Synergy-based delivery system for combating sexually-transmitted bacterial infections: liposomal azithromycin-in-chitosan hydrogel

Assoc. Prof. Dr. Z. Vanić1), University Zagreb/Croatia

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Associate Prof. Dr. Željka VanićDepartment of Pharmaceutical Technology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia

People involved


Topical vaginal therapy presents the non-invasive and direct delivery of drugs to the site of action at lower doses while escaping adverse side effects caused by systemic drug administration. To achieve efficient, safe and targeted vaginal therapy of sexually-transmitted bacterial diseases and recurrent bacterial vaginosis, we are proposing development of a biodegradable and biocompatible synergy-based delivery system containing liposomal azithromycin incorporated into chitosan hydrogel. Such formulation would allow combating of bacteria and bacterial vaginosis biofilm by dual antimicrobial action of liposomally encapsulated azithromycin and chitosan-based hydrogel. Encapsulation into liposomes is expected to increase azithromycin solubility and allow fusion with bacterial cells, resulting in improved localized effect even at lower doses in comparison with the free drug. Incorporation into the chitosan hydrogel would enhance the residence time of liposomal azithromycin on vaginal mucosa and further increase the antimicrobial action against bacteria and bacterial vaginosis biofilm by the antimicrobial activity of the chitosan itself. Using biodegradable and biocompatible synergy-acting antibacterial delivery system presents a novel approach for the targeting sexually-transmitted genital infections and combating bacterial resistance.

The objective of this project is the development of efficient, safe, and stable liposome-based delivery system containing azithromycin destined for improved topical vaginal therapy of sexually-transmitted diseases and bacterial vaginosis. The system utilizes the synergy between antimicrobial efficacy of chitosan as a suitable vehicle for vaginal administration and liposomal formulation able to enhance the bioavailability of azithromycin through the superior properties of phospholipids as drug carrier.

Benefit for the community

The proposed research has a great significance for improvement of public health. It is addressing two important health issues: targeting biofilm-related infections and women’s health. The search for new modes for targeting infection diseases, particularly biofilm-related, in a form of nanomedicine, is focus of many EU calls. Similarly, efficient combating of sexually transmitted infections is focus of many national health policies. Unfortunately, the incidence of sexually transmitted diseases is increasing and with antimicrobial resistances among antibiotics used becoming alarmingly serious issue. We are expecting that our novel concept of biodegradable and biocompatible synergy-based delivery system will enable advanced topical therapy of sexually-transmitted bacterial infections at lower doses than by systemic action. It will highlight the importance of phospholipid-based nanomedicine, confirming its potential not only in targeting the biofilm-related infections via parenteral and skin route but also via vaginal route. Additionally, the proposed technology is rather simple, scalable and appropriate for industrial implementation.


In the first part of the project, we have optimized liposome composition, and we have obtained vesicles with desired encapsulation efficiency, bilayer rigidity/elasticity, size and surface properties. All the prepared conventional and elastic liposomes were characterized for physicochemical properties and in vitro azithromycin release, where three specific compositions (CL-3, PGL-2, and DPGL-2) were selected as optimal liposomes for further research. Suitable viscosity of liposomes for vaginal application was achieved by incorporation into chitosan hydrogel of appropriate pH value. Selected liposomes-in-hydrogel formulations were investigated for texture properties and in vitro azithromycin release.

In the second part of the project in vitro release studies from liposomal hydrogels were completed as well as storage stability studies. We have performed ex vivo permeation studies of optimized liposomes and demonstrated localization of the drug in the vaginal tissue in comparison to control (free drug solution). In vitro antibacterial studies of azithromycin-loaded liposomes tested on several E. coli strains, both planktonic and biofilm, showed increased activity of PGL-2 and CL-3 formulations in comparison to free azithromycin and DPGL-2 against planktonic bacteria and prevention of biofilm formation. In vitro cytotoxicity studies proved azithromycin-loaded CL-3 as the most tolerable nano-formulation with the cervical cells, while DPGL-2, even without the entrapped drug (empty liposomes) were shown to exhibit cytotoxic effects, particularly at higher lipid concentrations.

Taking into considerations satisfactory entrapment of azithromycin, the prolonged drug release profile, appropriate physical stability, antimicrobial and cytotoxicity studies, as well as increased local drug effect (based on ex vivo permeation studies), CL-3 and PGL-2 would be appropriate for the further investigations.

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Publications derived from the project:
Vanić Ž, Rukavina Z, Manner S, Fallarero A, Uzelac L, Kralj M, Amidžić Klarić D, Bogdanov A, Raffai T, Virok DP, Filipović-Grčić J, Škalko-Basnet N, 2019
Azithromycin-liposomes as a novel approach for localized therapy of cervicovaginal bacterial infections
Int. J. Nanomed. 14, 5957-5976
Bogdanov A, Janovák L, Vraneš J, Meštrović T, Ljubin-Sternak S, Cseh Z, Endrész V, Burián K, Vanić Ž, Virok DP, 2021
Liposomal Encapsulation Increases the Efficacy of Azithromycin against Chlamydia trachomatis
Pharmaceutics 14, 36
Čačić A, Amidžić Klarić D, Keser S, Radiković M, Rukavina Z, Jøraholmen MW, Uzelac L, Kralj M, Škalko-Basnet N, Šegvić Klarić M, Vanić Ž, 2023
A Novel Approach for the Treatment of Aerobic Vaginitis: Azithromycin Liposomes-in-Chitosan Hydrogel
Pharmaceutics 15, 1356
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