The aim of the current study was to analyse the particle size distribution of a liposome dispersion, which contained small egg phosphatidylcholine vesicles and had been prepared by high-pressure homogenisation, by various size analysis techniques. Such liposomes were chosen since they can be looked at as a prototype of drug nano-carriers. Three sub-micron particle size analysis techniques were employed: (1) fixed-angle quasi-elastic laser light scattering or photon correlation spectroscopy (PCS), (2) size exclusion chromatographic (SEC) fractionation with subsequent (off-line) PCS size-analysis and quantification of the amount of particles present in the sub-fractions, and (3) field-flow-fractionation coupled on-line with a static light scattering and a refractive index (RI)-detector. When designing liposome-based drug carrier systems, a reliable and reproducible analysis of their size and size distribution is of paramount importance: Not only does liposome size influence the nanocarrier's in-vitro characteristics such as drug loading capacity, aggregation and sedimentation but also it is generally acknowledged that the pharmacokinetic behaviour and biodistribution of the carrier is strongly size-dependent. All three approaches of liposome size analysis used here were found to yield useful results, although they were not fully congruent. PCS indicated either a broad, mono-modal, log-normal size distribution in the range of below 20 to over 200 nm in diameter, or alternatively, a bimodal distribution with two discrete peaks at 30 to 70 nm and 100 to over 200 nm. Which of the two distribution models represented the best fit depended primarily on the data collection times used. In contrast, both fractionating techniques revealed a size distribution with a large, narrow peak well below 50 nm and a minor, broad, overlapping peak or tail extending to over 100 nm in diameter. The observed differences in liposome size distribution may be explained by the inherent limitations of the different size analysis techniques, such as the detection limit and the fact that PCS is overemphasizing bigger particle sizes.

The increased prevalence of chronic wounds requires novel treatment options. The aim of this study was to develop a beta-glucan (βG)-loaded nanofiber wound dressing. Nanofibers were prepared using the needle-free Nanospider™ technology, an electrospinning method which enables the production of nanofibers at an industrial scale. The βG was selected as active ingredient based on its confirmed wound healing potential in both animals and humans. Hydroxypropyl methylcellulose (HPMC) and polyethylene oxide (PEO) were included as copolymers. Rheological profiles of spinning solutions containing HPMC, PEO, βG, ethanol and water, were optimized. The nanofiber formation was confirmed by Field Emission Scanning Electron Microscopy (FE-SEM), and both nanofibers with (βG-nanofibers) or without βG (NoβG-nanofibers) were evaluated by their swelling index and FT-IR spectroscopy. The formulations, active ingredient and excipients were tested for their possible in vitro toxicity in keratinocytes. Finally, the wound healing potential of the nanofibers was tested in externally induced excisional wounds in male diabetic db/db mice. Three different doses of βG-nanofibers and the βG-free, NoβG-nanofibers, were evaluated for their in vivo wound healing efficacy. All nanofiber-treatments provided improved wound healing as compared to the negative control (water). All βG-nanofiber treated groups exhibited significantly improved wound healing as compared to the NoβG-nanofiber treated group, indicating the potential of βG-nanofibers as wound dressing.

A liposomes-in-hydrogel system as an advanced wound dressing for dermal delivery of curcumin was proposed for improved chronic wound therapy. Curcumin, a multitargeting poorly soluble active substance with known beneficial properties for improved wound healing, was incorporated in deformable liposomes to overcome its poor solubility. Chitosan hydrogel served as a vehicle providing superior wound healing properties. The novel system should assure sustained skin delivery of curcumin, and increase its retention at the skin site, utilizing both curcumin and chitosan to improve the therapy outcome. To optimize the properties of the formulation and determine the effect of the liposomal charge on the hydrogel properties, curcumin-containing deformable liposomes (DLs) with neutral (NDLs), cationic (CDLs), and anionic (ADLs) surface properties were incorporated in chitosan hydrogel. The charged DLs affected the hydrogel’s hardness, cohesiveness, and adhesiveness. Importantly, the incorporation of DLs, regardless of their surface charge, in chitosan hydrogel did not decrease the system’s bioadhesion to human skin. Stability testing revealed that the incorporation of CDLs in hydrogel preserved hydrogel´s bioadhesiveness to a higher degree than both NDLs and ADLs. In addition, CDLs-in-hydrogel enabled the most sustained skin penetration of curcumin. The proposed formulation should be further evaluated in a chronic wound model.

Limitations of the anticancer drug product Taxotere® have encouraged researchers to entrap the active ingredient docetaxel (DTX) into nanocarriers such as liposomes. However, until now no DTX-liposome formulation has reached the clinic. Hence, in the present study, different Soy-PC based DTX-liposome formulations were screened in an attempt to identify lipid-compositions with promising DTX-entrapment (DTX-EE). Various other quality attributes, such as vesicle size and morphology, poly dispersity index (PDI), zeta potential (ZP), stability and in vitro drug release were also investigated. In an initial study, the inclusion of charged lipids within the liposome bilayer was observed to have a positive effect on DTX-EE. Thus, cationic DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and anionic DMPG (1,2-Dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) lipids were selected for further investigations. With anionic DMPG, only a temporary rise in EE was gained with ≥ 20% (w/w) DMPG in Soy-PC lipid-based liposomes, whereas a concentration-dependent increase in EE was observed with cationic DOTAP. A DTX-EE > 95% was obtained with only 5% (w/w) DOTAP in Soy-PC, while neutral liposomes formed from Soy-PC alone, gave 41.5% DTX-EE. In the stability study, a DOTAP concentration > 10% (w/w) in Soy-PC was found to facilitate a stable DTX-EE > 90% after 12 weeks storage. The positive effect of cationic lipids on the EE was confirmed when replacing cholesterol (CHOL), initially shown to suppress DTX-entrapment, with cationic 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]Cholesterol (DC-CHOL). Here, DTX-EE was improved from 29.8% to 92.0% (w/w) with 10% (w/w) CHOL and DC-CHOL in Soy-PC, respectively. Finally, PEGylation of DOTAP-liposomes with DSPE-PEG2000 and DSPE-PEG750 reduced the DTX-EE relative to DOTAP-liposome with no PEGylation. As with the DMPG-liposomes, a temporarily raised affinity between DTX and liposomes was obtained with anionic DSPE-PEGylation of Soy-PC liposomes, however, this effect was not maintained after 4 weeks storage. However, in a dialysis set-up, cationic DOTAP-liposomes released DTX to a higher extent than PEGylated liposomes. Thus, the optimal formulation with regard to storage stability and in vivo performance need to be investigated further, applying conditions that are closer to mimic the in vivo-situation. Applying the Dual Asymmetric Centrifugation (DAC) method in liposome production appears favourable due to its good reproducibility. The observed increase in DTX entrapment with cationic lipids or PEGylation appears scalable into pilot manufacturing scale.

The topical administration of exogenous human epidermal growth factor (hEGF) is a promising approach for improved chronic wound therapy. To develop therapeutically superior hEGF formulation, we prepared hEGF-containing neutral (NDLs), cationic (CDLs) and anionic (ADLs) deformable liposomes (DLs), respectively, since it is expected that the liposomal surface charge can affect both the liposomal physicochemical properties, their skin penetration potential and therapeutic efficacy of liposome-associated drug. All prepared liposomes were of similar size (300-350 nm) with high hEGF load (~80% entrapment efficacy). Among the studied DLs, ADLs were found to be most promising for sustained release of hEGF, as assessed in vitro using the polyamide membrane. Ex vivo studies revealed that all DLs were excellent systems for skin therapy with hEGF and no penetration of hEGF through the full thickness human skin was detected. ADLs provided a depot exhibiting the highest hEGF retention onto the human skin surface. ADLs also revealed enhanced mitogenic activities in human fibroblasts compared to both NDLs and CDLs after 48 hrs treatment. Moreover, hEGF-containing ADLs significantly enhanced mitogenic activity in fibroblast as compared to activity of hEGF solution (positive control). Similar trends were observed in human keratinocytes after 24 hrs of treatment. We proved that the liposomal surface charge affects the therapeutic potential of hEGF-containing liposomes. hEGF-containing ADLs can be a promising nanosystem-based formulation for localized therapy of chronic wounds.