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 ofMycobacterium smegmatisInfection

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-snglycero-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 mg/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.

Effect of surface charge of nanohybrids on the intracellular bacterial killing efficiency

“…Vancomycin is the most commonly used antibiotic for S. aureus infections; it inhibits the biosynthesis of peptidoglycan and the assembly of NAM-NAG-polypeptide in the peptidoglycan chain [5,28]. Overall, our experimental data indicated that the encapsulation of Doxy or Vanco into the LPHNPs or BNPs signi cantly reduced the antibacterial e cacy, but may have prolonged activity by extending the time of action of a single dose as compared to that for free antibiotics [29,33] [5,34]. This improved antibiotic entrapment ability of LPHNPs can be explained by the ability of lipids to act as a molecular fence, which prevents drug leakage in the external phase during the solvent evaporation process, consistent with the results of Zhang et al (2008) [16].…”

“…The encapsulation of Doxy or Vanco within the polymeric matrix protected the drug from degradation. Doxycycline is a broad-spectrum tetracycline-based antibiotic, which exerts bactericidal action by inhibiting protein synthesis [29,33].…”

“…The bacteria and macrophages were obtained from our laboratory resources. The bacterial cultures were methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA 1683) and Mycobacterium smegmatis (ATCC 607) [5,33]. The mouse leukemic monocyte macrophage cells RAW 264.7 (ATCCTIB-71) and macrophage cells J774A.1 (ATCC TIB-67) were used for all cell culture experiments [2,34].…”

Novel Synthesis and Characterization of Doxycycline‐Loaded Gold Nanoparticles: The Golden Doxycycline for Antibacterial Applications

“…Previously, Kumar and co‐workers have studied the process of loading of DOXY onto chitosan nanoparticles to improve drug delivery and treatment efficacy . The effectiveness of a gradient loading system is highly dependent on the chemistry of the drug being loaded . Gradient‐loading systems in response to a pH gradient, have been developed for other antibacterial drugs, but differ from the method described in this paper.…”

“…We developed a novel‐loading methodology, resulting in pegylated nanoparticles with higher drug loading and longer retention times than passive aqueous capture . Our loading efficiency is 10–40 times more efficient than the previously published methods …”