Lipid for protein delivery
https://onlinelibrary.wiley.com/doi/abs/10.1002/ange.201311245
86: N,N-Bis(2-hydroxyethyl)ethylene diamine) : 10G https://www.sigmaaldrich.com/catalog/product/aldrich/268879, 10G $87.80
2,2'-(3-aminopropylazanediyl)diethanol
87: N-(3-aminopropyl)diethaneamine, N,N-Bis(2-hydroxyethyl)-1,3-diaminopropane, 25g http://www.tcichemicals.com/eshop/en/us/commodity/A1280/ 25g $99.00
Cas: 4985-85-7
1,2-Epoxyhexadecane: https://www.sigmaaldrich.com/catalog/product/aldrich/260215, http://www.tcichemicals.com/eshop/en/us/commodity/E0316
Cas: 7320-37-8
Lauryl acrylate, 14 C
Sigma: 447315-100ML https://www.sigmaaldrich.com/catalog/product/aldrich/447315?lang=en®ion=US, 447315-100ML, $42.25
Tetradecyl Acrylate, 14 C,
COM490020904-25G $101.25
TCI: 25mL http://www.tcichemicals.com/eshop/en/gb/commodity/T2265/, 25mL 33.00 GBP
Octadecyl acrylate :409693-250G  https://www.sigmaaldrich.com/catalog/product/aldrich/409693?lang=en®ion=US, 409693-250G $38.75 
N,N'-dimehtylpropane-l,3-diamine (left), 2-(decyldisulfanyl)ethyl acrylate (right)
https://patents.google.com/patent/WO2014134445A1
Synthesis of lipidoids. Lipidoids were synthesized and characterized as previously described by our group 1 . Briefly, in a 5 mL Telfon-lined glass screw-top vial, 1, 2-epoxyohexadecane and varying amines were mixed at molar ratios of 2.4:1 (epoxide:amine), followed by a two-day reaction at 80 oC without solvent.
The lipidoids with different tail lengths were purified from the crude reaction mixtures by column chromatography and tested for their ability to deliver β-gal DNA to HeLa cells. The
screening results showed that most of the pure lipidoids in the refined library supported DNA delivery into the HeLa cell with expression of β-gal (Figure 1); however the efficiency
varied depending on the lipidoid structure. Lipidoids derived from amine 86 and 87 with two medium-length tails (e.g., C14 or C15 for acrylamide and acrylate, respectively, and C14 or
C16 for epoxide) displayed high transfection efficiencies, in some cases higher than Lipofectamine 2000
The lipidoids with amide linkers
displayed substantially higher delivery efficiency than those with ester and hydroxyl groups. The delivery efficiency also varied with the amine molecules utilized for lipidoids synthesis,
for example, the acrylamide and epoxide conjugated with amine 87 (14N-87 and 16C-87) delivered DNA more efficiently than those conjugated with amine 86 (14N-86 and 16C-86),
while for the lipidoids synthesized from acrylate, the reverse results were observed (Figure 1).
To further investigate the structure-activity relationship of these lipidoids for DNA delivery, we selected lipidoids prepared from the reaction of amine 87 with acrylamide (14N-87),
epoxide (16C-87), and acrylate (14O-87) (Scheme 1B), which displayed high delivery efficiency.
14O-87 is the best
Formulating EC16-1/protein nanoparticles for in vivo protein delivery: EC16-1/protein nanoparticles were formulated by a thin film hydration method. Briefly, EC16-1, cholesterol, and
DOPE were mixed at a weight ratio of 16:4:1 in chloroform, and the organic solvent was evaporated under vacuum. To formulate EC16-1/protein nanoparticles, thin layer film that had
formed was re-hydrated with PBS to make a cloudy solution. The solution was then mixed with saporin at a weight/weight ratio of 15:1 (EC16-1:saporin) or RNase A-Aco at a weight/weight ratio of 3:2 (EC16-1: RNase A-Aco), followed by 30 minutes of incubation at room temperature. The EC16-1/protein complex was post-modified with mPEG2000-ceramide C16/DSPE-PEG2000-Biotin (molar/molar = 1:1, 25% weight percentage in total compared to lipidoid), followed by a further 60 minutes of incubation at room temperature. The final EC16-1 concentration in all formulations was kept at 1 mg/mL. For empty EC16-1 nanoparticles, the protein was replaced with PBS. RNase AAco or saporin was similarly formuated with cholesterol, DOPE, and C16-ceramide-PEG in the absence of EC16-1 to evaluate the vital role lipidoids play in facilitating protein delivery. The formuated nanoparticles were then characterized by DLS analysis (Brookhaven ZetaPALS, NY) or TEM imaging (FEI, OR).
PNAS
[edit]http://www.excellgen.net/eln/images/a/a2/Synthesis_of_protein_delivery_lipid.png
Lipid Nanoparticle Formulation.
[edit]Lipid was formulated into nanoparticles for all delivery applications. The as-synthesized lipid was mixed with cholesterol, DOPE, and C16-PEG2000-Ceramide (Avanti Polar Lipids) at a wt/wt ratio of 16:4:1:1. Briefly, lipid, cholesterol, and DOPE were mixed in a chloroform solution in a 0.5-dr vial. The organic solvent was evaporated under vacuum and further dried over night to form a thin layer film on the vial. The lipid film was hydrated using a mixed solution of ethanol/ sodium acetate buffer (200 mM, pH = 5.2, vol:vol = 9:1). The above solution was added dropwise to an aqueous solution of C16-PEG2000-ceramide, and the resulted solution was incubated at 37 °C for 30 min before subjecting to dialysis against water or PBS in 3500 molecular weight cut-off (MWCO) cassettes (Pierce/ Thermo Scientific).
Delivery of proteins complexed with cationic lipids in cell culture
Cultured cells were plated in 48-well format (250 µL volume) in Dulbecco’s Modified Eagle’s Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ~70% confluence the next day. Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery. Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 µM protein (based on a 275 µL final volume) with 0.2–2.5 µL commercially available cationic lipids in 25 µL OPTIMEM media (Life Technologies) according to the manufacturer’s protocol for normal plasmid transfection, including incubation time. 25 µL of the OPTIMEM mixture containing cationic lipids and protein was then added to cells in 250 µL of full serum media. For Cas9 delivery in vitro, purified sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent in 25 µL OPTIMEM and treating cells in 250 µL of full serum media. All complexing steps were performed at room temperature. For all protein deliveries that lasted longer than 12–16 hours, media containing lipid complexes was replaced with fresh full serum media without antibiotics after 12–16 hours unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for optimal gene activation either 4 hours (TALE) or 12–16 hours (dCas9-VP64) after delivery, and for gene modification (Cas9) 48 hours after delivery. Delivery of proteins complexed with cationic lipids in cell culture
Cultured cells were plated in 48-well format (250 µL volume) in Dulbecco’s Modified Eagle’s Media plus GlutaMAX (DMEM, Life Technologies) with 10% FBS (“full serum media”) and antibiotics at a cell density necessary to reach ~70% confluence the next day. Full serum media was replaced with the same media but containing no antibiotics at least one hour before delivery. Delivery of Cre and TALE proteins was performed by combining 1 nM to 1 µM protein (based on a 275 µL final volume) with 0.2–2.5 µL commercially available cationic lipids in 25 µL OPTIMEM media (Life Technologies) according to the manufacturer’s protocol for normal plasmid transfection, including incubation time. 25 µL of the OPTIMEM mixture containing cationic lipids and protein was then added to cells in 250 µL of full serum media. For Cas9 delivery in vitro, purified sgRNA was incubated with Cas9 protein for 5 min before complexing with the cationic lipid reagent in 25 µL OPTIMEM and treating cells in 250 µL of full serum media. All complexing steps were performed at room temperature. For all protein deliveries that lasted longer than 12–16 hours, media containing lipid complexes was replaced with fresh full serum media without antibiotics after 12–16 hours unless otherwise noted. Cells were assayed for recombination 48 hours after delivery, for optimal gene activation either 4 hours (TALE) or 12–16 hours (dCas9-VP64) after delivery, and for gene modification (Cas9) 48 hours after delivery.
https://patents.google.com/patent/WO2014134445A1
Compositions 31 and 32 were prepared using a thin film hydration method described below. Compound 1-016B or 1-018B, cholesterol, and DOPE were mixed at a weight ratio of 16:4: 1 in chloroform, which was then evaporated under vacuum, leaving a thin film. Re-hydrating the thin film in PBS yielded a solution of 1-016B or 1-018B at a concentration of 1 mg/mL. Saporin was added (1-016B or 1-018B solution : saporin = 15: 1 by weight). The mixture was incubated for 15 minutes at room temperature followed by addition of mPEG2000-ceramide C16/DSPE-PEG2000-Biotin (purchased from Avanti Polar Lipids, weight/weight = 8: 1, PEG 10% by weight of l-Ol 6B, this component not shown in the table above). The mixture was again incubated for 15 minutes to yield a nanocomplex composition, Composition 3 lor 32. See the table above for the weight ratios between Compound 1-016B or 1-018B, cholesterol, DOPE, and saporin. These two compositions thus prepared were subjected to an in vivo assay described in Example 47 below.