The isolated perfused human skin flap model: A missing link in skin penetration studies?

Manuscript. Published version available in http://dx.doi.org/10.1016/j.ejps.2016.10.003 Development of effective (trans)dermal drug delivery systems requires reliable skinmodels to evaluate skin drug penetration. The isolated perfused human skin flap remainsmetabolically active tissue for up to 6 h during in vitro perfusion. We introduce the isolated perfused human skin flap as a close-to-in vivo skin penetration model. To validate the model's ability to evaluate skin drug penetration the solutions of a hydrophilic (calcein) and a lipophilic (rhodamine) fluorescence marker were applied. The skin flaps were perfused with modified Krebs- Henseleit buffer (pH 7.4). Infrared technology was used to monitor perfusion and to select a well-perfused skin area for administration of the markers. Flap perfusion and physiological parameters were maintained constant during the 6 h experiments and the amount of markers in the perfusate was determined. Calcein was detected in the perfusate, whereas rhodamine was not detectable. Confocal images of skin cross-sections shoved that calcein was uniformly distributed through the skin, whereas rhodamine accumulated in the stratum corneum. For comparison, the penetration of both markers was evaluated on ex vivo human skin, pig skin and cellophanemembrane. The proposed perfused flapmodel enabled us to distinguish between the penetrations of the two markers and could be a promising close-to-in vivo tool in skin penetration studies and optimization of formulations destined for skin administratio

Deformable liposomes for skin therapy with human epidermal growth factor: The effect of liposomal surface charge

Accepted manuscript version, licensed CC BY-NC-ND 4.0. Published version available at https://doi.org/10.1016/j.ejps.2018.10.005.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

Nanocarriers for tailored skin delivery: More than just the carriers?

Skin diseases are among the top 5 most leading diseases causing non-fatal health burden worldwide and strategies to assure more effective treatments are urgently needed. Dermal therapy is very attractive due to the direct administration of the drug at the diseased skin site thus minimizing systemic side effects associated with the oral and parenteral routes. However, the great variety within the skin conditions can be a challenge in the development of effective dermal therapies. Based on the skin disease, the drug action is often required at different depths within the skin. The failure to penetrate the skin layers might result in sub-therapeutic drug levels at the targeted skin site and unsuccessful treatments. Phospholipid-based nanocarriers have great potential to overcome the current limitations in dermal therapy by assuring controlled and sustained drug delivery and promoting drug/substance transport in the deeper skin layers. The nanocarrier properties can be tailored and exploited to optimize skin drug delivery. In the present study we optimized nanocarriers for tailored skin drug delivery. A closer-to-in vivo skin penetration model was developed to select nanocarriers with specific skin-targeted drug delivery potential. Deformable liposomes were found the most promising nanocarriers delivering model substances in the deeper skin layers while avoiding systemic absorption. Further optimization of the selected nanocarrier was performed by exploring the effect of the liposomal surface charge on dermal delivery. The sustained skin penetration of drug/active substances for liposomally-associated drugs/substances was influenced by both the liposomal surface charge and physicochemical properties of the nanocarrier-associated drug/substance. The enhancement of the biological activities of both human epidermal growth factor and curcumin when incorporated in the liposomal system as compared to both active substances in solutions was found to be dependent on the liposomal surface charge. Positively charged deformable liposomes embedded in chitosan hydrogel enabled stable bioadhesive hydrogel providing a sustained skin penetration of curcumin. The developed liposomal hydrogel formulation has a potential to be further evaluated as advanced wound dressing

Curcumin-In-Deformable Liposomes-In-ChitosanHydrogel as a Novel 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

Mucus-PVPA (mucus Phospholipid Vesicle-based Permeation Assay): An artificial permeability tool for drug screening and formulation development

The mucus layer covering all mucosal surfaces in our body is the first barrier encountered by drugs before their potential absorption through epithelial tissues, and could thus affect the drugs’ permeability and their effectiveness. Therefore, it is of key importance to have in vitro permeability models that can mimic this specific environment. For this purpose, the novel mucus phospholipid vesicle-based permeation assay (mucus-PVPA) has been developed and used for permeability screening of drugs and formulations. The model proved to be stable under the chosen conditions and demonstrated the ability to discriminate between compounds with different chemical structures and properties. Overall, a decrease in drug permeability was found in the presence of mucus on top of the PVPA barriers, as expected. Moreover, mucoadhesive (chitosan-coated) and mucopenetrating (PEGylated) liposomes were investigated in the newly developed model. The mucus-PVPA was able to distinguish between the different liposomal formulations, confirming the penetration potential of the tested formulations and the related drug permeability. The mucus-PVPA model appears to be a promising in vitro tool able to mimic the environment of mucosal tissues, and could therefore be used for further drug permeability screening and formulation development

Going skin deep: a direct comparison of penetration potential of lipid-based nanovesicles on the isolated perfused human skin flap model

Phospholipid-based nanocarriers are attractive drug carriers for improved local skin therapy. In the present study, the recently developed isolated perfused human skin flap (IPHSF) model was used to directly compare the skin penetration enhancing potential of the three commonly used nanocarriers, namely conventional liposomes (CLs), deformable liposomes (DLs) and solid lipid nanoparticles (SLNs). Two fluorescent markers, calcein (hydrophilic) or rhodamine (lipophilic), were incorporated individually in the three nanosystems. The nanocarrier size ranged between 200 and 300 nm; the surface charge and entrapment efficiency for both markers were dependent on the lipid composition and the employed surfactant. Both carrier-associated markers could not penetrate the full thickness human skin, confirming their suitability for dermal drug delivery. CLs exhibited higher retention of both markers on the skin surface compared to DLs and SLNs, indicating a depo formation. DLs and SLNs enabled the deeper penetration of the two markers into the skin layers. In vitro and ex vivo skin penetration studies performed on the cellophane membrane and full thickness pig/human skin, respectively, confirmed the findings. In conclusion, efficient dermal drug delivery can be achieved by optimization of a lipid nanocarrier on the suitable skin-mimicking model to assure system’s accumulation in the targeted skin layer

The isolated perfused human skin flap model: A missing link in skin penetration studies?

Development of effective (trans)dermal drug delivery systems requires reliable skinmodels to evaluate skin drug penetration. The isolated perfused human skin flap remainsmetabolically active tissue for up to 6 h during in vitro perfusion. We introduce the isolated perfused human skin flap as a close-to-in vivo skin penetration model. To validate the model's ability to evaluate skin drug penetration the solutions of a hydrophilic (calcein) and a lipophilic (rhodamine) fluorescence marker were applied. The skin flaps were perfused with modified Krebs- Henseleit buffer (pH 7.4). Infrared technology was used to monitor perfusion and to select a well-perfused skin area for administration of the markers. Flap perfusion and physiological parameters were maintained constant during the 6 h experiments and the amount of markers in the perfusate was determined. Calcein was detected in the perfusate, whereas rhodamine was not detectable. Confocal images of skin cross-sections shoved that calcein was uniformly distributed through the skin, whereas rhodamine accumulated in the stratum corneum. For comparison, the penetration of both markers was evaluated on ex vivo human skin, pig skin and cellophanemembrane. The proposed perfused flapmodel enabled us to distinguish between the penetrations of the two markers and could be a promising close-to-in vivo tool in skin penetration studies and optimization of formulations destined for skin administratio