Michael Unbehauen

Tailored dendritic core-multishell nanocarriers for efficient dermal drug delivery: A systematic top-down approach from synthesis to preclinical testing

Drug loaded dendritic core-multishell (CMS) nanocarriers are of especial interest for the treatment of skin diseases, owing to their striking dermal delivery efficiencies following topical applications. CMS nanocarriers are composed of a polyglycerol core, connected by amide-bonds to an inner alkyl shell and an outer methoxy poly(ethylene glycol) shell. Since topically applied nanocarriers are subjected to biodegradation, the application of conventional amide-based CMS nanocarriers (10-A-18-350) has been limited by the potential production of toxic polyglycerol amines. To circumvent this issue, three tailored ester-based CMS nanocarriers (10-E-12-350, 10-E-15-350, 10-E-18-350) of varying inner alkyl chain length were synthesized and comprehensively characterized in terms of particle size, drug loading, biodegradation and dermal drug delivery efficiency. Dexamethasone (DXM), a potent drug widely used for the treatment of inflammatory skin diseases, was chosen as a therapeutically relevant test compound for the present study. Ester- and amide-based CMS nanocarriers delivered DXM more efficiently into human skin than a commercially available DXM cream. Subsequent in vitro and in vivo toxicity studies identified CMS (10-E-15-350) as the most biocompatible carrier system. The anti-inflammatory potency of DXM-loaded CMS (10-E-15-350) nanocarriers was assessed in TNFα supplemented skin models, where a significant reduction of the pro-inflammatory cytokine IL-8 was seen, with markedly greater efficacy than commercial DXM cream. In summary, we report the rational design and characterization of tailored, biodegradable, ester-based CMS nanocarriers, and their subsequent stepwise screening for biocompatibility, dermal delivery efficiency and therapeutic efficacy in a top-down approach yielding the best carrier system for topical applications.

Stratum corneum targeting by dendritic core-multishell-nanocarriers in a mouse model of psoriasis

Inflammatory disorders of the skin pose particular therapeutic challenges due to complex structural and functional alterations of the skin barrier. Penetration of several anti-inflammatory drugs is particularly problematic in psoriasis, a common dermatitis condition with epidermal hyperplasia and hyperkeratosis. Here, we tested in vivo dermal penetration and biological effects of dendritic core-multishell-nanocarriers (CMS) in a murine skin model of psoriasis and compared it to healthy skin. In both groups, CMS exclusively localized to the stratum corneum of the epidermis with only very sporadic uptake by Langerhans cells. Furthermore, penetration into the viable epidermis of nile red as a model for lipophilic compounds was enhanced by CMS. CMS proved fully biocompatible in several in vitro assays and on normal and psoriatic mouse skin. The observations support the concept of CMS as promising candidates for drug delivery in inflammatory hyperkeratotic skin disorders in vivo.

Localization of dexamethasone within dendritic core-multishell (CMS) nanoparticles and skin penetration properties studied by multi-frequency electron paramagnetic resonance (EPR) spectroscopy.

Graphical abstract Figure. No Caption available. Abstract The skin and especially the stratum corneum (SC) act as a barrier and protect epidermal cells and thus the whole body against xenobiotica of the external environment. Topical skin treatment requires an efficient drug delivery system (DDS). Polymer‐based nanocarriers represent novel transport vehicles for dermal application of drugs. In this study dendritic core‐multishell (CMS) nanoparticles were investigated as promising candidates. CMS nanoparticles were loaded with a drug (analogue) and were applied to penetration studies of skin. We determined by dual‐frequency electron paramagnetic resonance (EPR) how dexamethasone (Dx) labelled with 3‐carboxy‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxy (PCA) is associated with the CMS. The micro‐environment of the drug loaded to CMS nanoparticles was investigated by pulsed high‐field EPR at cryogenic temperature, making use of the fact that magnetic parameters (g‐, A‐matrices, and spin‐lattice relaxation time) represent specific probes for the micro‐environment. Additionally, the rotational correlation time of spin‐labelled Dx was probed by continuous wave EPR at ambient temperature, which provides independent information on the drug environment. Furthermore, the penetration depth of Dx into the stratum corneum of porcine skin after different topical applications was investigated. The location of Dx in the CMS nanoparticles is revealed and the function of CMS as penetration enhancers for topical application is shown.

Rhamnolipids form drug-loaded nanoparticles for dermal drug delivery

Graphical abstract Figure. No Caption available. Abstract Bacterial biosurfactants are nature’s strategy to solubilize and ingest hydrophobic molecules and nutrients using a fully biodegradable transport system. Eight structurally defined rhamnolipids were selected and investigated as potential drug carrier systems. Depending on the molecular structures defining their packing parameters, the rhamnolipids were found to form spherical nanoparticles with precisely defined average sizes between 5 and 100 nm, low polydispersity, and stability over a broad concentration range as revealed from dynamic light scattering and electron microscopy. As rhamnolipids were tolerated well by the human skin, rhamnolipid nanoparticles were considered for dermal drug delivery and thus loaded with hydrophobic drug molecules. Using the drug model, Nile red, dexamethasone, and tacrolimus nanoparticles charged with up to 30% drug loading (w/w) were obtained. Nanoparticles loaded with Nile red were investigated for dermal drug delivery in a Franz cell using human skin. Fluoresence microscopy of skin slices indicated the efficient penetration of the model drug into human skin, both into the stratum corneum and although to a lesser extent into the lower epidermis. Rhamnolipid nanocarriers were found to be non‐toxic to primary human fibroblasts in a proliferation assay and thus are considered candidates for the dermal delivery of drugs.

Investigation of the cutaneous penetration behavior of dexamethasone loaded to nano-sized lipid particles by EPR spectroscopy, and confocal Raman and laser scanning microscopy

Graphical abstract Figure. No Caption available. Abstract An improvement of the penetration efficiency combined with the controlled release of actives in the skin can facilitate the medical treatment of skin diseases immensely. Dexamethasone (Dx), a synthetic glucocorticoid, is frequently used for the treatment of inflammatory skin diseases. To investigate the penetration of nano‐sized lipid particles (NLP) loaded with Dx in comparison to a commercially available base cream, different techniques were applied. Electron paramagnetic resonance (EPR) spectroscopy was used to monitor the penetration of Dx, which was covalently labeled with the spin probe 3‐(Carboxy)‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxy (PCA). The penetration into hair follicles was studied using confocal laser scanning microscopy (CLSM) with curcumin‐loaded NLP. The penetration of the vehicle was followed by confocal Raman microscopy (CRM). Penetration studies using excised porcine skin revealed a more than twofold higher penetration efficiency for DxPCA into the stratum corneum (SC) after 24 h incubation compared to 4 h incubation when loaded to the NLP, whereas when applied in the base cream, almost no further penetration was observed beyond 4 h. The distribution of DxPCA within the SC was investigated by consecutive tape stripping. The release of DxPCA from the base cream after 24 h in deeper SC layers and the viable epidermis was shown by EPR. For NLP, no release from the carrier was observed, although DxPCA was detectable in the skin after the complete SC was removed. This phenomenon can be explained by the penetration of the NLP into the hair follicles. However, penetration profiles measured by CRM indicate that NLP did not penetrate as deeply into the SC as the base cream formulation. In conclusion, NLP can improve the accumulation of Dx in the skin and provide a reservoir within the SC and in the follicular infundibula.

Drug distribution in nanostructured lipid particles

&NA; The targeted design of nanoparticles for efficient drug loading and defined release profiles is even after 25 years of research on lipid‐based nanoparticles still no routine procedure. It requires detailed knowledge about the interaction of the drug with the lipid compounds and about its localisation and distribution in the nanoparticle. We present here an investigation on nano‐sized lipid particles (NLP) composed of Gelucire and Witepsol as solid lipids, and Capryol as liquid lipid, loaded with Dexamethasone, a glucocorticoid used in topical treatment of inflammatory dermal diseases. The interactions of Dexamethasone, which was spin‐labelled by 3‐(Carboxy)‐2,2,5,5‐tetramethyl‐1‐pyrrolidinyloxy (DxPCA), with its microenvironment are monitored by EPR spectroscopy at 94 GHz at low temperatures. The mobility of the spin‐labelled drug was probed by X‐band EPR at room temperature. In order to relate the magnetic and dynamic parameters deduced from EPR to the local environment of the spin probe in the NLP, investigations of DxPCA in the individual lipid compounds were carried out. The magnetic parameters reflecting the polarity of DxPCAs environment as well as the parameters describing the mobility of the drug reveal that in the case of colloidal dispersions of the lipids and also the NLP DxPCA is attached to the surface of the nanoparticles. Although the lipophilic drug is almost exclusively associated with the NLP in aqueous solution, dilution experiments show, that it can be easily released from the nanoparticle. Graphical abstract Figure. No caption available.

Dendritic Core-Multishell Nanocarriers in Murine Models of Healthy and Atopic Skin

Dendritic hPG-amid-C18-mPEG core-multishell nanocarriers (CMS) represent a novel class of unimolecular micelles that hold great potential as drug transporters, e.g., to facilitate topical therapy in skin diseases. Atopic dermatitis is among the most common inflammatory skin disorders with complex barrier alterations which may affect the efficacy of topical treatment.Here, we tested the penetration behavior and identified target structures of unloaded CMS after topical administration in healthy mice and in mice with oxazolone-induced atopic dermatitis. We further examined whole body distribution and possible systemic side effects after simulating high dosage dermal penetration by subcutaneous injection.Following topical administration, CMS accumulated in the stratum corneum without penetration into deeper viable epidermal layers. The same was observed in atopic dermatitis mice, indicating that barrier alterations in atopic dermatitis had no influence on the penetration of CMS. Following subcutaneous injection, CMS were deposited in the regional lymph nodes as well as in liver, spleen, lung, and kidney. However, in vitro toxicity tests, clinical data, and morphometry-assisted histopathological analyses yielded no evidence of any toxic or otherwise adverse local or systemic effects of CMS, nor did they affect the severity or course of atopic dermatitis.Taken together, CMS accumulate in the stratum corneum in both healthy and inflammatory skin and appear to be highly biocompatible in the mouse even under conditions of atopic dermatitis and thus could potentially serve to create a depot for anti-inflammatory drugs in the skin.

Biodegradable Core–Multishell Nanocarriers: Influence of Inner Shell Structure on the Encapsulation Behavior of Dexamethasone and Tacrolimus

We here present the synthesis and characterization of a set of biodegradable core–multishell (CMS) nanocarriers. The CMS nanocarrier structure consists of hyperbranched polyglycerol (hPG) as core material, a hydrophobic (12, 15, 18, 19, and 36 C-atoms) inner and a polyethylene glycol monomethyl ether (mPEG) outer shell that were conjugated by ester bonds only to reduce the toxicity of metabolites. The loading capacities (LC) of the drugs, dexamethasone and tacrolimus, and the aggregate formation, phase transitions, and degradation kinetics were determined. The intermediate inner shell length (C15) system had the best overall performance with good LCs for both drugs as well as a promising degradation and release kinetics, which are of interest for dermal delivery.

Spin-labeling of Dexamethasone: Radical Stability vs. Temporal Resolution of EPR-Spectroscopy on Biological Samples

Abstract Spin-labeling active compounds is a convenient way to prepare them for EPR spectroscopy with minimal alteration of the target molecule. In this study we present the labeling reaction of dexamethasone (Dx) with either TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PCA (3-(carboxy)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy) with high yields. According to NMR data, both labels are attached at the primary hydroxy group of the steroid. In subsequent spin-stability measurements both compounds were applied onto HaCaT cells. When the signal of Dx-TEMPO decreased below the detection limit within 3 h, the signal of Dx-PCA remained stable for the same period of time.

Protease-mediated Inflammation: An In Vitro Human Keratinocyte-based Screening Tool for Anti-inflammatory Drug Nanocarrier Systems

Abstract Background: Refined encapsulation approaches in dermatotherapy gain increased interest. There is need of reproducible in vitro systems representing disease features to screen drug delivery systems for preclinical assessment. Inflammatory human skin diseases are commonly accompanied by abnormal epidermal differentiation and barrier impairment. Serine proteases (SPs) and their inhibitors play a critical role in such dysfunctional differentiation. SPs also initiate cellular pathways via activation of protease-activated receptors, which contribute to inflammation. Thus, function and activity of SPs should be considered for the design of new therapies of such disorders. Objectives: Herein, we established a novel simplified cell culture model, based on SP-mediated inflammation suitable to assess nanocarriers loaded with anti-inflammatory drugs. Methods: SP-mediated inflammation and the regulatory effect of free or encapsulated dexamethasone were determined by measuring interleukin-6 and interleukin-8 in culture medium of HaCaT (human adult low calcium temperature)-keratinocytes. Additionally, radical formation was analyzed by electron paramagnetic resonance spectroscopy. Cellular uptake of core-multishell nanocarriers was investigated by fluorescence microscopy. Cytotoxicity of all additives was determined by a viability assay. Results: SP-Stimulation of keratinocytes resulted in increased radical production and release of inflammatory cytokines without affecting cell viability. Induced inflammation was successfully downregulated by addition of free or encapsulated dexamethasone. Conclusion: SP-addition can be used as inflammatory stimulus in cell culture to mimic effects of aberrant enzymatic activities found in skin of atopic dermatitis patients. The set-up is appropriate as a preliminary test to examine the effectiveness of new molecules or delivery-systems to counteract serine protease-mediated inflammatory processes prior to skin studies.