The skin is the largest and most easily accessible organ of the human body and transdermal drug delivery represents an attractive alternative over other drug administration routes. Since one of the most important functions of the skin is the protection of the body against the influence of the external macro and micro environment, passive molecular transport through the skin is practically impossible. Quite a few enhancement techniques have been developed so far to overcome the stratum corneum barrier and to facilitate transdermal drug delivery and they include various passive (penetration enhancers, liposomes) and active approaches (electroporation, iontophoresis, microneedles). However, to ensure successful transport of molecules through the skin or into the skin cells it is necessary to carefully choose the method or their combination and to optimize it for a specific target application. The main purpose of my research work was gaining new insights into the mechanisms and the underlying physics of some of the transdermal enhancement methods, as well as to compare them and use them in combination where appropriate. Our focus was on three different physical enhancement methods: i) Electroporation: increases the permeability of the outermost layer of the skin (stratum corneum), and/or cell membranes in lower skin layers when exposed to electric pulses, due to local changes in the microstructure of the cell membrane lipids. These changes depend on the magnitude, duration and number of pulses and may be the size of a few nano to micrometers, also up to a few 100 micrometers (local transport regions – LTR). For electrically charged molecules, electric pulses also provide driving force to push molecules into the skin (electrophoresis); ii) Laser microablation: the exposure of the skin to a fractional laser beam in order to create micro-channels in the stratum corneum; iii) Ultrasound: the use of low-frequency ultrasound (20-40 kHz) in order to increase skin permeability by creating aqueous pathways in the skin by cavitation (sonoporation), skin heating and mechanical effects (sonophoresis). Apart from the physical enhancement methods, we added a passive transdermal enhancement method: the encapsulation of molecules in nano-delivery systems (liposomes and ethosomes), and used combinations of different physical approaches with nano-delivery systems. The first part of the experimental work of my doctoral thesis was carried out in vitro on dermatomed pigs' ear skin, in a two-compartment Franz diffusion cell system, where electroporation was used to enhance transdermal transport of flourecent molecule calcein. We focused on comparison of different combinations of square wave electric pulses using short high voltage pulses (HV) and longer low voltage pulses (LV). Our results show, that the calcein transport through the skin significantly increased using more long LV pulses, while the efficiency of short HV pulses compared to the passive diffusion only was negligibly small. We also showed that the combination of HV pulses, followed by LV pulses do not work in synergy as we hypothesized. Surprisingly, when LV pulses are preceded by HV pulses, calcein delivery was lowered compared to LV pulses alone. We explained the mechanism with a numerical model that is based on the development of local transport regions (LTR). In a further study we were using Green skin pore electroporator for pulse delivery (together with electrodes, both developed exclusively for use on skin) and Patent Blue dye later observed by cryo-histology to assess the distribution of dye in the skin. We have shown that a uniform distribution of the dye can be observed primarily in the upper layers of the skin, when using protocols suitable for possible clinical use. We conclude that electroporation is suitable for the dermal molecular delivery and also for the applications that need to ensure the introduction of molecules not only in the skin tissue but also inside the cells. Further, using the same experimental system, Er:YAG laser with fractional output beam profile was used as enhancement method for transdermal drug delivery of three model molecules of different sizes: FITC-dextrans with average molecular weights of 4 kDa, 10 kDa and 20 kDa. The Er:YAG laser is used for controlled removal of the thin dead outer layer of the skin, the stratum corneum, taking advantage of the ablative effects of laser light on tissue while causing minimal thermal damage, thus sparing viable underlying layers (epidermis, dermis). Further, laser beam of fractional lasers is split into microbeams, so even larger portion of viable skin tissue is spared. First, we used protocols among which pulse duration was varied while keeping pulse energy constant (shorter pulses of equal energy mean higher peak power). Following that, we kept the duration of the pulses constant while varying their energy. We showed that the energy of the delivered pulses is the most important parameter for the size/depth of the microchannels, while differing pulse duration/power dictates the extent of thermally-altered tissue and with that the partitioning of the permeant into tissue. Also, laser pulse parameters have different impacts on the molecules of different sizes and physico-chemical properties, therefore each molecule and each application requires optimization of the laser protocols. Further, we introduced a different experimental system using ex vivo full thickness pigs' ear skin, in order to move one step closer to the potential in vivo conditions. We developed and optimized electroporation (application of square wave electric pulses) and sonoporation (application of low-frequency ultrasound) protocols and investigated the effectiveness of a combination of methods to further enhance transdermal molecular transport. The results showed a statistically significant increase in calcein transport into the skin using already 6x100 short high voltage electrical pulses (amplitude of 200 V and duration 100 μs), or 5 minutes of low-frequency ultrasound exposure. Later, to achieve a synergistic effect, the combination of the methods was delivered to skin so that one method is directly followed by the other one. Surprisinglly, the results showed no evident improvement over a single method. The mechanism of action of both methods is the creation of aqueous pathways in the stratum corneum leading to increased skin permeability. However, when used in combination (regardless of the order of methods), the second method was unsuccessful in adding many new aqueous pathways in the stratum corneum, as it acted preferentially near the sites of the existing ones. In the last part of the doctoral thesis, we used previously developed protocols to verify the transdermal enhancement efficay of calcein encapsulated in two different nano-delivery systems. Liposomes showed to be a successfull dermal enhanceres of the calcein, where they can serve as a sort of reservoir of the molecules. On the other hand, the ethosomes proved to be extremely successful as dermal and transdermal molecular enhancers and can deliver the molecules also into the lower skin layers.
Our research work contains the combination of theoretical and experimental approaches to shed light on the mechanisms of three physical dermal and transdermal drug delivery enhacement methods used separately and some in combination, as well as a passive approach: different encapsulation techniques. The knowledge gained in this field represents new knowledge that will further contribute to development of new methods for dermal and transdermal drug delivery enhancement tehniques.
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