A new preparation method for preparation of liposomes-in-hydrogels primed for treatment of skin diseases

Poster presented at the Liposome Research Days 2014: Living Innovation, at the Technical University of Denmark, Copenhagen, 4-7 August 2014Liposomes are spherical vesicles that form spontaneously when phospholipids are dispersed in an aqueous medium. However, to get liposomes with a more unimodal size distribution, liposomes are usually processed further to reduced size and lammellarity, typically by extrusion, sonication or homogenization. Liposome formulations for topical application needs a vehicle to assure the desirable retention and adhesion of the drug-loaded liposomes onto the skin, and for this, hydrogels are regarded as promising systems. The hydrogel “soluble beta-glucan” (SBG) has caught our attention since it also promotes wound healing on its own. Dual asymmetric centrifuge (DAC) utilizes a unique combination of two contra rotating movements of the sample-holder, which results in shear forces that efficiently homogenize and gives a size reduction of liposomes. We have investigated the use of DAC for both use in liposome size reduction and for further mixing of liposomes into hydrogels

Successful co-encapsulation of benzoyl peroxide and chloramphenicol in liposomes by a novel manufacturing method - dual asymmetric centrifugation

Accepted manuscript version. Published version available at http://dx.doi.org/10.1016/j.ejps.2016.11.017 Encapsulation of more than one active pharmaceutical ingredient into nanocarriers such as liposomes is an attractive approach to achieve a synergic drug effect and less complicated dosing schedules in multi-drug treatment regimes. Liposomal drug delivery in acne treatment may improve drug efficiency by targeted delivery to pilosebaceous units, reduce adverse effects and improve patient compliance. We therefore aimed to co-encapsulate benzoyl peroxide (BPO) and chloramphenicol (CAM) into liposomes using the novel liposome processing method – dual asymmetric centrifugation (DAC). Liposomes were formed from soybean lecithin, propylene glycol and distilled water (2:1:2 w/v/v ratio), forming a viscous liposome dispersion. Liposomes containing both drugs (BPO-CAM-Lip), single-drug (BPO-Lip and CAM-Lip), and empty liposomes were prepared. Drug entrapment of BPO and CAM was determined by a newly developed HPLC method for simultaneous detection and quantification of both drugs. Encapsulation of around 50% for BPO and 60% for CAM respectively was obtained in both single- drug encapsulated formulations (BPO-Lip and CAM-Lip) and co-encapsulated formulations (BPO- CAM-Lip). Liposome sizes were comparable for all liposome formulations, ranging from 130 to 150 nm mean diameter, with a polydispersity index < 0.2 for all formulations. CAM exhibited a sustained release from all liposomal formulations, whereas BPO appeared retained within the liposomes. BPO retention could be attributed to its poor solubility. However, HaCaT cell toxicity was found dependent on BPO released from the liposomes. In the higher concentration range (4% v/v), liposomal formulations were less cytotoxic than the corresponding drug solutions used as reference. We have demonstrated that DAC is a fast, easy, suitable method for encapsulation of more than one drug within the same liposomes

A new preparation method for preparation of liposomes-in-hydrogels primed for treatment of skin diseases

Liposomes are spherical vesicles that form spontaneously when phospholipids are dispersed in an aqueous medium. However, to get liposomes with a more unimodal size distribution, liposomes are usually processed further to reduced size and lammellarity, typically by extrusion, sonication or homogenization. Liposome formulations for topical application needs a vehicle to assure the desirable retention and adhesion of the drug-loaded liposomes onto the skin, and for this, hydrogels are regarded as promising systems. The hydrogel “soluble beta-glucan” (SBG) has caught our attention since it also promotes wound healing on its own. Dual asymmetric centrifuge (DAC) utilizes a unique combination of two contra rotating movements of the sample-holder, which results in shear forces that efficiently homogenize and gives a size reduction of liposomes. We have investigated the use of DAC for both use in liposome size reduction and for further mixing of liposomes into hydrogels

Development of targeted liposome drug delivery vehicle

Coating of liposomes with polyethylene glycol (PEG) has proven to prolong the circulation time of liposomes in the blood stream. PEG prevents the binding of opsonins and subsequent uptake of the liposomes by mononuclear phagocytic system (MPS). The reduction in clearance of PEGylated liposomes from the circulation improve the bioavailability of the liposomes in the blood and increase the chance of liposomes being accumulated in tumor tissue by the enhanced permeability and retention effect (EPR). The aim of this study was therefore to investigate the incorporation and retention ability of PEGylated liposome formulations of the anticancer agent Camptothecin (CPT), and further try to develop an immunoliposomal formulation of CPT targeting the EGFR receptors on the surface of colorectal cancer cells. The results from the incorporation and retention studies showed that the formulation consisting of 79 % egg phosphatidylcholine (EPC), 20 % 1,2-di-oleyl-3- trimethylammonium-propane (DOTAP) and 1 % PEG conjugated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) clearly showed the highest incorporation of CPT and the most stable retention ability in different media including medium containing erythrocytes. Formulations with dimethyldioctadecylammonium (DDAB) and 4-(Dodecyloxy)-benzoic acid (DB) demonstrated lower incorporation ability and were slightly more unstable in regard to retention of CPT compared to the formulation with DOTAP. Based on these results, the PEGylated formulation with DOTAP was chosen as the basis for the immunoliposomal formulation. Bovine serum albumin (BSA) and EGFR antibodies were conjugated to the liposome surface by the inclusion of 1 % maleimide terminated DSPE-PEG into the liposome membrane. The loss of CPT from the liposomes observed during conjugation was however significant. In conclusion the presence of PEG on the liposome surface and DOTAP in the liposome bilayer seems to give the most promising PEGylated CPT formulation, which could possibly be a candidate for further in vivo studies. For the immunoliposomes, the attachment of antibodies on the surface was successful. However, due to loss of CPT during the conjugation process the method used is not optimal for this CPT liposome formulation, and further studies are needed to find a more suitable preparation method or a more stable immunoliposome formulation

Dermal Application of Chloramphenicol - The Effect of Liposomes and Chitosan Hydrogel Formulations on ex vivo Permeation and Antimicrobial Activity

Liposomes solubilize lipophilic drugs in the phospholipid membrane, and entrap hydrophilic drugs in its aqueous core. These vesicles provide a sustained- and targeted- drug release, and protect from degradation. Liposomes retain the drug onto/in the skin and improve skin drug deposition. Due to their liquid nature, a secondary vehicle, such as a hydrogel, is needed to obtain a necessary retention and bioadhesion onto the skin surface. Chloramphenicol (CAM) is an antimicrobial drug that, due to its bone marrow toxicity, is mainly applied in the treatment of eye and ear infections. When applied dermally to treat skin infections, systemic absorption should be avoided. The chitosan (CS) hydrogel with inherent antimicrobial activity might improve the antimicrobial activity. In this study, we investigated the effect of both the liposomal carrier and the CS-hydrogel on the retention and permeation of CAM through pig skin ex vivo. Four different formulations were compared; CAM aqueous solution (CAM-Sol), CAM in a liposome dispersion (CAM-Lip), CAM dissolved in chitosan hydrogel (CAM-CS) and CAM in a liposome-in-hydrogel formulation (CS-CAM-Lip). Finally, the antimicrobial activity of CSCAM-Lip and CAM-Sol was compared

Development and optimization of a new processing approach for manufacturing topical liposomes-in-hydrogel drug formulations by dual asymmetric centrifugation

Objectives: The objective of the present study was to utilize dual asymmetric centrifugation (DAC) as a novel processing approach for the production of liposomes-in-hydrogel formulations. Materials and Methods: Lipid films of phosphatidylcholine, with and without chloramphenicol (CAM), were hydrated and homogenized by DAC to produce liposomes in the form of vesicular phospholipid gels with a diameter in the size range of 200-300 nm suitable for drug delivery to the skin. Different homogenization processing parameters were investigated along with the effect of adding propylene glycol (PG) to the formulations prior to homogenization. The produced liposomes were incorporated into a hydrogel made of 2.5 % (v/v) soluble β-1,3/1,6-glucan (SBG) and mixed by DAC to achieve a homogenous liposomes-in-hydrogel-formulation suitable for topical application. Results and Discussion: CAM-containing liposomes with a vesicle diameter of 282 ± 30 nm and polydispersity index (PI) of 0.13 ± 0.02 were successfully produced by DAC after 50 minutes centrifugation at 3500 rpm, and homogenously (< 4 % content variation) incorporated into the SBG hydrogel. Addition of PG decreased the necessary centrifugation time to 2 minutes and 55 seconds, producing liposomes of 230 ± 51 nm and PI of 0.25 ± 0.04. All formulations had an entrapment efficiency of approximately 50%. Conclusions: We managed to develop a relatively fast and reproducible new method for the production of liposomes-in-hydrogel formulation by DAC

Old drug, new wrapping – A possible comeback for chloramphenicol?

The antimicrobial drug chloramphenicol (CAM) exhibits activity against resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). However, its use has been limited due to its toxicity. As the threat of antibiotic resistance continues to grow, a promising approach might be to increase the use of historical antimicrobial agents that demonstrate clinical efficacy, but are hampered by toxicity. We therefore aimed to prepare a liposome-in-hydrogel system for dermal delivery of CAM. Chitosan (CS) was used as the hydrogel vehicle due to its antimicrobial activity and excellent biocompatibility. All critical preparation steps were carried out by dual centrifugation (DC). The DC-method proved to be fast and simple, and organic solvents were avoided in all processing steps. Liposomes with high drug entrapment (49–56%), low polydispersity and a size of approximately 120 nm were produced. Mixing of liposomes into CS-hydrogel by DC produced a homogenous liposomes-in-hydrogel system. Bioadhesive properties were good and comparable to plain CS-hydrogel formulations. Ex vivo permeation studies using pig skin indicated a sustained release of CAM and limited skin permeation. The in vitro antimicrobial activity of CAM in the new liposome-in-hydrogel formulation was similar or better as compared to CAM in solution. Thus, the new formulation was considered highly promising

Electrospun Amphiphilic Nanofibers as Templates for In Situ Preparation of Chloramphenicol-Loaded Liposomes

The hydration of phospholipids, electrospun into polymeric nanofibers and used as templates for liposome formation, offers pharmaceutical advantages as it avoids the storage of liposomes as aqueous dispersions. The objective of the present study was to electrospin and characterize amphiphilic nanofibers as templates for the preparation of antibiotic-loaded liposomes and compare this method with the conventional film-hydration method followed by extrusion. The comparison was based on particle size, encapsulation efficiency and drug-release behavior. Chloramphenicol (CAM) was used at different concentrations as a model antibacterial drug. Phosphatidylcoline (PC) with polyvinylpyrrolidone (PVP), using ethanol as a solvent, was found to be successful in fabricating the amphiphilic composite drug-loaded nanofibers as well as liposomes with both methods. The characterization of the nanofiber templates revealed that fiber diameter did not affect the liposome size. According to the optical microscopy results, the immediate hydration of phospholipids deposited on the amphiphilic nanofibers occurred within a few seconds, resulting in the formation of liposomes in water dispersions. The liposomes appeared to aggregate more readily in the concentrated than in the diluted solutions. The drug encapsulation efficiency for the fiber-hydrated liposomes varied between 14.9 and 28.1% and, for film-hydrated liposomes, between 22.0 and 77.1%, depending on the CAM concentrations and additional extrusion steps. The nanofiber hydration method was faster, as less steps were required for the in-situ liposome preparation than in the film-hydration method. The liposomes obtained using nanofiber hydration were smaller and more homogeneous than the conventional liposomes, but less drug was encapsulated