title: Assessment of free fatty acids and cholesteryl esters delivered in liposomes as novel class of antibiotic.
abstract: BACKGROUND
  Healthcare associated infections (HAI) with multidrug-resistant (MDR) bacteria continue to be a global threat, highlighting an urgent need for novel antibiotics. In this study, we assessed the potential of free fatty acids and cholesteryl esters that form part of the innate host defense as novel antibacterial agents for use against MDR bacteria.
  
  
  METHODS
  Liposomes of six different phospholipid mixtures were employed as carrier for six different fatty acids and four different cholesteryl esters. Using a modified MIC assay based on DNA quantification with the fluoroprobe Syto9, formulations were tested against Gram-positive and Gram-negative bacteria implicated in HAI. Formulations with MIC values in the low μg/mL range were further subjected to determination of minimal bactericidal activity, hemolysis assay with sheep erythrocytes, and cytotoxicity testing with the human liver cell line HepG2. The potential for synergistic activity with a standard antibiotic was also probed.
  
  
  RESULTS
  Palmitic acid and stearic acid prepared in carrier 4 (PA4 and SA4, respectively) were identified as most active lipids (MIC against MDR Staphylococcus epidermidis was 0.5 and 0.25 μg/mL, respectively; MIC against vancomycin resistant Enterococcus faecalis (VRE) was 2 and 0.5 μg/mL, respectively). Cholesteryl linoleate formulated with carrier 3 (CL3) exhibited activity against the S. epidermidis strain (MIC 1 μg/mL) and a Pseudomonas aeruginosa strain (MIC 8 μg/mL) and lowered the vancomycin MIC for VRE from 32-64 μg/mL to as low as 4 μg/mL. At 90 μg/mL PA4, SA4, and CL3 effected less than 5 % hemolysis over 3 h and PA4 and CL3 did not exhibit significant cytotoxic activity against HepG2 cells when applied at 100 μg/mL over 48 h.
  
  
  CONCLUSIONS
  Our results showed that selected fatty acids and cholesteryl esters packaged with phospholipids exhibit antibacterial activity against Gram-positive and Gram-negative bacteria and may augment the activity of antibiotics. Bactericidal activity could be unlinked from hemolytic and cytotoxic activity and the type of phospholipid carrier greatly influenced the activity. Thus, fatty acids and cholesteryl esters packaged in liposomes may have potential as novel lipophilic antimicrobial agents.
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
authors.forename: Annie H
authors.initials: AH
authors.lastname: Cheung Lam
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Sandoval
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
authors.forename: Ritambhara
authors.initials: R
authors.lastname: Wadhwa
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
authors.forename: Janine
authors.initials: J
authors.lastname: Gilkes
authors.affiliation: Molecular Express, Inc., Rancho Dominguez, CA, USA.
authors.forename: Thai Q
authors.initials: TQ
authors.lastname: Do
authors.affiliation: Molecular Express, Inc., Rancho Dominguez, CA, USA.
authors.forename: William
authors.initials: W
authors.lastname: Ernst
authors.affiliation: Molecular Express, Inc., Rancho Dominguez, CA, USA.
authors.forename: Su-Ming
authors.initials: SM
authors.lastname: Chiang
authors.affiliation: Molecular Express, Inc., Rancho Dominguez, CA, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Kosina
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA.
authors.forename: H
authors.initials: H
authors.lastname: Howard Xu
authors.affiliation: Molecular Express, Inc., Rancho Dominguez, CA, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Fujii
authors.affiliation: Department of Biological Sciences, California State University Los Angeles, 5151 State University Drive, Los Angeles, CA, 90032, USA. eporter@calstatela.edu.
authors.forename: Edith
authors.initials: E
authors.lastname: Porter
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002788:Cholesterol Esters / Q000494:pharmacology*; D003428:Cross Infection / Q000188:drug therapy; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004338:Drug Combinations; D004339:Drug Compounding; D024901:Drug Resistance, Multiple, Bacterial; D004357:Drug Synergism; D013293:Enterococcus faecalis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D004912:Erythrocytes / Q000187:drug effects; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006461:Hemolysis / Q000187:drug effects; D056945:Hep G2 Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008826:Microbial Sensitivity Tests; D009930:Organic Chemicals; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D012756:Sheep; D013212:Staphylococcus epidermidis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D014640:Vancomycin / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D002788:Cholesterol Esters; D004269:DNA, Bacterial; D004338:Drug Combinations; D005230:Fatty Acids, Nonesterified; D005456:Fluorescent Dyes; D008081:Liposomes; D009930:Organic Chemicals; C103389:SYTO 9; D014640:Vancomycin
keywords: Antibiotic; Antimicrobial lipids; Drug delivery; HAI; Innate immunity; Liposomes; Multidrug-resistance