K.D. Kramer

Nanoparticles for skin penetration enhancement--a comparison of a dendritic core-multishell-nanotransporter and solid lipid nanoparticles.

Nanosized particles are of growing interest for topical treatment of skin diseases to increase skin penetration of drugs and to reduce side effects. Effects of the particle structure and size were studied loading nile red to dendritic core-multishell (CMS) nanotransporters (20-30 nm) and solid lipid nanoparticles (SLNs, 150-170 nm). Interaction properties of CMS nanotransporters with the dye molecules--attachment to the carrier surface or incorporation in the carrier matrix--were studied by UV/Vis and parelectric spectroscopy. Pig skin penetration was studied ex vivo using a cream for reference. Interactions of SLN and skin were followed by scanning electron microscopy, internalisation of the particles by viable keratinocytes by laser scanning microscopy. Incorporating nile red into a stable dendritic nanoparticle matrix, dye amounts increased eightfold in the stratum corneum and 13-fold in the epidermis compared to the cream. Despite SLN degradation at the stratum corneum surface, SLN enhanced skin penetration less efficiently (3.8- and 6.3-fold). Viable human keratinocytes showed an internalisation of both nanocarriers. In conclusion, CMS nanotransporters can favour the penetration of a model dye into the skin even more than SLN which may reflect size effects.

Cyproterone acetate loading to lipid nanoparticles for topical acne treatment: particle characterisation and skin uptake.

PurposeTopical cyproterone acetate (CPA) treatment of skin diseases should reduce side effects currently excluding the use in males and demanding contraceptive measures in females. To improve skin penetration of the poorly absorbed drug, we intended to identify the active moiety and to load it to particulate carrier systems.Materials and MethodsCPA metabolism in human fibroblasts, keratinocytes and a sebocyte cell line as well as androgen receptor affinity of native CPA and the hydrolysis product cyproterone were determined. CPA 0.05% loaded solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), a nanoemulsion and micropheres were characterized for drug-particle interaction and CPA absorption using human skin ex-vivo.ResultsNative CPA proved to be the active agent. Application of CPA attached to SLN increased skin penetration at least four-fold over the uptake from cream and nanoemulsion. Incorporation into the lipid matrix of NLC and microspheres resulted in a 2–3-fold increase in CPA absorption. Drug amounts within the dermis were low with all preparations. No difference was seen in the penetration into intact and stripped skin.ConclusionWith particulate systems topical CPA treatment may be an additional therapeutic option for acne and other diseases of the pilosebaceous unit.

Influences of opioids and nanoparticles on in vitro wound healing models

For efficient pain reduction in severe skin wounds, topical opioids may be a new option - given that wound healing is not impaired and the vehicle allows for slow opioid release, since long intervals of painful wound dressing changes are intended. We investigated the influence of opioids on the wound healing process via in vitro models, migration assay and scratch test. In fact, morphine, hydromorphone, fentanyl and buprenorphine increased the number of migrated HaCaT cells (spontaneously transformed keratinocytes) twofold. In the scratch test, morphine accelerated the closure of a monolayer wound (scratch). As possible slow release application forms are nanoparticulate systems like solid lipid nanoparticles (SLN) and dendritic core-multishell (CMS) nanotransporters, we evaluated the effect of unloaded nanoparticles on HaCaT cell migration, too. CMS nanotransporters did not inhibit migration, SLN even enhanced it (twofold). Applying morphine plus unloaded nanoparticles reduced morphine effects possibly due to uptake into CMS nanotransporters and adsorption to the surface of SLN. In contrast to SLN, TGF-beta1 was taken up by CMS nanotransporters, too. Both nanoparticles are tolerable by skin and eye as derived from Episkin-SM(TM) skin irritation test and HET-CAM assay. No acute toxic effects were observed either. In conclusion, opioids as well as the investigated nanoparticulate carriers conform the essential conditions for topical pain reduction.

Drug Release and Skin Penetration from Solid Lipid Nanoparticles and a Base Cream: A Systematic Approach from a Comparison of Three Glucocorticoids

Solid lipid nanoparticles (SLNs) can enhance drug penetration into the skin, yet the mechanism of the improved transport is not known in full. To unravel the influence of the drug-particle interaction on penetration enhancement, 3 glucocorticoids (GCs), prednisolone (PD), the diester prednicarbate (PC) and the monoester betamethasone 17-valerate (BMV), varying in structure and lipophilicity, were loaded onto SLNs. Theoretical permeability coefficients (cm/s) of the agents rank BMV (–6.38) ≧ PC (–6.57) > PD (–7.30). GC-particle interaction, drug release and skin penetration were investigated including a conventional oil-in-water cream for reference. Both with SLN and cream, PD release was clearly superior to PC release which exceeded BMV release. With the cream, the rank order did not change when studying skin penetration, and skin penetration is thus predominantly influenced by drug release. Yet, the penetration profile for the GCs loaded onto SLNs completely changed, and differences between the steroids were almost lost. Thus, SLNs influence skin penetration by an intrinsic mechanism linked to a specific interaction of the drug-carrier complex and the skin surface, which becomes possible by the lipid nature and nanosize of the carrier and appears not to be derived by testing drug release. Interestingly, PC and PD uptake from SLN even resulted in epidermal targeting. Thus, SLNs are not only able to improve skin penetration of topically applied drugs, but may also be of particular interest when specifically aiming to influence epidermal dysfunction.

SLN for topical application in skin diseases--characterization of drug-carrier and carrier-target interactions.

The modes of drug-particle interactions considerably influence drug delivery by nanoparticulate carrier systems and drug penetration into the skin. The exact mechanism of the drug loading and its release are still ambiguous. Therefore, the loading process, the interaction of the agent and the lipid matrix of solid lipid nanoparticles (SLNs) as well as the uptake of the loaded agent by skin lipids were analysed by electron spin resonance (ESR) and parelectric spectroscopy (PS) using spin probes (TEMPO, TEMPOL, and CAT-1) as model drugs differing in their lipophilicity. The spin probes were closely attached to the particles lipid surface (TEMPO) or located in the layers of the surfactant (CAT-1), respectively. Furthermore, two distinct sub-compartments on the SLN were found. To simulate the processes at the phase boundary SLN dispersion/skin, skin lipid mixtures were prepared and the transfer process of the spin labels was followed by ESR tomography. Transfer rates were related to the lipophilicity of the spin probe, the lipid mixture and the applied pharmaceutical formulation, SLN dispersion and aqueous solution, respectively. In particular, SLN accelerated in particular the distribution of the lipophilic agents.

Stimulating effects of modulated 150 MHz electromagnetic fields on the growth of Escherichia coli in a cavity resonator

Abstract A simple and quick screening method is described for investigating the effects of modulated and unmodulated 150 MHz electromagnetic fields on the growth of Escherichia coli . The experiments were performed in a cavity resonator which allowed simultaneous monitoring of the influence of five different field intensities. The impact of temperature fluctuations and the way in which they were taken into account in the experimental set-up are discussed. Screening was performed at modulation frequencies of 72, 217 and 1100 Hz and also with unmodulated electromagnetic fields. Maximum field values of 1.6 kV m −1 for the electrical field and 5.4 μT for the magnetic induction were applied. Cells exhibited identical growth behaviour with the various modulation frequencies and in unmodulated fields. Therefore, the modulation frequency could not be responsible for the observed growth effect. Frequency or intensity windows were not observed. There are hints that the growth stimulation observed at higher field intensities is partly caused by a microthermal effect on the cellular level and not by a general temperature increase of the suspending medium.

The lack of effects of nonthermal RF electromagnetic fields on the development of rat embryos grown in culture.

Rat embryos (9.5 days old) were exposed for up to 36 h to various radio frequency (RF) electric and magnetic fields (modulation frequency: 16, 60, 120 Hz; electric field strength: 60, 600 V/m; magnetic induction: 0.2, 2.0 microT). A resonator technique was used to generate standing waves thus fulfilling three conditions: The site of maximum electric and magnetic oscillations could be separated, the field strengths were known exactly and a high homogeneity over the sample volume was achieved. In each frequency region the transmitter power levels were set to give specific absorption rate (SAR) values spreading from far below to far above the values met in the field of telecommunication (0.2, 1.0 and 5.0 W/kg). The criteria used to examine the embryos on day 11.5 for possible structural effects consisted of a scoring system, photographs, histology using both light and electron microscopy and determination of the protein content. All these data have been taken as sets of different intermediate frequency (IF) amplitude modulation of the RF carriers. Neither the electric nor the magnetic fields tested interfered significantly with the normal growth and differentiation of the embryos in vitro.

Generation of homogeneous electric and magnetic fields at radio frequencies in a cavity resonator

Abstract A standing wave in a rectangular cavity resonator has the advantage of offering positions where purely electric or purely magnetic oscillating fields can be found. Such a cavity with inner dimensions of 1.41 m × 1.41 m × 0.25 m can be excited by a comb of frequencies; we have selected the lowest eigenfrequency to be 150 MHz. We can calculate the amplitudes of the vectors E and B from the transmitter power, the loaded quality factor and the reflection coefficient. Samples can be inserted through a slit of dimensions 1–3 cm along the whole length without disturbing the cavity wall currents and almost without additional electric loading and/or detuning of the oscillation pattern. The relatively low value of 30 for the loaded quality factor guarentees that low frequency modulation on the radio-frequency carrier is not disturbed by up to 100 kHz of sinusoidal or square wave information. Depending on the sample dimensions, the average values E and B and their root mean square deviations Δ E and Δ B are well known. During each run both the carrier and modulation amplitudes can be monitored via an undercritically coupled induction loop. The feeding of the transmitter power into the cavity by a small 50 Ω rod antenna at position of maximum E can easily be adapted to give zero reflection by varying the rod length about its optimum value of 18 cm to compensate for small additional electric loading by the sample media; slight detuning of the resonance frequency as caused by large samples of high dielectric constant can be compensated by varying of the insertion gap width.

Improving Topical Non-Melanoma Skin Cancer Treatment: In vitro Efficacy of a Novel Guanosine-Analog Phosphonate

Actinic keratosis, a frequent carcinoma in situ of non-melanoma skin cancer (NMSC), can transform into life-threatening cutaneous squamous cell carcinoma. Current treatment is limited due to low complete clearance rates and asks for novel therapeutic concepts; the novel purine nucleotide analogue OxBu may be an option. In order to enhance skin penetration, solid lipid nanoparticles (SLN, 136-156 nm) were produced with an OxBu entrapment efficiency of 96.5 ± 0.1%. For improved preclinical evaluation, we combined tissue engineering with clinically used keratin-18 quantification. Three doses of 10-3 mol/l OxBu, dissolved in phosphate-buffered saline as well as loaded to SLN, were effective on reconstructed NMSC. Tumour response and apoptosis induction were evaluated by an increase in caspase-cleaved fragment of keratin-18, caspase-7 activation as well as by reduced expression of matrix metallopeptidase-2 and Ki-67. OxBu efficacy was superior to equimolar 5-fluorouracil solution, and thus the drug should be subjected to the next step in preclinical evaluation.

Action of a high-frequency magnetic field on the cartilage matrix in vitro☆

Abstract In an attempt to elucidate the action of magnetic fields on matrix macromolecules (collagens and proteoglycans) in embryonal chondrogenic tissue, organoid cultures of limb bud blastemal cells from mouse embryos (day 12) were placed in an electromagnetic chamber that offered separately both electric and magnetic oscillating fields producing uniform magnetic fields around the cultures and in the culture medium. To simulate inflammatory conditions, the cultures were treated with bacterial lipopolysaccharides from day 4 of the culture onwards. After different exposure periods (once for 4 h, once for 24 h and 4 h daily for 7 days), collagen types I and II and proteoglycans were determined in the cultures and the activity of metalloproteinases (MMPs) was measured in the culture medium. The results showed that high-frequency magnetic fields caused pronounced enhancement of collagens (type II > type I) in the cultures exposed once for 4 h, and elevation of proteoglycans in cultures exposed once for 24 h. The activity of MMPs, however, was inhibited by magnetic fields, mostly in the cultures exposed to magnetic fields for 4 h daily for 7 days. It is concluded that high-frequency magnetic fields exert anabolic effects on embryonal chondrogenic tissue in vitro.