Palmitic acid

Ethyl acetate, iron(II) sulphate, isopropanol, dimethyl sulfoxide (DMSO), methanol and chloroform were purchased from Thermo Fisher Scientific (Leicester, UK), while low-density lipoprotein (LDL), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), bovine serum albumin (BSA) and thiobarbituric acid (TBA) were acquired from Merck (Darmstadt, Germany). Quercetin, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase assay kit and formaldehyde were from Sigma-Aldrich, Merck (St. Louis, MO, USA). Cell culture medium such as Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS) and penicillin-streptomycin were from Nacalai Tesque (Tokyo, Japan). Palmitic acid and Oil Red O (ORO) were from Acros Organics (New Jersey, NJ, USA). Lastly, phosphate buffer saline (PBS) was from Oxoid (Basingstoke, UK).

Flucloxacillin sodium (FS) monohydrate was purchased from Aspen Pharmacare Australia Pty Ltd. (St Leonards, NSW, Australia). Eudragit^® EPO was gifted by Evonik (Evonik Industries, Essen, Germany). Palmitic acid was purchased from Acros Organics (Morris Plains, NJ, USA), ethanol was purchased from Chem-Supply Pty Ltd. (Gillman, SA, Australia), and acetone was purchased from RCI Labscan (Bangkok, Thailand). Potassium dihydrogen phosphate was purchased from Unilab (Sydney, NSW, Australia). Unless specified, all chemicals and reagents were of analytical grade. This study utilised five solvent systems comprising ethanol:acetone at the following volume ratios: 1:0, 0.75:0.25, 0.5:0.5, 0.25:0.75, and 0:1.

Stearic acid (97%), myristic acid (99%), oleic acid (extra pure), and palmitoleic acid (> 98%) were purchased from Exir (Austria). Palmitic acid (98%) and linoleic acid (99%) were purchased from Acros Organic (USA). Cholesterol (> 99%) and butylhydroxytoluene (98%) were purchased from Sigma (Germany). Also, 1,8-cineole (99%) was supplied by Alfa Aesar (Germany). Diazepam was supplied by Changzhou Siyao Pharmaceutical Company (Changzhou, China). All other solvents and reagents were of analytical or HPLC grade.

Pure standard of caffeine, myristic acid (99%), palmitic acid (99%), acetyl chloride (98%), trolox (97%), and ethanol (96 vol%, not denaturated) were obtained from ACROS ORGANICS. Acetonitrile, ethyl acetate, and potassium hydroxide were supplied by Fisher Chemical (Waltham, MA, USA). Pure standard of 3-caffeoylquinic acid (CAS 327-97-9), oleic acid (99%), stearic acid (98%), Vitamin E acetate (97%), DPPH (95%), and cyclohexane were purchased from Alfa Aesar (Haverhill, MA, USA). Caprylic acid (99%) and arachidic acid (99%) were obtained from Sigma Aldrich (St. Louis, MO, USA). Capric acid (99%) and lauric acid (99%) were obtained from Interchim (Montlucon, France). Methanol (≥99.9%) was obtained from Honeywell (Nawabash, IN, USA). Hydrochloric acid (37%) was obtained from Roth. Linoleic acid (99%), and n-hexane (>99.9%) were obtained from Fluka. All solvents and reagents were of analytical grade and used as received.

Methanol (Optima grade), formic acid (Optima grade), ammonium acetate, hexane, acetonitrile, and ammonium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Oxalyl chloride, oleic acid, erucic acid, petroselaidic acid, heptane HPLC grade, methyl-tert-butyl-ether HPLC grade, isopropanol HPLC grade, acetic acid, and anhydrous dichloromethane were from Sigma Aldrich (St Louis, MO, USA). N, N-dimethylformamide, heptadecanoic acid, and eicosanoic acid were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Lauric acid, myristic acid, palmitic acid, stearic acid, and docosanoic acid were purchased from Acros Organics (New Jersey, USA). Elaidic acid was from MP Biomedical Inc. (Solon, OH, USA) and linoleamide was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). Stearoyl ethanolamine, oleoylglycine, linoleoylglycine, palmitoylglycine, arachidonoylglycine, and arachidoylglycine were purchased from Cayman Chemicals (Ann Arbor, MI, USA). 1 monopalmitoyl-rac-glycerol (MAG) and tristearin (TAG) were from Sigma (St. Louis, MO) 99% purity and 1,2-dipalmitoyl-rac-glycerol (DAG) was from MP Biomedicals (Solon, Ohio).

As previously reported [19 , 20 ], fatty acids and PLL were used to synthesize fatty acid-PLLs. The saturated fatty acids used were caproic acid (C₆, Sigma-Aldrich), caprylic acid (C₈, Acros Organics), capric acid (C₁₀, Sigma-Aldrich), lauric acid (C₁₂, Acros Organics), myristic acid (C₁₄, Acros Organics), and palmitic acid (C₁₆, Acros Organics), whereas the unsaturated fatty acids used were palmitoleic acid (C_16:1, Fluka), oleic acid (C_18:1, Sigma-Aldrich), and linoleic acid (C_18:2, Sigma-Aldrich). The cationic α-PLL (Alamanda Polymers; molecular mass around 21,000 Da) is a polypeptide composed of repeated structural units of 100 lysine residues and PLL was analyzed by gas chromatography and nuclear magnetic resonance for molecular mass determination and polydispersity index.
Each fatty acid was activated by ethyl chloroformate (ECF), which acts as a coupling agent by activating the carboxylic group of the fatty acids. The intermediate compound was linked to the ε-amine group of the lysine residue through amide-bond formation [21 (link)]. This was then purified by dialysis in buffer solutions and lyophilized for analysis. Thus, the final compounds were called fatty acid-PLLs. For example, lauric acid linked to PLL was called lauryl-PLL (see Figure 1).