Carnauba wax nanosuspensions with TIO2 as sunscreen – Influence of different grades of carnauba wax

Dahl K, Müller-Goymann CC
Institut für Pharmazeutische Technologie, TU Braunschweig

To prevent both short-term and long-term skin damage from excessive sun exposure the development of highly effective sun protection products is a growing demand. A nanosuspension composed of titanium dioxide as inorganic sunscreen within a matrix of carnauba wax and decyl oleate in a 2:1 ratio has previously been reported to yield high sun protection factors (SPF) in vitro. The carnauba wax-decyl oleate nanoparticles as a carrier system for encapsulated titanium dioxide offer many advantages like avoiding organic filters and thus potential intrinsic irritation. Since carnauba wax is a natural product with varying composition, the present contribution aims at the influence of different grades of carnauba wax on the physicochemical properties of the nanosuspensions.

Methods: Nanosuspensions were manufactured by dispersing a lipid phase into an aqueous phase using high-pressure homogenization. Particle size distribution (PIDS technique) and contact angle measurements were performed to characterize the systems. The visualization of the nanoscale particles was made with transmission electron microscopy (TEM) after freeze fracture of the nanosuspensions. In vitro sun protection factors representing an indicator of the UVA/UVB protective property of the sunscreen formulation were determined with an SPF analyzer and calculated from the monochromatic protection factor, the solar irradiance and the erythemal constants.

Results: In all the cases the SPF were higher than 50. According to COLIPA recommendations (European Cosmetic, Toiletry and Perfumery Association, 2006) the formulations were rated as „high to very high protection“. Contact angle measurements did not show any noticeable differences between the various carnauba wax grades. From particle size distributions, the D50 values of almost all formulations were in the nanoscale range. The D90 values, however, were in the micrometer range with remarkable differences between the systems. A close contact between carnauba wax and titanium dioxide crystals was confirmed by TEM. The spherical wax particles exhibited a size of about 200 to 400 nm with encapsulated crystals of titanium dioxide in the rutile modification. In conclusion, differences between various carnauba wax grades were determined concerning particle size measurements only, whereas all the other properties, especially SPF values, were not affected.

(1) Institut für Pharmazie, Freie Universität Berlin, (2) Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité Universitätsmedizin Berlin, (3) Fachbereich Physik, Freie Universität Berlin, (4) Institut für Pharmazie, Pharmazeutische Technologie, Friedrich Schiller Universität Jena, 5Institut für Chemie und Biochemie, Freie Universität Berlin

Background: As a prerequisite for Electron Paramagnetic Resonance (EPR) spectroscopic measurements of the skin, the application of stable free radicals, so called nitroxides, is necessary. Several nitroxides are commercially available but only a few can be used for in vivo measurements. Furthermore, due to their physical/chemical properties the stability and skin penetration is limited. Depending on the desired application, different nitroxides have to be used. Therefore, the use of various nanometer scaled transport systems, which were developed for pharmaceutics and cosmetics to enhance penetration and storage of drugs applied to the skin seems promising.

Objectives: In this study, the amphiphilic and reactive nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) was used in different lipid formulations to study the stabilising effect of the storage systems. Furthermore, the penetration enhancing effect on the hydrophilic nitroxide PCA (3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyoxy) was investigated using polymer and lipid based carrier as penetration enhancing systems. The studies were performed ex vivo and also in vivo, when the use of the respective carrier system was permitted.

Methods: First, the transport systems were measured at W-band frequency to gain an insight into the nitroxide location within the carrier system, as well as information about its microenvironment. Then, the nitroxides were applied to the skin within the formulation or in the respective solvent ex vivo on porcine skin and in vivo on the forearms of human volunteers, when possible. The used nano systems were: invasomes, nanostructured lipid carriers and core-multishell nanotransporters.

Results: Stabilisation of the nitroxide TEMPO could be demonstrated ex vivo and in vivo for both lipid based carrier systems with an increase in measurement time of approximately 2-fold for invasomes. The penetration enhancing effect of the carriers when applied with PCA could be demonstrated, which compared to free PCA was up to 3-fold.

Conclusion: EPR spectroscopy can be used to evaluate the stabilising and storage effect of nano carriers as well as their ability to enhance skin penetration of hydrophilic compounds. Summarising the results, comparative studies of different nano carriers become feasible.

Derivates of betulin facilitate wound closure - High significant acceleration of epithelialisation of superficial wounds by triterpen-dry-extract from the outer bark of birch (birch cork)

Hoffmann M (1), Bross F (1), Metelmann HR (2), Podmell F (2), Schumann H (1), Brandner JM (3), Scheffler A
(4)

(1) Department of Dermatology, Albert-Ludwigs-University Freiburg, Department of Dermatology at Dermatology at the University Medical Center Freiburg, (2) Department of Maxillofacial Surgery Ernst-Moritz-Arndt-University Greifswald, Department of Maxillofacial Surgery at Greifswald University, (3) University Medical Center Hamburg-Eppendorf, Hamburg, (4) Birken AG, Niefern-Öschelbronn

Background: A newly designed purified dry extract of birch cork (TE) with > 80% betulin (m/m) respectively > 90% triterpen, in combination with oils, provides a semi-firm gel (oleogel), which can emulgate more water than its proper weight (tensid-free pickering-emulsion). Such an oleogel showed in vitro, ex vivo and in vivo the following relevant effects: (i) stimulation of cell division of basall cells of the skin (KI67); (ii) expression of early and late differentiation markers (keratin 10, involucrin, transglutaminase); (iii) specific activation of terminal differentiation (apoptotic staining at the junction between stratum granulosum and stratum corneum, Wölfle et al JID 2010) and (iv) acceleration of epithelialisation in an ex vivo wound-healing porcine model.

Study design: Prospective, randomized, controlled, multi-centric, blind-evaluated phase II study of compatibleness and acceleration of epithelialisation of superficial wounds by an oleogel consisting of 10%TE in sunflower oil (called Sericare®). As wound model, the split thickness skin graft (SSG) donator site (0,3 mm) was chosen. For an intra-individual approach, one half of the donator site wound was dressed with a non-adhesive silicon-coated bandage alone, whereas the other half used the TE-oleogel under an identical bandage. Blinded macro images at every bandage-change of both outer thirds of the wound were compared to determine which wound surface showed stronger epithelialisation. The resulting score of all decisions per patient determined which wound half experienced better epithelialisation and therefore faster wound healing. Additionally the surgeon collected information, such as degree of re-epithelialisation, sensitivity and pain to touch as well as pruritus of the wound. The cosmetic result after 3 months was also evaluated. To obtain satisfactory results for a statistic superiority rate of the oleogels of 0.67 or an effectiveness strength of 80%, originally some 50 patients were needed to be reviewed. In order to adjust the number of cases, an intermediary appraisal of the results was made after 24 cases. To be sure of a significant superiority of the oleogel after 20 cases, a p < 0,0038 had to be achieved.

Results: 1. Superior epithelialisation in the blinded evaluation of oleogel versus bandage alone: wound closure was faster for 20 cases with oleogel application; in 2 cases it was faster with bandage alone; and for another 2 cases equal results were observed. The faster re-epithelialisation under oleogel was with a p < 0,0001 highly significant. A higher number of cases wasn’t required.
2. Almost two times faster epithelialisation under oleogel application as compared to con-ventional wound healing. Touch pain was reduced under oleogel, whereas pruritus was slightly increased at oleogel site.
3. Better cosmetic results after three months in 15 out of 19 oleogel treated wounds. 4/19 wounds showed identical results. One patient could not be examined after 3 months because of cancer-death. Four patients didn’t take part in the check-up of results.

Effects of INLB321-CD on immortalized human keratinocyte cell line and modified organotypic co-culture

Kolditz F (1), Krausze J (2), Heinz DW (2), Niemann HH (3), Müller-Goymann CC (1)

(1) Institut für Pharmazeutische Technologie, TU Braunschweig, (2) Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, (3) Departement of Chemistry, Bielefeld University

Internalin B (InlB) is an invasion protein of Listeria which facilitates its uptake into host cells by activating the receptor tyrosine kinase c-Met. It was proposed that activation via receptor dimerization is mediated through an InlB dimer. The dimerized fragment of Internalin B, InlB321-CD¹ (crystal dimer), was designed to stabilize the InlB dimer in solution. In binding studies and in in vitro scatter assays [1], InlB321-CD revealed to be a stronger agonist than monomeric InlB321 and Internalin B.

In human skin, mainly epithelial cells express the c-Met receptor which controls amongst others proliferation and migration. After being stimulated by its endogenous agonist hepatocyte growth factor (HGF), which is secreted by e.g. dermal fibroblasts, this receptor plays an important role in the regeneration of the epidermis.

The present project aims at investigating the influence of dimeric as well as monomeric InlB321 on the epidermal regeneration in a scratch assay. For the cultivation of an epidermal monolayer, a HaCaT cell line consisting of human dermal immortalized keratinocytes was used. The scratch assay was performed on a HaCaT monolayer. Scratching the confluent monolayer with a pipette simulated a superficial epidermal wound. In addition to the scratch assay on the HaCaT monolayer, a proliferation assay was performed with HaCaT seeded onto a 3D collagen gel with incorporated fibroblasts.

In a previous study such a three-dimensional bio-engineered skin from tissue culture experiments had been found to be an appropriate model for studying epidermal regeneration after traumatization with sodium dodecyl sulfate [2]. But it might be not optimal for proliferation assays with InlB because HGF is secreted by the fibroblasts being integral part of the skin construct and may cloud any proliferative effects of low InlB321-CD concentrations. Hence, for comparison, the fibroblasts in the collagen gel were or were not killed prior to the InlB321-CD supplementation to compare the effects.

Methods: A confluent monolayer of HaCaT was scratched with a pipette tip. Afterwards, incubation with 0.5 nM InlB321-CD, 1 nM InlB321, 0.5 nM HGF or just medium was carried out, respectively. The cultivation with medium served as negative control whereas that with HGF served as positive control. The alterations of the scratch areas were documented with micrographs and quantified with an imaging software system.
Premature co-cultures (4 days after seeding HaCaT onto the collagen gel) with dead or viable fibroblasts were treated for 24 hours with 0.5 nM InlB321-CD or medium, respectively. The proliferation of keratinocytes was measured with an MTT assay.

Results: 24 h after scratching, the cells treated with 0.5 nM dimeric InlB321 showed a smaller gap compared to those incubated with medium. The cells treated with 0.5 nM HGF as positive control reduced the ‘wound’ gap as well. 1 nM monomeric InlB321 also stimulated the HaCaT cell line.

The proliferation assay carried out with premature organotypic co-cultures that contained killed human dermal fibroblasts (HDF) showed a significant mitogenic effect of InlB321-CD on HaCaT cell line versus medium. In contrast to that, premature co-cultures comprising viable HDF cells did not benefit from InlB321-CD supplementation significantly.

[1] Ferraris DM et al: Ligand-Mediated Dimerization of the Met Receptor Tyrosine Kinase by the Bacterial Invasion Protein InlB. J Mol Biol 395 (2010) 522-532

[2] Weber C, Müller-Goymann CC: The usefulness of a 3D skin constructs in the detection of regenerative effects after previous SDS damage. J Drug Del Sci Technol 19 (2009) 337-342

Visualization of fluorescent nanoparticles in pig skin

Paulus A (1), Schäfer UF (1), Lehr C-M (1,2) , Hansen S (1,2)

(1) Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbrücken,
(2)Helmholtz Institute of Drug Delivery, Helmholtz-Centre for Infectious Research, Saarbrücken

Current strategies of needle-free vaccination via the dermal route are mainly based on reduction of the barrier function of the stratum corneum [1]. Hair follicles represent an alternative route for transdermal drug delivery without damaging the skin. The trans-follicular route is investigated as route for vaccine delivery due to accumulation of Langerhans cells around the hair bulbs [2]. The infundibulum of the hair follicle presents an excellent reservoir for the nanoparticles, which, as was shown in the last years penetrate better into the hair follicle than non-particle formulations [3].

Therefore, the aim of the study was to visualize the uptake of fluorescence labeled nanoparticles into the hair follicles of pig skin. The particles were applied using massage. After incubation time, samples were taken using different techniques: longitudinal cryosections, punch biopsies and cyanoacrylate biopsies. Confocal laser scanning microscopy was used for examination being an efficient method to visualize the particles on skin [4].

The first challenge was to detect the nanoparticles in presence of the high auto fluorescence of fresh pig ear skin. A method was successfully established to detect the fluorescence nanoparticles using confocal laser scanning microscopy. This method allows the differentiation between skins auto florescence and fluorescent signal of the particles.

By means of this method particles can be visualized simultaneously with skin and hair morphology. Among the skin sectioning techniques examined, cyanoacrylate biopsies were shown best suitable for visualizing nanoparticles in the follicular content. The developed approach in this study is a successful method for investigation of the uptake of nanoparticles into the hair follicles.

[1] Combadière B, Mahé B: Particle-based vaccines for transcutaneous vaccination. Comparative Immunology, Microbiology and Infectious Diseases 31 (2008) 293-315

[2] Babiuk S, Baca-Estrada M, Babiuk LA, Ewen C, Foldvari M: Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery. J Control Release 66 (2000) 199-214

[3] Lademann, J, Richter H et al: Nanoparticles – an efficient carrier for drug delivery into the hair follicles. Eur J Pharm Biopharm 66 (2007) 159-164

[4] Toll, R, Jacobi U et al: Penetration profile of microspheres in follicular targeting of terminal hair follicles. J Invest Dermatol 123 (2004) 168-176

UVB-induced DNA damage, generation of reactive oxygen species and inflammation is effectively attenuated by the flavonoid luteolin in vitro and in vivo

Wölfle U (1), Esser PR (2,3), Simon-Haarhaus B (1), Martin SF (2), Lademann J (4), Schempp CM (1)

(1) Competence Center skintegral, Department of Dermatology, University Medical Center Freiburg, (2) Allergy Research Group, Department of Dermatology, University Medical Center Freiburg, (3) Faculty of Biology, University of Freiburg, (4) Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité Universitätsmedizin Berlin

Ultraviolet (UV) radiation induces DNA damage, oxidative stress and inflammatory processes in human keratinocytes resulting in skin inflammation, photoaging and photocarcinogenesis. The flavonoid luteolin is one of the most potent antioxidative plant polyphenols.

We investigated the UV-protective and antioxidant properties of luteolin in human keratinocytes in vitro, ex vivo and in vivo. Spectrophotometric measurements revealed extinction maxima of luteolin in the UVB and UVA range. UV transmission below 370 nm was < 10%. In human skin, luteolin effectively reduced the formation of UVB-induced cyclobutane pyrimidine dimers. The free radical scavenging activity of luteolin was assessed in various cell-free and cell-based assays.

In the cell-free DPPH assay the half-maximal effective concentration (EC50) of luteolin (12 µg/ml) was comparable to Trolox (25 µg/ml) and N-acetylcysteine (32 µg/ml). In contrast, in the H2DCFDA-assay performed with UVB irradiated keratinocytes, luteolin (EC50 3 µg/ml) was much more effective compared to Trolox (EC50 12 µg/ml) and N-acetylcysteine (EC50 847 µg/ml). Luteolin also inhibited both UVB-induced skin erythema and the upregulation of cyclooxygenase-2 and prostaglandin E2 production in human skin via interference with the MAPK pathway.

These data suggest that luteolin may protect human skin from UVB-induced damage by a combination of UV-absorbing, DNA protective, antioxidant and anti-inflammatory properties.