3.8. In summary
Table 1 and Table 2 summarize the results obtained in the experiments, comparing the different skin architectural features of human and mouse, and the effect of different volumes injection on both species, respectively.
Table 1. Main structural and thickness differences between mouse and human skin*
*Values are 𝑥̅ ± SD of the skin layers measurements of OCT scans from mouse and human. Ten measurements were taken for each skin layer and injection depth.
Thickness (µm) Human Mouse
Epidermis 83.4 ± 14.6 15.6 ± 2.8
Dermis Papillary 215 ± 35.6 298.8 ± 36.4
Reticularis 425.8 ± 107.5
Injection depth 229 ± 63,7 184 ± 20,5
Hypodermis 296.2 ± 46.2
Panicculus carnosus 148.7 ± 15.9
Table 2. Features of injection in human and mouse skin according to the 2%
(w/v) MB volume injected
Volume of 2% MB injected (µl)
Features of injection
Human Mouse
50
Skin with disruptions. Skin tissue mostly free of disruptions.
100
Tissue disruptions higher in size and number than those observed following the administration of 50 µl.
No visible disruptions of the tissue. Formation of a very pronounced blebbing area.
200
Larger disruptions visible in the skin tissue.
Big blebbing area at the injection site. Low number of small disruptions in the tissue.
4 Discussion
The data acquired both via OCT and histological techniques permits to visualize the main structural differences between the human and mouse skin, despite the first ones being more exact and accurate. These alteration occurred as the skin manipulation aiming at the histological analysis was indeed a long process, which most probably affected the final characteristics of this tissue in several aspects, like the creation of artefacts. These artefacts are, for example, the aspect of the mouse D layers being detached from the ones below (Figures 13 and 25). Another point is that the H&E staining did not provided satisfying results (Figures 9 and 13) as the haematoxylin did not stain the tissue properly, affecting the capability of identifying additional skin structural features in the histological pictures and then to make a full characterization of the tissue different layers. Moreover, additional training and expertise on the equipment used to prepare these samples, and higher number of samples would most probably lead to more accurate observations and conclusions.
In the human control, the main layers that constitute the skin can be visualized - EP, D, HP – including some of their specialized structures, like the hair follicles.
Starting by the histological data (Figure 9), the EP was very well stained with the eosin, having a dark-red colour. It is mainly due to the characteristics of this part of the tissue, which is mainly constituted by a squamous keratinized stratified epithelium (3), the SC.
Due to its keratinized morphology, it prevents excessive water loss and the entrance of some drugs. By its turn, the D appeared to be slightly light-red stained because of its major content in collagen and elastin fibres, which provides the skin its strength and extensibility. On the other hand, the RD layer showed some purple points stained by haematoxylin, that could be explained by the presence of some cells, like fibroblasts and macrophages, although these were not identified or distinguished in the presented figures.
Similar observations apply to the mouse skin histological picture (Figure 13). It is also possible to distinguish the main skin structural layers, namely the EP, D, hypodermis.
In addition to it, it is visible the striated muscle layer PC, which is well-defined in human, with exception of the neck and the ADV. This last layer is a connective tissue composed by loose collagen fibres that allows for skin flexibility (11).
Although the skin structure layers may be similar in their function and content, they differ considerably in size, being the human skin the thickest. This could be easily demonstrated by the direct comparison of the control pictures (Figures 9 and 13). The mouse skin structure layers were indeed very thin, being almost impossible to distinguish the reticular dermis (RD) from the papillary one. It is also possible to observe some hair follicles and sebaceous glands.
The OCT data acquired also demonstrates in a different way the structural layers for both skin structures (Figures 12 and 15). It is easier to distinguish all the skin structures, particularly in the mouse, due to the definition of the pictures obtained. Comparing the human with the mouse skin, as seen in the histological data (Figures 9 and 13), the first one is much thicker. According to the literature, the thickness of the thinnest skin, which is considered to be at the abdomen in human and the back in mouse, has the mean values of, 19.2 µm and 16.4 µm for SC, 46.9 µm and 12.6 µm for EP, higher than 2000 µm and 784.7 µm for D, in human and mouse, respectively (11) (12). The values obtained using the OCT data acquired (Table 1) can be compared to the ones reported in the literature, because the mouse skin used in this assay was also obtained from the back of the animal, whereas the human breast skin has a thickness similar to the skin of the abdominal region. Because the mouse skin is thinner, it is easier to visualize all the layers in OCT images than in the histological data. However, the measured values, despite being very similar, are slightly lower that the ones reported in the literature, which could be explained by the donor’s age of the samples used. As the human ones were collected from people in middle age – elderly, it is needed to take in account that skin thickness decreases with age. The same apply to mice skin(13).
It was also measured the injection depth on both species (Table 1). The values obtained were greater than the epidermal thickness, which are indicative of an adequate EP disruption for a successful drug delivery using the MJ600®.
Considering the D, there are very few anatomical differences other than thickness between human and mouse. At the same time, the rete ridges at the DEJ are much more prominent in human skin than in mouse. As their main function is to reduce the existence of some frictional stress, it does not exist in mouse, once its skin has also an hair protection (11).
Despite being easy to identify in mouse because of the PC muscle presence, the human hypodermis is almost non-visible in the human OCT pictures. It could be explained by its large content in white adipose tissue adipocytes, which are cells filled with a vacuole of fat (11). These characteristics makes the HP areflective (Annex A1).
The skin of all mammals has a barrier function against the entrance of substances, loss of water and protection from mechanical stress, together with a thermoregulatory, endocrine and immunological role (vitamin D synthesis, antigen presentation, production of cytokines). It also protects from mechanical stress (stretch and compression), as well as from UV radiation. Moreover, it acts as a sensory and autonomic organ that can perform some socio-sexual communication (11). Despite this similarity, some experiments realized within the TDD field have shown that the skin structure (SC, thickness, number of cell layers, number and area of hair follicle openings for mm2) relate to drug permeability across this organ. It was demonstrated that a thinner skin and a higher pelage density, promotes a higher permeability (12), which make the skin of rodents generally more permeable and fragile (15).
Nonetheless, those same studies reported that the SC thickness would not be the only factor affecting permeability, but also some differences in the skin structure itself (12) (14), like the density of hair follicles and SC cell composition, as well as the structure organization. In the human specimen, the SC is composed of multiple layers of keratinocytes, while in the mouse skin is typically one cell layer, which is responsible for the main differences in thickness. Apart from it, the corneocytes in the mouse skin have a columnar arrangement, which contrast with the human oblique arrangement:
whereas the first one offers a relatively short route of diffusion, the last is a tortuous ingress route for drugs (15).
Despite the MN being able to surpass the SC barrier, permitting a successful drug delivery across the skin, it could be also affected by the skin morphological properties, which was observed by the injection experiments performed using the MJ600® with the MB solution. Comparing the two situations, human and mouse, it was noted a larger spreading area in the mouse skin, coupled with a bleb formation at the surrounding area of the injection site. By its turn, the MB spot in the human skin was almost constant in time (Figures 18 and 19). This fact can be explained by the delivery of the injected solution to different layers of the skin and by taking in account that both tissues had different volumes. As the human skin is thicker, the injected solution could spread to
deeper layers, whereas in the mouse it may be forced to spread longitudinally.
Moreover, there is another difference in this experiment: it was used a whole mouse skin whose sub-cutaneous tissue would have been intact, while the subcutaneous tissue in ex vivo human skin had been removed.
Apart from the experimental set up, it is important to consider the differences within the skin biomechanical properties of distinct species, which depend on the characteristics of their corresponding fibrils in the D layer. Human and mouse skin have anisotropic, nonlinear, heterogeneous and viscoelastic properties, depending on the donor age and hydration level. As the matrix of collagen and elastin fibrils provides strength and flexibility to the skin, some interspecies differences, like the diameter, degree of crosslinking, strength, rate of degradation, density as well as the dermal layer thickness, will affect the skin mechanical behaviour when submitted to a load application. When a load is applied, the dermal fibrils tend to cross themselves, thus increasing the stiffness of the tissue (16). Some studies reported that the human skin can undergo a greater initial strain before stiffening, while in the mouse it happens earlier, stating also that in this animal the anisotropy is more elevated (8), conferring a greater extensibility to this organ. Considering that the MB injected volume with the MN would create a significant load force (a hydrostatic pressure) capable of inducing a stress-strain response in the skin, it is possible to analyse the results obtained accordingly. In the case of the mouse skin, because of its greater extensibility, the bleb was formed, with the volume of solution injected spreading to a larger area. It was also observed that elasticity effect as the bleb completely disappeared, as the skin returned to its original shape. On the contrary, the human skin not only did not suffer any change in its shape, but also the MB spreading in the tissue was constant over time, being this difference a result of its lower anisotropic behaviour, which leads to a greater initial strain (8). Although this explanation could be acceptable, it is important to consider the different skin layers targeted by the injected solution and the different volumes presented by both tissues. As the human skin is thicker, the injected solution could spread to deeper layers, whereas in the mouse it may be forced to spread longitudinally.
Moreover, there is another big difference in this experiment: it was used a whole mouse skin whose sub-cutaneous tissue would have been intact, while the subcutaneous tissue in ex vivo human skin had been removed.
In Figure 18 it is possible to observe a peculiar MB spreading through the skin tissue, without a non-uniform circular spreading from the injection site area, which is indicative that the MN injection reached some vessels, from where the MB solution injected was flowing through. In fact, it was rarely seen when performing injections in human skin, but it was never observed in mice, due to the higher vascularization of human skin compared with the mouse one (3).
In this study, mouse and human skin structure visualizations were also performed using the OCT, together with histological data, after different MB volume injection (50, 100 and 200 μl), using the MJ600®. There were considerable differences between human and mouse skin structural behaviour. Starting by the histological data, its analysis showed the presence of some tissue disruptions whose dimensions and number increased for higher volumes of the injected MB solution. Differently from it, the histological pictures obtained from mouse showed almost no changes. Both results were consistent with the OCT data. Similarly, it was observed disruptions even with the injection of the smallest volume in the human skin, which increased both in number and size, being almost absent in the mouse. However, it was in the OCT real time acquired scans that the real structural changes in mouse skin could be observed and analysed. These scans obtained right after the MN injection permit to visualize the bleb phenomena visible at naked eye previously described in a very detailed way. They also confirm that the tissue disruptions observed in the histological picture correspondent to the 200 µl injection really occurred, removing the possibility of being only findings caused by the skin histological preparation process.
Considering the disruptions as a consequence of a high hydrostatic pressure generated by the delivery of the MB blue solution that may have created pores in cells’ plasma membrane, thus disrupting the local tissue architecture (7), there are additional key points that lead to the different biomechanical behaviour of both skin species. To point it out, the human skin is less extensible than the mouse one, which confers to the skin the ability to undergo a greater initial strain caused by the hydrostatic pressure of the injected solution. It makes the dermal fibres to straight in direct contact with the cells that could reach their maximum strain value, and then to surpass it, making them to fragment. On the other hand, as the mouse skin is more extensible and anisotropic (16), the hydrostatic pressure triggers an earlier initial tissue response to the same loads, allowing it to expand (the blebbing phenomena), as the volume of the solution disperses
throughout it, without leading to cell disruption, and maintaining also the ability to return to its original shape. However, this is not observed when the initial load is very high, as it was witnessed after the injection of the 200 of µl 2% (w/v) MB solution. In this case, small disruptions occurred.
In the light of this study, it is possibly to understand how the gene delivery performed in mouse by Dul M et al was not successful, being the transfection effective only in the human skin, using small formulation volumes (50 µl). The pDNA intracellular delivery is effective only when it enters the cells by the pores created in their membrane, made by the hydrostatic pressures originated from the formulation volume and flux through the skin structure. For this reason, as the mouse skin has the capability of extending, avoiding this way the disrupting action caused by the volume injected, there was no creation of pores in the cell membranes, and therefore, the intracellular delivery of the pDNA was highly limited. If the human skin would have been used in those experiments, the pDNA transfection would most probably have been successfully achieved, once its biomechanical skin properties would allows for the hydrostatic pressure and therefore, the intracellular delivery of the pDNA was highly limicreation of temporary pores in cell membranes following the application of MJ600®.
Although this research project allowed for important and useful results that provide additional understanding on the impact of the skin of different animal species on the outcome of novel TDD systems, it would be of interest to study the effect of another variables, such as the formulation viscosity. This study would improve the understanding and knowledge on how the different formulation parameters, coupled with the different biomechanical and architectural skin differences, would affect the way by which new development studies involving drug delivery using hollow MNs would be conducted.
5 Conclusion
This is the first study that compares the effect of different formulation volumes using hollow MN delivery, in human and mouse skin. The main structural differences and skin biomechanical behaviour were explored using conventional histological techniques, as well as using high technology applied to dermatological skin studies and diagnosis tool, the OCT system. Both methods used showed that human skin has properties remarkably different from the mouse one, which are of high importance for the development of new formulations for drug delivery using hollow MNs, especially the MJ600® ones. At the same time, it was possible to remark that the histological technique provided results that were confusing and not very reliable, which contrasted with the accurate and efficient data obtained using the OCT technology, principally when analysing the mouse skin.
As stated by other previous studies, the mouse skin is not the most appropriate model to study the drug delivery through the human skin, due to their different biomechanical properties and architecture. In fact, despite being capable of surpassing the SC barrier which is the most compelling obstacle, the effect of the volume injected was very different from one specie to another. With this in mind, drug delivery studies through the skin, even using MNs, should be performed using human skin.
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