title: A Novel Loading Method for Doxycycline Liposomes for Intracellular Drug Delivery: Characterization of In Vitro and In Vivo Release Kinetics and Efficacy in a J774A.1 Cell Line Model of Mycobacterium smegmatis Infection.
abstract: Doxycycline (doxy) is used in treating intracellular and extracellular infections. Liposomal (LE) antibiotics allow low-frequency dosing and extended efficacy compared with standard (STD) formulations. We developed a novel sulfuric acid-loading method for doxycycline liposomes (LE-doxy). We hypothesized that a single s.c. injection of LE-doxy would be detectable in serum for at least 2 weeks at concentrations equal to or better than STD-doxy and would be bactericidal in an in vitro Mycobacterium smegmatis infection of J774A.1 macrophage cells. Liposomes were encapsulated by sulfuric acid gradient loading, and release kinetics were performed in vitro and in vivo. LE-doxy made using 8.25 mg/ml doxycycline loaded for 24 hours achieved 97.77% capture in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 43.87% in sphingomyelin (sphing). Rats were injected s.c. with 50 mg/kg LE-doxy or 5 mg/kg STD-doxy, and serial blood samples were collected. Pharmacokinetics were analyzed using high-performance liquid chromatography. Liver and injection site skin samples were collected at euthanasia (4 weeks postinjection). Minimal histologic tissue reactions occurred after injection of STD (nonliposomal), DPPC, or sphing-doxy. DPPC-doxy had slightly faster in vitro leakage than sphing liposomes, although both were detectable at 264 hours. The mean residence time for DPPC was the highest (111.78 hours), followed by sphing (56.00 hours) and STD (6.86 hours). DPPC and sphing-doxy were detectable at 0.2 μg/ml in serum at 336 hours postadministration. LE-doxy was not toxic to J774A.1 cells in vitro and produced inhibition of viable Mycobacterium smegmatis at 24 and 48 hours. LE-doxy will require further testing in in vivo infection models.
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.) franklin@rarc.wisc.edu.
authors.forename: Rebekah K
authors.initials: RK
authors.lastname: Franklin
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Marcus
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Adel M
authors.initials: AM
authors.lastname: Talaat
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Butch K
authors.initials: BK
authors.lastname: KuKanich
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Ruth
authors.initials: R
authors.lastname: Sullivan
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Krugner-Higby
authors.affiliation: Pharmacology, Clinical, Analytical, and Toxicological Services and the Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas (B.K.K.); and Departments of Surgical Sciences (L.A.K.-H.), Pathobiological Sciences (R.S.), and Comparative Biosciences (A.M.T., S.A.M.); School of Veterinary Medicine (R.S.); School of Pharmacy (T.D.H.); and Research Animal Resources Center, University of Wisconsin, Madison, Wisconsin (R.K.F., L.A.K.-H., R.S.).
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Heath
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000818:Animals; D000900:Anti-Bacterial Agents / Q000008:administration & dosage* / Q000737:chemistry / Q000627:therapeutic use; D002460:Cell Line; D002626:Chemistry, Pharmaceutical; D004318:Doxycycline / Q000008:administration & dosage* / Q000737:chemistry / Q000627:therapeutic use; D004339:Drug Compounding; D016503:Drug Delivery Systems; D005260:Female; D007279:Injections, Subcutaneous; D008081:Liposomes; D008297:Male; D008826:Microbial Sensitivity Tests; D009165:Mycobacterium Infections, Nontuberculous / Q000188:drug therapy* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects; D010316:Particle Size; D051381:Rats; D013109:Sphingomyelins / Q000737:chemistry; D013464:Sulfuric Acids / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D008081:Liposomes; D013109:Sphingomyelins; D013464:Sulfuric Acids; D015060:1,2-Dipalmitoylphosphatidylcholine; D004318:Doxycycline