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-inhydrogel-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.

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 (2:1:2w/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 150nm 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.

Conventional liposome preparation methods bear many limitations, such as poor entrapment efficiencies for hydrophilic drugs, batch size limitations, and limited options for aseptic manufacturing. Liposome preparation by dual centrifugation (DC) is able to overcome most of these limitations. DC differs from normal centrifugation by an additional rotation of the samples during the centrifugation process. Thus, the direction of the centrifugal forces changes continuously in the sample vials. The consequential powerful sample movements inside the vials result in powerful homogenization of the sample. Since this "in-vial" homogenization is optimal for viscous samples, semisolid "vesicular phospholipid gels" (VPGs) are preferred intermediates in the liposome manufacturing by DC. The DC method easily enables aseptic preparation and is gentler as compared to other methods, such as high-pressure homogenization. The method allows very small samples to be prepared, and VPG batches down to 1-5 mg scale have been prepared successfully. VPGs have several applications; they are attractive as depot formulations, or as stable storage intermediates, and can be easily transferred into conventional liposomal formulations by simple dilution. Here, we aim to present the novel DC-liposome technique; the concept, advantages, and limitations; and provide an overview of the experiences of liposome preparation by DC so far.

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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 120nm 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.