Skip to main content
SpringerLink
Log in

Inhalable Levofloxacin Liposomes Complemented with Lysozyme for Treatment of Pulmonary Infection in Rats: Effective Antimicrobial and Antibiofilm Strategy

  • Research Article
  • Published:
AAPS PharmSciTech Aims and scope Submit manuscript

Abstract

Treatment of bacterial infections becomes increasingly complicated due to increasing bacterial resistance and difficulty in developing new antimicrobial agents. Emphasis should be laid on improvising the existing treatment modalities. We studied the improved antimicrobial and antibiofilm activity of levofloxacin (LFX) and lysozyme (LYS) in microbiological studies. LFX at sub-minimum inhibitory concentration with LYS eradicated > 85% of preformed biofilm. LFX was actively loaded into the liposomes using pH gradient method and was spray-dried with LYS solution. Percent entrapment of LFX in liposome was > 80% and prolonged cumulative release of 85% LFX at the end of 12 h. In vitro lung deposition study and solid-state characterization for spray dried LFX liposome in combination with LYS (LFX liposome-LYS) was performed. Co-spray dried product had mass median aerodynamic diameter ranging < 5 μm. In pharmacodynamic study, Staphylococcus aureus infected rats were treated with LFX liposome-LYS. Lungs, bronchoalveolar lavage fluid (BALF), and nasal fluid were evaluated for microbial burden. Expression of cytokine levels in BALF and serum were also studied by ELISA. In addition, mRNA expression for lung inflammatory mediators and lung myeloperoxidase activity were carried out. Further, lungs and histological changes were observed grossly. Untreated infected rat lungs demonstrated higher mRNA expression for inflammatory markers, cytokine levels, and microbial load compared to vehicle control. Conversely, LFX liposome-LYS significantly abated these adverse repercussions. Histology findings were also in agreement of above. Acute toxicity study revealed safeness of LFX liposome-LYS. Our findings confirm LFX liposome-LYS exhibited prolonged, improved antibiofilm and antimicrobial efficacy in treating S. aureus infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12

Similar content being viewed by others

References

  1. Lister JL, Horswill AR. Staphylococcus aureus biofilms: recent developments in biofilm dispersal. Front Cell Infect Microbiol. 2014;4(178):1–9.

    Google Scholar 

  2. Zhou QT, Leung SSY, Tang P, Parumasivam T, Loh ZH, Chan HK. Inhaled formulations and pulmonary drug delivery systems for respiratory infections. Adv Drug Deliv Rev. 2015;85:83–99.

    Article  CAS  PubMed  Google Scholar 

  3. Waters V, Smyth A. Cystic fibrosis microbiology: advances in antimicrobial therapy. J Cyst Fibros. 2015;14(5):551–60.

    Article  CAS  PubMed  Google Scholar 

  4. Zakaria AS, Melake NA, Baky NA, El Rasheed NM, Ibrahim NH. In vitro and in vivo studies of antibacterial effect of ceftriaxone moxifloxacin combination against methicillin resistant Staphylococcus aureus biofilms formed on biomedical implants. Afr J Microbiol Res. 2012;6(25):5399–409.

    CAS  Google Scholar 

  5. Archer N, Mazaitis M, Costerton J, Leid J, Powers M, Shirtliff M. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence. 2011;2:445–59.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Liu H, Zhao Y, Zhao D, Gong T, Wu Y, Han H, et al. Antibacterial and anti-biofilm activities of thiazolidione derivatives against clinical staphylococcus strains. Emerg Microbes Infect. 2015;4(1):e1. https://doi.org/10.1038/emi.2015.1.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Estela CRL, Alejandro PR. Biofilms: a survival and resistance mechanism of microorganisms. In: Pana M, editors. Antibiotic resistant bacteria—a continuous challenge in the new millennium. InTech; 2012. ISBN: 978–953–51-0472-8.

  8. Donlan RM. Role of biofilms in antimicrobial resistance. ASAIO J. 2000;46(6):S47–52.

    Article  CAS  PubMed  Google Scholar 

  9. Davies D. Understanding biofilm resistance to antibacterial agents. Nat Rev Drug Discov. 2003;2(2):114–22.

    Article  CAS  PubMed  Google Scholar 

  10. Du J, Bandara H, Du P, Huang H, Hoang K, Nguyen D, et al. Improved biofilm antimicrobial activity of polyethylene glycol conjugated tobramycin compared to tobramycin in Pseudomonas aeruginosa biofilms. Mol Pharm. 2015;12(5):1544–53.

    Article  CAS  PubMed  Google Scholar 

  11. Hou Y, Wang Z, Zhang P, Bai H, Sun Y, Duan J, et al. Lysozyme associated liposomal gentamicin inhibits bacterial biofilm. Int J Mol Sci. 2017;18(4):784.

    Article  PubMed Central  Google Scholar 

  12. Bjarnsholt T, Ciofu O, Molin S, Givskov M, Høiby N. Applying insights from biofilm biology to drug development—can a new approach be developed? Nat Rev Drug Discov. 2013;12(10):791–808.

    Article  CAS  PubMed  Google Scholar 

  13. Martinelli A, Bakry A, D’Ilario L, Francolini I, Piozzi A, Taresco V. Release behavior and antibiofilm activity of usnic acid-loaded carboxylated poly (l-lactide) microparticles. Eur J Pharm Biopharm. 2014;88(2):415–23.

    Article  CAS  PubMed  Google Scholar 

  14. Murillo O, Domenech A, Garcia A, Tubau F, Cabellos C, Gudiol F, et al. Efficacy of high doses of levofloxacin in experimental foreign-body infection by methicillin-susceptible Staphylococcus aureus. Antimicrob Agents Chemother. 2006;50(12):4011–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Penta J, Jannu KK, Musthyala R. Antimicrobial studies of selected antibiotics and their combination with enzymes. Int J Pharm Pharm Sci. 2010;2(3):43–4.

    CAS  Google Scholar 

  16. Sakharkar MK, Jayaraman P, Soe WM, Chow V, Sing LC, Sakharkar KR. In vitro combinations of antibiotics and phytochemicals against Pseudomonas aeruginosa. J Microbiol Immunol Infect. 2009;42(5):364–70.

    CAS  PubMed  Google Scholar 

  17. Van Vuuren S, Suliman S, Viljoen A. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Lett Appl Microbiol. 2009;48(4):440–6.

    Article  PubMed  Google Scholar 

  18. Venkataraman M, Nagarsenker M. Silver sulfadiazine nanosystems for burn therapy. AAPS PharmSciTech. 2013;14(1):254–64.

    Article  CAS  PubMed  Google Scholar 

  19. Darouiche RO, Mansouri MD, Gawande PV, Madhyastha S. Antimicrobial and antibiofilm efficacy of triclosan and DispersinB® combination. J Antimicrob Chemother. 2009;64(1):88–93.

    Article  CAS  PubMed  Google Scholar 

  20. Seifeldeen DW. Evaluation of the combination of N-acetylcysteine and or sodium salicylate with ciprofloxacin on bacterial adhesion and biofilm formation on urinary catheters. IAJAA. 2012;2(1):1–12.

    Article  Google Scholar 

  21. Gao X, Yao G, Guo N, An F, Guo X. A simple and rapid high performance liquid chromatography method to determine levofloxacin in human plasma and its use in a bioequivalence study. Drug Discover Ther. 2007;1(2):136–40.

    CAS  Google Scholar 

  22. Aguilar MI. HPLC of peptides and proteins. vol. 251. New York: Springer; 2004. p. 3–8.

    Google Scholar 

  23. Liao YH, Brown MB, Martin GP. Turbidimetric and HPLC assays for the determination of formulated lysozyme activity. J Pharm Pharmacol. 2001;53(4):549–54.

    Article  CAS  PubMed  Google Scholar 

  24. Zhang X, Sun P, Bi R, Wang J, Zhang N, Huang G. Targeted delivery of levofloxacin-liposomes for the treatment of pulmonary inflammation. J Drug Target. 2009;17(5):399–407.

    Article  CAS  PubMed  Google Scholar 

  25. Pathak P, Dhawan V, Magarkar A, Danne R, Govindarajan S, Ghosh S, et al. Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: in silico modeling, in vitro and in vivo evaluation. Int J Pharm. 2016;509(1):149–58.

    Article  CAS  PubMed  Google Scholar 

  26. MacLeod DL, Barker LM, Sutherland JL, Moss SC, Gurgel JL, Kenney TF, et al. Antibacterial activities of a fosfomycin/tobramycin combination: a novel inhaled antibiotic for bronchiectasis. J Antimicrob Chemother. 2009;64:829–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Marier JF, Lavigne J, Ducharme MP. Pharmacokinetics and efficacies of liposomal and conventional formulations of tobramycin after intratracheal administration in rats with pulmonary Burkholderia cepacia infection. Antimicrob Agents Chemother. 2002;46(12):3776–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Omri A, Beaulac C, Bouhajib M, Montplaisir S, Sharkawi M, Lagace J. Pulmonary retention of free and liposome-encapsulated tobramycin after intratracheal administration in uninfected rats and rats infected with Pseudomonas aeruginosa. Antimicrob Agents Chemother. 1994;38(5):1090–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–8.

    Article  CAS  PubMed  Google Scholar 

  30. Naikwade SR, Bajaj AN, Gurav P, Gatne MM, Soni PS. Development of budesonide microparticles using spray-drying technology for pulmonary administration: design, characterization, in vitro evaluation, and in vivo efficacy study. AAPS PharmSciTech. 2009;10(3):993–1012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lee SH, Teo J, Heng D, Ng WK, Chan HK, Tan RB. Synergistic combination dry powders for inhaled antimicrobial therapy: formulation, characterization and in vitro evaluation. Eur J Pharm Biopharm. 2013;83(2):275–84.

    Article  CAS  PubMed  Google Scholar 

  32. Cisani G, Varaldo PE, Grazi G, Soro O. High-level potentiation of lysostaphin anti-staphylococcal activity by lysozyme. Antimicrob Agents Chemother. 1982;21(4):531–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chung W, Hancock RE. Action of lysozyme and nisin mixtures against lactic acid bacteria. Int J Food Microbiol. 2000;60(1):25–32.

    Article  CAS  Google Scholar 

  34. Pellegrini A, Thomas U, Fellenberg RV, Wild P. Bactericidal activities of lysozyme and aprotinin against Gram-negative and Gram-positive bacteria related to their basic character. J Appl Bacteriol. 1992;72(3):180–7.

    Article  CAS  PubMed  Google Scholar 

  35. Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetration limitation in Klebsiella pneumoniae biofilm resistance to ampicillin and ciprofloxacin. Antimicrob Agents Chemother. 2000;44(7):1818–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Wecke J, Lahav M, Ginsburg I, Giesbrecht P. Cell wall degradation ofStaphylococcus aureus by lysozyme. Arch Microbiol. 1982;131(2):116–23.

    Article  CAS  PubMed  Google Scholar 

  37. Samaranayake Y, Cheung B, Parahitiyawa N, Seneviratne C, Yau J, Yeung K, et al. Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces. Arch Oral Biol. 2009;54(2):115–26.

    Article  CAS  PubMed  Google Scholar 

  38. Quon BS, Goss CH, Ramsey BW. Inhaled antibiotics for lower airway infections. Ann Am Thorac Soc. 2014;11(3):425–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Cipolla D, Blanchard J, Gonda I. Development of liposomal ciprofloxacin to treat lung infections. Pharmaceutics. 2016;8(1):1–31.

    Article  Google Scholar 

  40. Cipolla D, Gonda I, Chan H-K. Liposomal formulations for inhalation. Ther Deliv. 2013;4(8):1047–72.

    Article  CAS  PubMed  Google Scholar 

  41. Simon L, Gauvin F, Amre DK, Saint-Louis P, Lacroix J. Serum procalcitonin and C-reactive protein levels as markers of bacterial infection: a systematic review and meta-analysis. Clin Infect Dis. 2004;39(2):206–17.

    Article  CAS  PubMed  Google Scholar 

  42. Feng X, Maze M, Koch LG, Britton SL, Hellman J. Exaggerated acute lung injury and impaired antibacterial defenses during Staphylococcus aureus infection in rats with the metabolic syndrome. PLoS One. 10(5):e0126906. https://doi.org/10.1371/journal.pone.0126906.

  43. Yao L, Berman JW, Factor SM, Lowy FD. Correlation of histopathologic and bacteriologic changes with cytokine expression in an experimental murine model of bacteremic Staphylococcus aureus infection. Infect Immun. 1997;65(9):3889–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Summah H, Qu J-M. Biomarkers: a definite plus in pneumonia. Mediat Inflamm. 2009;2009:1–9.

    Article  Google Scholar 

  45. Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood. 1998;92(9):3007–17.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research work was supported by Indian Council of Medical Research (ICMR), File No. AMR/4/2011- ECD-I, New Delhi, India.

Author information

Authors and Affiliations

  1. Department of Pharmaceutics, Bombay College of Pharmacy, Kalina, Santacruz (East), Mumbai, 400098, India

    Purnima V. Gupta, Abhijit M. Nirwane & Mangal S. Nagarsenker

  2. College of Pharmacy, University of Minnesota, Duluth, Minnesota, USA

    Abhijit M. Nirwane

Authors
  1. Purnima V. Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abhijit M. Nirwane
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mangal S. Nagarsenker
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mangal S. Nagarsenker.

Ethics declarations

Protocol of in vivo study was approved by the Institution Animal Ethics Committee Bombay College of Pharmacy and carried out in accordance with the guidelines of Committee for the Purpose and Supervision of Animal Experiments.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gupta, P.V., Nirwane, A.M. & Nagarsenker, M.S. Inhalable Levofloxacin Liposomes Complemented with Lysozyme for Treatment of Pulmonary Infection in Rats: Effective Antimicrobial and Antibiofilm Strategy. AAPS PharmSciTech 19, 1454–1467 (2018). https://doi.org/10.1208/s12249-017-0945-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1208/s12249-017-0945-4

KEY WORDS

Search

Navigation