Branched ionizable lipid for mRNA delivery

In recent years, messenger RNA (mRNA) has come into the spotlight as a versatile therapeutic with the potential to prevent and treat a staggering array of diseases. Billions of dollars have been invested into the commercial development of mRNA drugs, with ongoing clinical trials focused on vaccines (for example, influenza and Zika viruses) and cancer immunotherapy (for example, myeloma, leukemia, and glioblastoma). Although significant progress has been made in the design of in vitro transcribed mRNA that retains potency while minimizing unwanted immune responses, the widespread use of mRNA drugs requires the development of safe and effective drug delivery vehicles. Unfortunately, delivery vehicle development has been stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. [1]

The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure-function relationships and identify a previously unappreciated parameter-lipid nanoparticle surface ionization-that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O10 and 306Oi10 , induce substantial luciferase expression in mice following a single 0.75 mg kg-1 mRNA dose. These lipidoids, which have ten-carbon tails and identical molecular weights, vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that Oi10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched-tail lipids facilitate protein expression in animals.[2]

LNP pKa and surface ionization measurements. [2]
LNP surface pKa was determined as previously described [20]. Briefly, a buffer solution consisting of 150 mM sodium chloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 mM ammonium citrate was titrated to pH values ranging from 2 to 12 in increments of 0.5. In a black 96-well plate, 250 "L of each pH solution was aliquoted in triplicate. 5 "L of LNP at an mRNA concentration of 0.1 mg/mL was added to each well. 10 "L of a 0.16 mM stock solution of 2-(p-toluidinyl)naphthalene-6-sulphonic acid in DI water was added to each well such that the final concentration of TNS was 6 "M. Fluorescence intensity was measured using a BioTek Synergy H1 microplate reader at an excitation of 322 nm and an emission of 431 nm. The maximum fluorescence value was assumed to represent 100% amine protonation, while the minimum fluorescence was assumed to represent 0% amine protonation. The LNP surface pKa was determined to be the pH at which 50% amine protonation occurred. It should be noted that the absolute value of fluorescence values will depend on plate reader settings, including the gain. When comparing different nanoparticle formulations, be sure to use the same settings.

In Vitro mRNA delivery. [2]
HeLa cells (ATCC) were maintained at 37°C and 5% CO2 in high glucose Dulbecco’s Modified Eagles Medium supplemented with 10% fetal bovine serum (by volume), 20 U/mL penicillin and 20 U/mL streptomycin. Cells were seeded in white 96-well plates at a density of 15,000 cells per well in 180 "L of media 6 - 8 hours before transfection. Cells were transfected with LNPs carrying unmodified mRNA encoding firefly luciferase at a dose of 100 ng per well (0.81 nM). Lipofectamine MessengerMax was formulated according to the manufacturer’s instructions. Firefly luciferase activity was measured using the Bright-Glo! Luciferase Assay System 24 hours posttransfection.

In Vivo Biodistribution studies. [2]
All animal experiments were conducted using institutionally approved protocols (IACUC). Female C57BL/6 mice received tail vein injections of LNPs loaded with Cy5-labeled luciferase mRNA (m5C/#) at a dose of 0.75 mg/kg. Mice were sacrificed 1 hour post-injection, and the pancreas, spleen, liver, kidneys, heart and lungs were harvested and imaged for Cy5 fluorescence using an IVIS" imaging system at an excitation of 649 nm and an emission of 670 nm. Total radiant efficiency [(p/s)/("W/cm2 )] was determined by region of interest analysis using Living Image" software [2].

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Confocal Microscopy. [2]
Female C57BL/6 mice (Charles River Laboratories) received tail vein injections of LNPs carrying Cyanine 5 luciferase mRNA (m5C/#) (TriLink Biotechnologies) at a dose of 0.75 mg/kg. Mice were sacrificed 1 hour post injection, and the spleen and liver were harvested. Liver and spleen tissues were fixed in 4% formaldehyde in DI water for 24 hours followed by three washes in PBS. Tissues were sectioned and permeabilized with 0.1% Triton X100 in PBS for 3-5 mins. Following two PBS washes, samples were then stained with Alexa Fluor 488 Phalloidin (1:40 dilution) and Hoeschst 33342 (1:2000 dilution) overnight on a shaker. After two PBS washes, samples were mounted on glass slides and imaged on a Zeiss LSM 700 Confocal using a 63x objective.

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In Vivo Luciferase mRNA delivery. [2]
Female C57BL/6 mice at least 6 weeks of age were used. Mice received tail vein injections of naked mRNA (negative control) or LNPs carrying luciferase-encoding mRNA at a dose of 0.75 mg/kg (unless otherwise specified). For all in vivo experiments, mRNA was base-modified with 5- methoxyuridine (mo5U). Fifteen minutes prior to each imaging time point, mice received an intraperitoneal injection of 130 "L D-luciferin at a concentration of 30 mg/mL. Mice were anesthetized with isoflurane (4% for induction, 2% for maintenance, Patterson Veterinary) and imaged for bioluminescence on the ventral side with the IVIS. Alternatively, 15 minutes after D-luciferin administration, mice were sacrificed and the pancreas, spleen, liver, kidneys, heart and lungs were harvested and imaged for bioluminescence using the IVIS. Total luminescent flux (photons/second) was determined by region of interest analysis using the Living Image" software.

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In Vivo Erythropoietin mRNA delivery. [2]
Female C57BL/6 mice received tail vein injections of LNPs carrying erythropoietin mRNA (mo5U) at a dose of 0.75 mg/kg. At 3, 6, and 24 hours post injection, blood samples were collected via the submandibular vein. Serum was isolated by centrifuging blood samples in BD Microtainer Serum Separator Tubes at 14,000 RPM for 10 minutes. Following serum dilution 1:1000, erythropoietin concentrations were determined using Human Erythropoietin Quantikine IVD ELISA Kit.

[1]. Hajj K A. Lipid Nanoparticle Systems for the Delivery of Messenger RNA. Carnegie Mellon University, 2019.

[2]. Branched-Tail Lipid Nanoparticles Potently Deliver mRNA In Vivo due to Enhanced Ionization at Endosomal pH. Small. 2019 Feb;15(6):e1805097. doi: 10.1002/smll.201805097.

Recently, it was found that a class of lipid-like materials, known as lipidoids, can be formulated into lipid nanoparticles (LNPs) that potently deliver a variety of nucleic acids in vitro and in vivo. Here, we investigate the use of these materials for the delivery of messenger RNA both in a variety of cell types and in mice. By synthesizing a library of 400 lipidoids, we show that the material chemistry has a large impact on the mRNA delivery efficacy in vitro. Using a smaller library of 11 lipidoids, we show that LNPs formulated from these materials potently deliver mRNA to a variety of organs in mice. The two most potent materials from the library, 306O10 and 306Oi10, have 10-carbon tails and identical molecular weights, and vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one-carbon branch. Remarkably, this small difference in structure conferred a 10-fold improvement in 306Oi10 efficacy. The enhanced potency of this branched-tail lipidoid was attributed to its strong surface ionization upon entry into the endosomal compartment of target cells. We also show that delivery of the top LNP, 306Oi10, enabled higher levels of protein expression than two gold standard lipids, C12-200 and DLin-MC3-DMA in mice. We also demonstrate that this material is sufficiently potent to encapsulate and deliver three mRNAs of varying lengths within the same formulation. Furthermore, 306Oi10 co-delivered Cas9 mRNA and single guide RNAs (sgRNAs), facilitating CRISPR-mediated gene editing in the livers of mice. Intravenous delivery of this material also did not significantly increase serum cytokine or IgG levels, nor did it cause liver toxicity, as determined by histology. Finally, we explore the impact of mRNA nucleoside modifications on resultant protein expression in mice. Specifically, we probe the influence of the delivery vehicle on the efficacy of mRNA modifications by delivering a variety of modified mRNAs using four LNPs which target either the liver, spleen, or lungs. We demonstrate that the delivery vehicle has a large impact on the efficacy of modified mRNAs. We also show that the majority of protein expression enhancement occurs in the spleen due to enhanced transfection as well as increased translation in cells in this organ. [1]

C59H115N3O8

994.56

993.868

2322290-93-5

146204786

Colorless to light yellow ointment

17.2

0

11

56

70

1040

0

O=C(CCN(CCCN(CCCN(CCC(OCCCCCCCC(C)C)=O)CCC(OCCCCCCCC(C)C)=O)C)CCC(OCCCCCCCC(C)C)=O)OCCCCCCCC(C)C

BPPZRZOKGADTQE-UHFFFAOYSA-N

InChI=1S/C59H115N3O8/c1-52(2)32-22-14-10-18-26-48-67-56(63)36-44-61(45-37-57(64)68-49-27-19-11-15-23-33-53(3)4)42-30-40-60(9)41-31-43-62(46-38-58(65)69-50-28-20-12-16-24-34-54(5)6)47-39-59(66)70-51-29-21-13-17-25-35-55(7)8/h52-55H,10-51H2,1-9H3

8-methylnonyl 3-[3-[3-[bis[3-(8-methylnonoxy)-3-oxopropyl]amino]propyl-methylamino]propyl-[3-(8-methylnonoxy)-3-oxopropyl]amino]propanoate

306Oi10; 2322290-93-5; SCHEMBL21504882; 306Oi10?;

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, avoid exposure to moisture.

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.)