treated mice would have had an even higher bile acid mean and would have been more statistically significant from the PBS-treated mice. Outliers were determined via the ROUT method using GraphPad Prism although limitations such as our small sample size were considered. Bile acid levels commonly range from 0 to 6 μmol/L; however, our results were likely not biologically possible (>130 μmol/L).

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In vivo animal assays for D-Lin-MC3-DMA focus on evaluating the efficacy and distribution of LNP-siRNA systems. Standard protocol: 1) Animal preparation: use 6-8 week old female C57BL/6 mice. 2) LNP-siRNA formulation: prepare LNPs containing D-Lin-MC3-DMA (molar ratio MC3/DSPC/cholesterol/PEG-lipid = 50/10/38.5/1.5) using microfluidic technology, encapsulating siRNA targeting a specific gene (e.g., Factor VII or YAP/TAZ). 3) Administration: inject intravenously via the tail vein at a volume of 10 mL/kg, with siRNA doses ranging from 0.005 to 1 mg/kg. 4) Endpoints: euthanize animals 24-72 hours post-administration, collect tissues such as liver, measure target mRNA levels by RT-qPCR, or assess protein expression by ELISA/Western blot. 5) Tissue distribution: trace using fluorescently labeled siRNA (e.g., Cy5-siRNA) or measure D-Lin-MC3-DMA concentration in tissues by LC-MS/MS.

The pharmacokinetic properties of D-Lin-MC3-DMA, as a component of lipid nanoparticles, differ significantly from conventional small molecule drugs. Following intravenous administration as LNPs, the particles are opsonized by apolipoprotein E in plasma and actively target the liver via low-density lipoprotein receptor-mediated endocytosis. The lipid’s neutral charge at physiological pH helps prolong circulation time and reduce non-specific uptake. In vivo, D-Lin-MC3-DMA-conjugated siRNA exhibits a tissue distribution profile similar to cholesterol-conjugated siRNA, with unique accumulation in vascular compartments. LNPs are typically sized between 80-120 nm, an ideal range to avoid rapid renal clearance and promote passive hepatic targeting. Public data on the terminal half-life and metabolic pathways of D-Lin-MC3-DMA are limited; however, the lipid is designed to be biodegradable to avoid excessive accumulation in the liver.

The toxicity of D-Lin-MC3-DMA and related LNP formulations is closely associated with formulation composition, dose, administration route, and target tissue. In preclinical studies, optimized D-Lin-MC3-DMA LNPs (such as the formulation used in Onpattro) achieved an ED50 as low as 0.005 mg/kg in mouse models with no significant toxicity observed at therapeutic doses. However, an important toxicological finding is that high tissue accumulation levels (>20 pmol/mg tissue) of D-Lin-MC3-DMA-covalently conjugated siRNA lead to non-specific modulation of gene expression. This effect is likely caused by unbound free lipid, indicating potential risks associated with direct covalent conjugation strategies that require fine-tuning to balance enhanced endosomal escape with minimized toxicity. In vitro, D-Lin-MC3-DMA liposomes loaded with siRNA (40 nM) did not induce cytotoxicity or hemolysis. D-Lin-MC3-DMA is for research use only and is not intended for human use.

[1]. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine. 2017 May;13(4):1377-1387.

[2]. Comparison of DLin-MC3-DMA and ALC-0315 for siRNA Delivery to Hepatocytes and Hepatic Stellate Cells. Mol Pharm. 2022;19(7):2175-2182.

Lipid nanoparticles (LNPs) containing distearate phosphatidylcholine (DSPC) and ionizable amino lipids (such as dilinoleoylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA)) are effective siRNA delivery vectors in vivo. This article explores the application of similar LNP systems as plasmid DNA (pDNA) transfection reagents. Results showed that replacing DSPC with unsaturated phosphatidylcholine (PC) and DLin-MC3-DMA with the related lipid DLin-KC2-DMA yielded highly efficient transfection reagents for HeLa cells in vitro. Furthermore, these formulations exhibited excellent transfection performance in various mammalian cell lines, with transfection efficiencies approaching 90% in primary cell cultures. These transfection levels are comparable to or higher than those of Lipofectamine, with significantly reduced toxicity. Finally, robust reporter gene expression was achieved by microinjecting LNP-eGFP into chicken embryo limb buds. The conclusion is that lipid nanoparticle (LNP) systems containing ionizable amino lipids can serve as efficient and non-toxic plasmid DNA (pDNA) delivery systems for in vitro and in vivo gene expression. In summary, the results of this study indicate that pDNA LNP formulations containing ionizable cationic lipids can serve as efficient in vitro transfection reagents with significantly lower toxicity than commercial reagents such as Lipofectamine. LNP systems containing DLin-KC2-DMA and unsaturated helper lipids are also effective systems for in vitro and in vivo transfection of primary cells. These systems are expected to have important applications in in vitro transfection of developmental tissues and have the potential for gene editing applications. [1]

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Ionizable cationic lipids are crucial for the efficient delivery of RNA by lipid nanoparticles (LNPs). DLin-MC3-DMA (MC3), ALC-0315, and SM-102 are currently the only ionizable cationic lipids that have been clinically approved for RNA therapy. ALC-0315 and SM-102 are structurally similar lipids used in SARS-CoV-2 mRNA vaccines; MC3 is used in siRNA therapy to knock down transthyretin protein in hepatocytes. Hepatocytes and hepatic stellate cells (HSCs) are ideal targets for RNA therapy because they synthesize a variety of plasma proteins, including those affecting blood clotting. Although lipid nanoparticles (LNPs) preferentially accumulate in the liver, evaluating the ability of different ionizable cationic lipids to deliver RNA carriers to different cell populations is crucial for designing RNA-LNP therapies with minimal hepatotoxicity. Here, we directly compare the ability of LNPs containing ALC-0315 or MC3, respectively, to knock down coagulation factor VII (FVII) in hepatocytes and ADAMTS13 in HSCs. In mice, ALC-0315 lipid nanoparticles (LNPs) administered at a dose of 1 mg/kg siRNA showed a 2-fold and 10-fold increase in knockdown of FVII and ADAMTS13, respectively, compared to MC3 lipid nanoparticles. At high doses (5 mg/kg), ALC-0315 lipid nanoparticles increased hepatotoxicity markers (ALT and bile acids), while the same dose of MC3 lipid nanoparticles did not. These results indicate that ALC-0315 lipid nanoparticles can effectively mediate siRNA-mediated knockdown of target proteins in mouse hepatocytes and hepatic stellate cells (HSCs), although hepatotoxicity markers may be observed at high doses. This study provides a preliminary comparison that can serve as a reference for developing ionizable cationic lipid nanoparticle therapies with maximum efficacy and minimum toxicity. This work validates the concept that ionizable cationic lipid nanoparticles can be used to target and knock down HSC-specific targets such as ADAMTS13. The results showed a difference in efficacy between MC3 and ALC-0315, two of three clinically approved ionizable cationic lipids used in the LNP delivery platform. This direct comparative insight may help optimize RNA-based drugs to modulate the expression of proteins that are previously unavailable in vivo for research and therapeutic purposes. [2]

C43H79NO2

642.0929

641.611

C, 80.43; H, 12.40; N, 2.18; O, 4.98

1224606-06-7

1224606-06-7 ; 1258299-72-7 (deleted)

49785164

Colorless to light yellow liquid

0.886±0.06 g/cm3(Predicted)

670.2±43.0 °C(Predicted)

13.647

0

3

36

46

687

0

O(C(CCCN(C)C)=O)C(CCCCCCCC/C=C\C/C=C\CCCCC)CCCCCCCC/C=C\C/C=C\CCCCC

NRLNQCOGCKAESA-KWXKLSQISA-N

InChI=1S/C43H79NO2/c1-5-7-9-11-13-15-17-19-21-23-25-27-29-31-33-35-38-42(46-43(45)40-37-41-44(3)4)39-36-34-32-30-28-26-24-22-20-18-16-14-12-10-8-6-2/h13-16,19-22,42H,5-12,17-18,23-41H2,1-4H3/b15-13-,16-14-,21-19-,22-20-

(6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate

D-Lin-MC3-DMA; MC 3; RV-28; MC3; DLin-MC3-DMA; (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate; Q0J6FQ6FKP; D-Lin-MC3-DMA (Excipient); RV 28; MC-3; RV28; DLin-MC3-DMA; Dlin-mc3-dma

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)

Ethanol : ~125 mg/mL (~194.68 mM)
DMSO : ~100 mg/mL (~155.74 mM)

Solubility in Formulation 1: 6.25 mg/mL (9.73 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: 5 mg/mL (7.79 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: 5 mg/mL (7.79 mM) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: ≥ 2.5 mg/mL (3.89 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 5: 2.5 mg/mL (3.89 mM) in 10% DMSO + 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 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 6: ≥ 2.5 mg/mL (3.89 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 7: ≥ 2.5 mg/mL (3.89 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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 (Please use freshly prepared in vivo formulations for optimal results.)