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• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
• • A—HUMAN NECESSITIES
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
  • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
• • A—HUMAN NECESSITIES
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  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
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  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D319/00—Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
  • C07D319/04—1,3-Dioxanes; Hydrogenated 1,3-dioxanes
  • C07D319/06—1,3-Dioxanes; Hydrogenated 1,3-dioxanes not condensed with other rings

Definitions

• the combinatorial library strategy has been shown to be effective for the development of cationic lipid-like (lipidoid) nanopartieles (LNPs) for drag delivery.
• Lipidoid molecules with various hydrophilic amine heads and hydrophobic tails have been synthesized and used to deliver small molecules, proteins and peptides, ribonucleoproteins (RNP), and nucleic acids (mRNA, siRNA, ASO, pDNA etc.), both in vitro and in vivo
• Lipidoid molecular design and nanoparticle supramolecuiar structure optimization strategies have achieved better delivery performances by improving delivery specificity, enhancing efficacy, and reducing side-effects.
• a library of reduction-responsive disulfide bond-containing lipidoid nanopartieles that can be degraded in the presence of glutathione (GSH) and other intracellular reducing agents was reported. These lipidoids were used for siRNA and protein delivery'.
• the concept of a stimuli -responsive combinatorial lipidoid library was further expanded from a biochemical trigger to a physical/external trigger. This was achieved through the integration of the o-ni trobenzyl ester group into the lipidoid tail structures. Photo-degradable lipidoid nanoparticles were then fabricated and used for small molecule drug delivery'.
• Each library ' of stimuli-responsive iipidoids has its own unique physicochemical properties. Creating and expanding these libraries helps to enrich our molecular toolbox for nano drag delivery applications.
• R Head Linker — R Lipid (i) ; and pharmaceutically acceptable salts thereof, wherein R Head is
• R a , R a ’, R a ”, and R a ”’ independently are R Lipid , H, C 1 -C 20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C 3 -C 20 cycloalkyl, C 1 -C 20 heteroalkyl, C1-C20 heterocycloalkyl, aryl, or heteroaryl, wherein R 3 and R a ’ or R a ” and R a ”’ are not both R Lipid ;
• Z is a C 1 -C 20 bivalent aliphatic radical, a C 1 -C 20 bivalent heteroaliphatie radical, a bivalent aryl radical, or a bivalent heteroaryl radical;
• Linker is an acid labile moiety that is cleavable under aqueous acidic conditions; each instance of R Lipid independently is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl or wherein:
• X is CH 2 , O, NR 30 , or S;
• R 30 is H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 alkynyl;
• U and V independently are S, Se, O, or CH 2 ; m is an integer selected from 1 to 3; n is an integer selected from 1 to 14; p is 0 or 1 ; q is an integer selected from 1 to 10; and t is 0, 1, or 2,
• lipidoid nanoparticles comprising a compound disclosed herein.
• FIG. 1 A is a schematic illustration of the acid-triggered degradation of lipidoid nanopanicles.
• Fig. IB is the synthetic route and an acidic pH-triggered hydrolysis reaction for R- 016CBA lipidoids.
• Fig. 1C shows the chemical structures of exemplar ⁇ ' amine head groups.
• Fig. 2A is a H NMR spectrum of 016CBA tail .
• Fig, 2B is a 13 C NMR spectrum of 016CBA tail
• Fig. 2C is a TI NMR spectra of 75-016CBA lipidoid.
• Fig. 2D is a 13 C NMR spectra of 75-016CBA lipidoid.
• Fig. 2E is a set of ESI-MS spectra of 75-016CBA and 76-016CBA
• Fig. 2F is a table of summary of chemical formulas and calculated and observed molecular weights of R-O16CBA lipidoids.
• Fig. 3A is a schematic illustration of an acid-induced hydrolysis reaction and tail cleavage of O16CBA lipidoids.
• Fig, 3B is a bar graph showing degradation efficacy of the cyclic benzylidene acetal group in 75-016CBA lipidoids at pH 4.5 and 7.0 for different incubation durations.
• Fig. 3C is a time series of 1 H NMR spectra of 75-016CBA lipidoids under pH 4.5.
• Fig. 3D is a time series of ⁇ NMR spectra of 75-016CBA lipidoids under pH 7.2.
• Fig. 3E is an ESI-MS spectra of 75-016CBA LNPs 24 h after incubation at pH 7.2, 6.0, 5.0, and 4.5.
• Fig. 4B are the average hydrodynamic diameters of R-016CBA LNPs in neutral (pH 7.2) and acidic (pH 5.0) solutions.
• Fig. 4C is a fluorescence emission spectra of NR/75-O 16CB A LNPs in pH 7.2 and 5.0 solutions.
• Fig. 5 is a bar graph showing intracellular GFP mRNA deliver ’ efficacies of R- O16CBA LNP formulations (R-G16CBA, R-Q16CBA-F1, and R-016CBA-F2). Lpf2k and naked mRNA were used as positive and negative controls.
• [R-016CBA] 7.4 pg/mL;
• Fig. 7 shows a synthetic route employed for the preparation of acid-degradable hydrophobic tail, O16CBA.
• Fig. 8 is a 1 HNMR spectrum of HexDMBA in DMSO-d 6.
• Fig. 9 is a 1 HNMR spectrum of HexDMBAH in DMSO-d 6.
• Fig. 10 is a set of ESI-MS spectra of cyclic benzyiidene acetal-containing lipidoids,
• Fig, 11 is a set of ESI-MS spectra of 76-O16CB A LNPs incubated under pH 7.2, 6.0, 5.0, and 4.5 after 24 h.
• Fig. 12 is a set of ESI-MS spectra of 77-O16CBA LNPs incubated under pH 7,2, 6.0, 5.0, and 4.5 after 24 h.
• Fig. 13 is a set of additional TEM images of 75-, 76-, and 77-O16CBA LNPs after 24 h incubation under pH 5.0.
• R Head is R a , R a ’, R a ”, and R a ”’ independently are R Lipid , H, C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 1 -C 20 alkynyl, C 3 -C 20 cycloalkyl, C 1 -C 20 heteroalkyl, C 1 -C 20 heterocycloalkyl, aryl , or heteroaryl, wherein R 3 and R a ’ or R a ” and R a , ” are not both R
• Z is a C 1 -C 20 bivalent aliphatic radical, a C 1 -C 20 bivalent heteroaliphatie radical, a bivalent aryl radical, or a bivalent heteroaryl radical;
• Linker is an acid labile moiety that is cleavable under aqueous acidic conditions; each instance of R Lipid independently is C 1 -C 20 alkyl, C 2 -C 20 alkenyl, C 2 -C 20 alkynyl or wherein:
• R 1 and R 2 independently are H, OH, NHR 30 , or SH;
• X is C H 2 , O, NR 30 , or S;
• R 30 is H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 alkynyl;
• U and V independently are 8, Se, O, or CH 2 ; m is an integer selected from 1 to 3; n is an integer selected from 1 to 14; p is 0 or 1 ; q is an integer selected from 1 to 10; and t is 0, 1, or 2,
• R Head is
• R a and R a' independently are R Lipid , H, or C 1 -C 20 alkyl
• R Head is derived from a compound selected from the group consisting of in certain embodiments
• R Head is derived from a compound selected from the group consisting of ⁇ 113 306 406
• each instance of R Llpld independently is C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyi .
• each instance of R Lipid independently is
• R* and R 2 are H.
• R 1 is H; and R 2 is
• R 3 and R 4 are H. In certain embodiments, R 3 and R 4 taken together form an oxo ( :::: Q) group.
• Z is CH 2 , O, or NR 30 . In certain embodiments, Z is CH 2 . In certain embodiments, Z is O. In certain embodiments, Z is NR 30 .
• U and V are independently -CH2- or -0-, In certain embodiments, U and V are independently --Ob-- or -0-, wherein U and V are not the same. In certain embodiments, U and V are independently -CH2- or -S-. In certain embodiments, U and V are both -CH2-. In certain embodiments, U and V are both -S-. In certain embodiments, m is 1 or 2.
• n is an integer selected from 4-12. In certain embodiments, n is an integer selected from 6-10.
• p is 0. In certain embodiments, p is 1.
• q is an integer selected from 2-8. In certain embodiments, q is an integer selected from 4-8.
• Linker is represented by formula II : wherein: W is O orNH; each of R 5 independently is hydrogen, halogen, nitro, cyano, amino, hydroxyl, alkoxy, alkyithio, alkyl, alkenyl, alkynyi, aralkyl, heteroaralkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl; r is an integer selected from 0 to 4;
• A is a 5- to 8-membered heterocycle; and R 6 is absent: or R 6 is alkylene or alkenylene;
• W is O, In certain embodiments, W is NH.
• R 5 is alkoxy, e.g., methoxy.
• r is 2.
• R 6 is absent. In certain embodiments, R 6 is methylene.
• A is a 6-membered heterocycle.
• A is unsubstituted.
• A is substituted with halogen, nitro, cyano, ammo, hydroxyl, alkoxy, alkylthio, alkyl, alkenyl, alkynyl, aralkyl, heteroaralkyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl .
• each instance of R Lipid independently is selected from the group consisting of «-pentyl, «-hexyl, «-heptyl, «-octyl.
• the compound is a compound of formula Ill: wherein
• R’ is derived from a compound selected from the group consisting of s is an integer selected from 1 to 4, as valency permits.
• pro vided are lipidoid nanoparticles, comprising a compound disclosed herein.
• the lipidoid nanoparticle further comprises cholesterol.
• the weight ratio of the compound to the cholesterol is about 2:1 to about 8:1.
• the weight ratio of the compound to the cholesterol Is about 4:1.
• the lipidoid nanoparticle further comprises DOPE, DSPC, DOPC; or DMG-PEG2K; wherein DSPC has the structure:
• DOPE has the structure:
• DOPC has the structure:
• DMG-PEG2K has the structure:
• the lipidoid nanoparticle further comprises DOPE.
• the weight ratio of the compound to the DOPE is about 4: 1 to about 1:1. In certain embodiments, the weight ratio of the compound to the DOPE is about 4:1 or about 1:1.
• the lipidoid nanoparticle disclosed herein further comprises an mRNA.
• the mRNA is green fluorescence protein (GFP) mRNA.
• tiie small molecule is selected from the group consisting of bortezomib, imatinib, gefitinib, erlotmib, afatinib, osimertinib, dacomitinib, daunorubicin hydrochloride, cytarabine, fluorouraeil, irinotecan hydrochloride, vincristine sulfate, methotrexate, paclitaxel, vincristine sulfate, epirubicin, docetaxel, cyclophosphamide, carboplatin, lenalidomide, ibrutinib, abiraterone acetate, enzalutamide, pemetrexed, palbociclib, niiotmib
• the lipidoid nanoparticle has a particle size of about 25 nm to about 1000 nm. In certain embodiments, the lipidoid nanoparticle has a particle size of about 50 nm to about 750 nm. in certain embodiments, the lipidoid nanoparticle has a particle size of about 200 nm to about 500 nm.
• compositions comprising a lipidoid nanoparticle disclosed herein, and one or more pharmaceutically acceptable carriers or excipients.
• HexDMBAH was synthesized using a similar procedure reported previously.
• HexDMBA (5.2 g, 19.5 mmol) and 1, 1, 1-tris(hydroxymethyl)ethane (6.7 g, 55.9 mmol) were dissolved in anhydrous tetrahydrofuran (2.00 mL). 5 A molecular sieves (30 g) and p-toluenesulfonic acid (0.44 g, 2.56 mmol) were then added. The reaction mixture was stirred at room temperature for 12 h, molecular sie ves were filtered out, and solvent was removed via rotary evaporation. HexDMBAH was purified by silica gel column chromatography, with hexane and ethyl acetate as the mobile phase. HexDMBAH was recovered as a white solid (5.4 g; yield -75 %), and its structure was confirmed by 1H NMR (Fig. 9).
• Lipidoids were synthesized from the 016CB.4 tail and amine heads via the Michael addition reaction, using our previously reported procedure.
• amines 75, 76, 77, 78, 80, 81, 82, and 93
• 016CBA 016CBA at a 1/2.2 molar ratio
• amines 113, 306, and 400
• 016CBA 016CBA at a 1/3.3 molar ratio.
• the crude products were purified using a Teledyne ISCO Chromatography purification system, with dichloromethane and methanol as the mobile phase.
• the lipidoids were characterized by 1H MR, l 3 C NMR (Figs. 2C and D), and ESI- MS (Figs. 2E and 10).
• the lipidoid nanoparticles without helper lipids were prepared by dissolving pure 016CBA lipidoids (75-016CBA etc.) in ethanol. Water was added as the selective solvent to trigger the self-assembly process with 10 min of somcation in an ultrasonic water bath. This was followed by dialysis (MWCO 3.5 kDa; Slide-A-Lyzer dialysis cassette; ThermoFisher Scientific) to remove the ethanol.
• GFP mRNA- (purchased from TriLink) loaded lipidoid nanoparticles were fabricated by mixing lipidoid nanoparticles (with or without helper lipids) and mRNA in PBS with a weight ratio of 10/1 (016CBA lipidoid/mRNA). The mixture was incubated at room temperature for 15 min before use.
• 48-well plates were seeded with HeLa ceils at an initial concentration of 2.0 k ceils per well dispersed in 250 pL of DMEM cell culture media and incubated for 24 h. 20 pL of the mRNA-loaded hpidoid nanoparticles were then added into each well The cells were incubated for another 2.4 h at 37 °C and 5% CO2 prior to flow cytometry analysis.
• 96-well plates were seeded with HeLa cells at an initial concentration of 5000 cells per well dispersed in 100 ⁇ L of DMEM cell culture media and incubated for 24 h. Lipidoid nanoparticles were then added into each well. The cells were incubated for another 2.4 h at 37 °C and 5% C02 before MTT reagent (5 mg/mL; in 30 pL PBS) was added. After 4 h incubation, the culture medium was carefully removed and 200 ⁇ L of DMSO was added to each well. After dissolving the fornazan with DMSO solution, the absorbance at 570 nm was determined using a microplate reader (Molecular Devices Spectra Max).
• the pH-responsive cyclic benzylidene acetal-containing hydrophobic tail, O16CBA was first synthesized through a multistep reaction (Fig. 7). Chemical structures of O16CBA (Figs. 2A and 2B) and its precursors (Figs. 8 and 9) were confirmed by NMR analysis.
• a combinatorial library' of cationic lipidoids was then synthesized through the Michael addition reaction by reacting acrylate-containing O16CBA tails with commercially available amine-containing head groups (75, 76, 77 etc.; Figs. IB and 1C). Lipidoids were nomenclated as R-O16CBA (where R represents the amine number) and chemical structures were confirmed by NMR (Figs. 2C and 2D) and ESI-MS spectra (Figs. 2E and 10). The summarized MS data (Fig.
• the cyclic benzylidene acetal moiety in the lipidoid tail can be cleaved through a hydrolysis reaction facilitated by acid. It has been previously reported that 2,4,6- trimethoxyphenyl groups containing cyclic acetal groups can be readily degraded at pH 5.2.3, The R-016CBA lipidoids synthesized in this study were thus expected to degrade in mild acidic conditions, dissociating the self-assembled nanoparticles (Figs. 1A and 1B).
• lipidoids containing two tails such as 75-016CBA and 76-016CBA
• the products R- O16CBA-1, R-O16CBA-0, and HexDMBA can form (Fig. 3A).
• Lipidoids containing more than two tails 113-, 306-, and 400-O 16CB A
• the acid- triggered lipidoid degradation process could be studied in real time.
• lipidoid nanoparticles Due to the self-assembly packing parameters of the lipidoid molecules and the seif- assembly procedures that w ere employed, almost all of our previsouly studied combinatorial lipidoid nanoparticles have the vescidar/liposomal structures.
• the supramolecular structures of lipidoid nanoparticles e.g. morphology, size, etc.
• Hie size and distribution of lipidoid nanoparticles can also be further optimized using microfluidics, mechanical extrusion, and other techniques.
• lipidoid nanoparticles were disrupted and amorphous aggregates around 130 nm resulted (Fig. 4A).
• the same acid-triggered morphological transformations were also observed in 76-016CBA and 77-016CBA LNPs with TEM images (Fig. 13).
• DLS measurements further indicated size variations after acid treatment.
• the hydrodynamic diameter of 75-016CBA decreased from 382 nm to 150 nm after 24 h of incubation in pH 5.0 solution (Fig. 4B). Decrease in average hydrodynamic size was also observed for 76- and 77-O16CBA, which was consistent with the TEM observation results.
• the hydrodynamic size of R-O16CBA measured by DLS was larger than the average size calculated from TEM images. This could be due to the fact that the TEM images were taken under dry status.
• a microenvironment polarity-sensiti ve fluorescent dye (Mile red) was incorporated into the hydrophobic lipidoid bilayer membrane of 75-O16CBA LNPs. Mile red has bright fluorescence emission in the nonpolar environment such as lipidoid bilayer, and reduced fluorescence in polar or aqueous solution. A decrease in fluorescence emission intensity (ca.
• Example 4 R-O16CBA LNPs for mRNA Delivery.
• R-O16CBA LNPs contain R-016CBA only; R-016CBA- F1 LNPs contain R-016CBA, cholesterol, and DOPE at a weight ratio of 4/1/1: R- 016CBA-F2 LNPs contain R-016CBA, cholesterol, and DOPE at a weight ratio of 4/1/4. Cholesterol and DOPE were added because previous studies have shown that these helper lipids can increase the stabilization of nanoparticles, membrane infusion, and cellular internalization.
• GFP mRNA was loaded into different nanopartide formulations by mixing mRNA and LNPs in PBS buffer (R-O16CBA/mRNA ::: 10/1; weight ratio). Most of the combinatial LNPs showed great stability during storage. Furthermore, the stability of LNPs can be further improved by adding small- and macrornolecular excipients (cholesterol, DOPE, PEG-DSPE etc.) into the formulations. It should be noted that in all of the following studies, freshly prepared LNPs were used unless otherwise noted.
• helper lipids greatly improved LNP delivery' efficacy, with multiple LNPs achieving GFP expression comparable to that of Lpf2k.
• all LNPs except for 93-O16CBA-F1 (-2% GFP+ cells), showed higher efficacies than their corresponding R-016CBA formulations.
• Nine of the R-016CBA-F1 formulations had >50% delivery efficiency, with 81-O16CBA-F1 being the highest in producing -93% GFP+ cells.
• Other top formulations such as 76-, 77-, 113-, 306-, and 400- 016CBA-F1 induced delivery ' efficacies of 78-83%.
• 81-Q16CBA-F2 was determined to he the most efficient in this formulated library' as ⁇ 78% GFP+ cells were recorded. Once again, 113-, 306-, and 400-016CBA-F2 were still among the top LNPs, as their delivery efficacies were determined to be 57-73%.
• helper lipids can improve most LNP deli very efficacies.
• R- 016CBA-F2 LNPs which had higher DOPE content, had comparable or slightly lower efficacies than R-016CBA-F1 LNPs.
• 81-016CBA-F1 and -F2 were determined to he the most efficient in the two formulation libraries, and three-tailed LNPs performed well both with and without helper lipids.
• helper lipids like cholesterol and DOPE would result in very similar to slightly lower cytotoxicity of the formulations, which is reasonable considering the excellent cell compatibility' of cholesterol and DOPE etc.
• Lpf2k is highly efficient for mRNA delivery', it is also rather toxic, as only -53% of cells w'ere viable after delivery.
• the newly developed R-016CBA LNPs were less toxic under the same conditions. 306- and 400-016CBA had -70% cell viabilities, and 81- and 113-O16CBA had >90% cell viabilities.

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