Mouse versus Human skin

Universidade de Lisboa Faculdade de Farmácia

Mouse versus Human skin

Comparing human and mouse skin when applying MicronJet

600 injections

Ana Francisca Madeira Martins

Mestrado Integrado em Ciências Farmacêuticas

2017

Cardiff University

School of Pharmacy and Pharmaceutical Sciences

Mouse versus Human skin

Comparing human and mouse skin when applying MicronJet

600 injections

Ana Francisca Madeira Martins

MSc in Pharmaceutical Sciences

2017

Universidade de Lisboa Faculdade de Farmácia

Mouse versus Human skin

Comparing human and mouse skin when applying MicronJet

600 injections

Ana Francisca Madeira Martins

Monografia de Mestrado Integrado em Ciências Farmacêuticas apresentada à Universidade de Lisboa através da Faculdade de Farmácia

Orientador:

Doutora Helena Florindo, Professora Auxiliar da Faculdade de Farmácia da Universidade de Lisboa

Co-Orientador:

2017

Cardiff University

School of Pharmacy and Pharmaceutical Sciences

Mouse versus Human skin

Comparing human and mouse skin when applying MicronJet

600 injections

Ana Francisca Madeira Martins

MSc in Pharmaceutical Sciences monography showed to Lisbon University by Faculty of Pharmacy

Supervisor:

Professor Helena Florindo, Professor at Faculty of Pharmacy, University of Lisbon

Co-Supervisor:

2017

Resumo

As microagulhas são uma tecnologia emergente, tendo em vista a veiculação de fármacos e de outras substâncias, tais como ADN plasmídico, através da pele humana, para efeito local e sistémico. Como a pele humana é difícil de obter, recorre-se frequentemente a modelos animais que permitam mimetizar esta barreira nos Humanos.

Contudo, diferenças estruturais e/ou fisiológicas inerentes a estes tecidos obtidos a partir de espécies distintas dificultam a correlação direta entre esses dados obtidos usando a pele de modelos animais, como ratinhos, mesmo quando as microagulhas são utilizadas como veículo. Desta forma, este estudo teve como objetivo investigar o efeito da injeção de diferentes volumes de uma solução de 2% (m/v) de azul de metileno (50 µl, 100 µl e 200 µl) nas peles humana e de ratinho, utilizando as microagulhas Micron Jet 600^®. Os dados foram obtidos através da análise histológica e de tecnologia OCT (Optical Coherence Tomography), sendo que estes últimos permitiram verificar alterações estruturais relevantes na pele obtida a partir de diferentes espécies, após administração do mesmo volume da solução de azul de metileno.

Palavras-chave: microagulhas, MicronJet 600^®, Optical Coherence Tomography, pele de ratinho, pele humana

Abstract

Microneedles are an emerging technology in clinical practice, being used as an alternative for the administration of drug and other bioactive substances, such as pDNA, through the human skin, aiming to achieve both the topical and the systemic effects.

Once it is very difficult to obtain human skin, animal models have been widely used to mimic the physiological and pathological skin conditions in Humans. However, structural and physiological differences between these tissues comprise the direct correlation of these results, including those obtained following the use of microneedles.

Thus, this study aims to investigate the impact of the administration of different volumes of a 2% (w/v) methylene blue solution (50 µl, 100 µl and 200 µl) in both mouse and human skin, using the Micron Jet 600^® hollow microneedle device. The data was obtained by histological analysis and OCT (optical coherence tomography) technology.

The OCT data evidenced allowed to find relevant structural differences and behaviour between both skins, using the same volume of the 2% (w/v) methylene blue solution.

Keywords: hollow microneedle, MicronJet 600^®, Optical Coherence Tomography, human skin, mouse skin

Abbreviations

ADV – adventitia D – dermis

DEJ - dermo-epidermal junction EP – epidermis

FDA – Food and Drug Administration H&E – haematoxylin and eosin MB – methylene blue

MJ600^® - Micron Jet 600^® MN – microneedle

OCT – optical coherence tomography SC – stratum corneum

TDD – transdermal drug delivery PC – panniculus carnosus

PD – papillary dermis RD – reticular dermis

Index

Resumo ... i

Abstract ... ii

Abbreviations ... iii

Figure Index ... v

Table Index ... vi

Video Index ... vii

1 Introduction ... 1

1.1 Objectives ... 4

2 Materials and methods ... 5

2.1 MN preparation for injection ... 5

2.2 Human skin ... 5

2.2.1 Ethics statement ... 5

2.2.2 Collection and processing of an ex vivo human skin ... 5

2.2.3 Preparation of frozen human skin for histological data acquisition ... 6

2.2.4 Preparation of frozen human skin for OCT data acquisition ... 7

2.3 Mouse skin sampling ... 8

2.3.1 Preparation of frozen mouse for histological data acquisition ... 8

2.3.2 Preparation of frozen mouse for OCT data acquisition ... 9

2.4 Data processing ... 9

3 Results ... 10

3.1 Control ... 10

3.1.1 Human ... 10

3.1.2 Mouse ... 13

3.2 MN injections ... 15

3.3 Human ... 15

3.4 Mouse ... 17

3.4 50 µl injection ... 19

3.4.1 Human ... 19

3.4.2 Mouse ... 21

3.5 100 µl injection ... 22

3.5.1 Human ... 22

3.5.2 Mouse ... 24

3.6 200 µl ... 25

3.6.1 Human ... 25

3.6.2 Mouse ... 27

4 Discussion ... 30

5 Conclusion ... 36

6 Bibliography ... 37

Annexes ... 40

A1. OCT terminology ... 40

A2. The Optical Coherence Tomography (OCT) system (9) ... 41

Figure Index

Figure 1. MJ600^®device inside a blister ... 3

Figure 2. A - MJ600^® capped B - MJ600^® device... 3

Figure 3. MJ600^® attached to a syringe ... 4

Figure 4. MJ600^® with 2% MB solution ready to use ... 5

Figure 5. Skin room laboratory where human skin experiments were realized ... 6

Figure 6. Cryostat FSE used for sectioning ... 7

Figure 7. Human skin ready for performing injections ... 7

Figure 8. OCT equipment ... 8

Figure 9. Human control skin sample stained with H&E...10

Figure 10. Structure of a human hair follicle. Blue staining due to the MB injection…11 Figure 11. OCT picture of human skin - control. ... 12

Figure 12. 3D human skin pictures obtained by OCT scanning treatment...13

Figure 13. Mouse control skin sample stained with H&E. ... 14

Figure 14. Mouse control skin picture obtained from an OCT scan. ... 14

Figure 15. 3D mouse skin pictures obtained by OCT scanning treatment………15

Figure 16. MJ600® approaching human skin right before injection obtained by OCT scanning ... 16

Figure 17. Human skin after being injected with 200 µl of 2% MB solution ... 17

Figure 18. Spreading of MB on mouse skin after 200 µl injection. A – right after injection B – 5 minutes after injection ... 17

Figure 19. Histological picture of mouse skin after 2% MB injection. ... 18

Figure 20. Three-dimensional view of injection sites on mouse skin A – Top view B – side view ... 19

Figure 21. Human skin after 50 µl MB injection. ... 19

Figure 22. Picture of human skin after 50 µl MB injection taken from an OCT scan. .20 Figure 23. Three-dimensional model of the human skin after 50 µl MB injection. .... 20

Figure 24. Mouse skin after 50 µl MB injection ... 21

Figure 25. Picture of mouse skin after 50 µl MB injection taken from an OCT scan. 21 Figure 26. 3-D picture of mouse skin after 50 µl MB injection………22

Figure 27. Human skin after 100 µl MB injection. ... 23

Figure 28 Picture of human skin after 100 µl MB injection taken from an OCT scan ... .23

Figure 29 A and B. Three-dimensional models of human skin after 100 µl MB injection ... 23

Figure 30. Mouse skin after 100 µl MB injection ... 24

Figure 31. Picture of mouse skin after 100 µl MB injection taken from an OCT scan. ... 24

Figure 32. Three-dimensional model of mouse skin after 100 µl MB injection... 25

Figure 33. Human skin after 200 µl MB injection. ... 25

Figure 34. Picture of human skin after 200 µl MB injection taken from an OCT scan ... 26

Figure 35. 3-D model of human skin after 100 µl MB injection. ... 26

Figure 36. Mouse skin after 200 µl MB injection ... 27

Figure 37. Picture of mouse skin after 200 µl MB injection taken from an OCT scan. ... 27

Figure 38. 3-D model of human skin after 100 µl MB injection. ... 28

Figure 39. OCT machine used for ophthalmology ... 41

Table Index

Table 1. Main structural and thickness differences between mouse and human skin . 28 Table 2. Features of injection in human and mouse skin according to the 2% (w/v) MB

volume injected ... 29

Video Index

Video 1. MB squirt from MJ600^®... 5

Video 2. MN injection on human skin ... 16

Video 3. MN injection reaching the skin vessels ... 16

Video 4. 50 µl MB injection on human skin ... 21

Video 5. 50 µl MB injection on mouse skin ... 22

Video 6. 100 µl MB injection on human skin ... 24

Video 7. 100 µl MB injection on mouse skin ... 25

Video 8. 200 µl MB injection on human skin ... 26

Video 9. 200 µl MB injection on mouse skin ... 28

1 Introduction

Transdermal drug delivery (TDD) systems are designed for drug delivery through the skin in order to reach the targeted site in the concentration required for the desired therapeutic effect. It has particular advantages that provide interesting features alternative to the oral route, for example by avoiding the first pass hepatic metabolism, and being easy to use thus improving patient compliance to therapy. For these reasons, drug delivery research has been actively developing products for the transdermal route in the last few years, despite the poor skin permeability for many drugs (1). To achieve successful TDD, it is important to understand the parameters that may affect the permeability of drugs through the skin, especially its particular features and structure.

The human skin is an organ with ca. 2m² area whose main functions consist of thermoregulation, immune defence and prevention of water loss. It can be divided into two main groups: glabrous (non-hairy), which is thicker and less permeable, being limited to areas such as the palms of the hands, soles of the feet and lips; whereas the remaining skin is non-glabrous. These regions also differ in their epidermal thickness, hair follicles density and in the number of sweat and sebaceous ducts.

All the skin surface is covered by a thin film comprised of sebum, some corneocyte debris and sweat, which forms an “acid-mantle” with antimicrobial activity, mainly due to the low pH (ca. 5 in average) (2).

It is also important to take into account the three principal layers of skin structure, which are, from the inner to the outermost, hypodermis (HP), dermis (D) and epidermis (EP), respectively. The first one, HP, has the main role on providing padding and protection, since it absorbs shocks from impacts, whereas the D ensures elasticity, plasticity, tensile strength, sensing ability, as well as, biochemical and immunological support to the EP.

In the dermo-epidermal junction (DEJ) there is an underlying blood supply that provides a large surface for nutrient and waste exchange. Moreover, the EP has a protective function, being constituted by Langerhans cells (antigen presenting cells), melanocytes and corneocytes. The latter has hydrophilic domains surrounded by a lipid- rich matrix, whose cellular packaging arrangement make the “brick and mortar structure”. For this reason, the stratum corneum (SC), the outermost layer of the EP, known to be 10-15 µm thick with 15-20 corneocyte layers (3), provides the major barrier for drug penetration across the skin. Under this circumstance, only a few

number of low molecular weight (< 500 Da) lipophilic drugs are able to reach the SC, having the potential to reach the targeted site. Owing to this, innovative approaches focused on overcoming the SC have been explored, such as microneedles (MN). These devices are among the most recently developed systems for drug delivery, consisting of a micro structured system with micro sized array projections.

The main advantages of MN are related to the absence of pain and needle phobia, also overcoming the need for a professional to perform the administration, which improves patient compliance to therapy but also reduce the cost associated to these therapeutics.

Moreover, these medical devices do not result in bleeding, which is very important in relation to sharps waste and also particular healthcare settings in which they must be used.

The drug delivery device mentioned above has been considerably studied for the last years for TDD, as a diagnostic tool (4) and for vaccination.

There are different teams focused on the MN subject within Europe, being one of them led by Professor James Birchall, at the School of Pharmacy and Pharmaceutical Sciences, Cardiff University, UK. His team has been involved in relevant published studies about MN. These studies include research on the use of MN for vaccination, nanoparticle delivery, gene therapy and issues related to their clinical administration and practice (5) (6). These studies have been performed on both mouse and human skin, using different types of MN, such as solid, coated and hollow MN.

Their most recently published data (7), in which I have been involved, reports the study of a gene delivery in human skin using the MN device MicronJet 600^® (MJ 600^®), being the first one to explore the potential of hollow MN devices for local skin gene delivery.

This study has shown an efficient and reproducible gene expression of exogenous naked pDNA in human skin by injecting a volume that is considered to be standard for intradermal administration (50 µl). Although these results were obtained using human skin, earlier experiments performed using mouse skin (data not published) did not achieve successful results. The cause for the limited outcomes may be mainly related to structural differences between both skins.

This research group has performed interesting research about the differences underlying the biomechanical properties of human and mice skin. They thus concluded that there are some limitations in using mouse skin as a human model (8). However, this study

per si does not allow the understanding of what could have caused such differences in the gene delivery between both skins. For this reason, it would be of interest to investigate the main anatomical differences between human and mouse skin, as well as their structural behaviour after an injection using Micron Jet 600^® (MJ600^®).

The hollow MN devices MJ600^® used in this study were provided by NanoPass Technologies Ltd. It is constituted by an array of three 600 µm length silicon MNs bonded to a tip of plastic adapter, which could be mounted on a standard hypodermic syringe. This device is packed in a blister sterilized by ethylene oxide (EtO) (Figure 1 and 2).

Figure 1. MJ600^®device inside a blister

This device is regulatory approved by FDA, being in commercial distribution since June 2016 (10). It has been also used in other studies related to the evaluation of MNs as a TDD device, as well as in the study of the skin behaviour upon the MN injections (7).

The MJ600^® is intended for intradermal injections of any substance or drug approved for delivery by transdermal route, but not for aspiration of liquids. It should not be used on skin abrasions, open wounds, cuts, scars neither on rashes, skin infections or any other area of damaged or diseased skin. A single use of the device is expected, as well as, its administration to the patient only by a health professional.

A B

Figure 2. A - MJ600^® capped B - MJ600^® device

Figure 3. MJ600^® attached to a syringe

Since MJ600^® is a device capable of disrupting the skin barrier in order to achieve the delivery of the formulation to its target site, there are some adverse reactions related to its use. Of particular interest are the small risk of micro bleeding and rarely local infection at the injection site, together with additional local adverse reactions, like local edema, erythema and discoloration of the injection site, even though those are most likely related to the injected substance.

There are also physicochemical properties of the formulations that can influence the success of a hollow MN injection, like the viscosity and volume. Since most of them depend on many factors, like the active pharmaceutical ingredient (API), the main structural differences between both human and mouse skin would be more accurately detected by only changing a volume of the solution to be injected. For this purpose, we performed a study in which different volumes of a 2% (w/v) methylene blue (MB) solution were injected. This dye is commonly used in tissue staining and is harmless, which facilitate its handling. It also permits a better volume spreading and therefore allows its visualization right after the injection, facilitating the histological data analysis. The skin was also observed using OCT after the injections, a non-invasive technique for morphological investigation of tissue that enables the visualization of architectural changes (9).

1.1 Objectives

In order to understand the reasons that caused different results in gene delivery when using both human and mouse skin, this project had the following objectives:

• Evaluate the main differences between human and mouse skin.

• Understand how those differences can impact DNA delivery, uptake and expression at both tissue and cellular levels using the hollow MN devices (MicronJet 600®) from NanoPass Technologies Ltd.

2 Materials and methods

All reagents and laboratory consumables were purchased from Fisher Scientific, UK, (Loughborough, UK), unless otherwise indicated.

2.1 MN preparation for injection

Extemporaneous 2% (w/v) MB solution was prepared by MB powder dissolution in deionized water, followed by its filtration. A 1 ml syringe was then filled with this solution and further inserted into the MN device at the time of the experiment (Figure 4). Before each injection, it was important to verify the squirt (Video 1) of the MN, which must be continuous and straight to be used in the experiment.

Figure 4. MJ600^® with 2% MB solution ready to use

Video 1. MB squirt from MJ600^®

2.2 Human skin

2.2.1 Ethics statement

Human skin samples were obtained from female patients undergoing breast reduction surgery or mastectomy under informed written patient consent and local ethical committee approval (South East Wales Research Ethics Committees Panel C, Reference: 08/WSE03/55).

2.2.2 Collection and processing of an ex vivo human skin

Excised human breast skin was collected immediately after surgery and transported to the laboratory in culture media, which consisted of Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 100unit mL-1 of penicillin and 100 µg mL-1 of

streptomycin (Life Technologies, UK) at 4ºC. In the laboratory, the samples were washed with sterile PBS. Afterwards the skin was frozen at – 20ºC until its usage. Data in this study was obtained from replicated samples obtained from two independent skin donors, aged 70 and 46 years old. Both skin samples were checked to be free from damage, scarring, moles, disease or other imperfections. All human skin was handled in a biosafety containment level otwo laboratory.

Figure 5. Skin room laboratory where human skin experiments were realized 2.2.3 Preparation of frozen human skin for histological data acquisition

The human skin was removed from storage at – 20ºC and allowed to defrost for 1 h.

Then the subcutaneous adipose tissue was removed by blunt dissection before being pinned, D side down, onto a cork dissection board ready for MN treatment. Sterile PBS solution was used during the process to keep the skin wet. An 8-mm biopsy punch was used to isolate areas of treated skin, which were put onto 10% (v/v) formaldehyde solution and stored overnight at 4ºC. On the following day, the skin samples were washed from formaldehyde by immersing it onto PBS solution and changing it 3 times, 30’ each. After the last PBS solution changing, the skin samples were put into cassettes embedded with optimum cutting temperature (OCT) media, D side down, and stored overnight at – 80 ºC. In the next day, skin samples embedded in OCT media blocks were sectioned using a Cryostat FSE (Thermo Scientific Shandon, UK). Skin sections (10 µm thickness) were collected onto Superfrost Plus microcope slides and observed with an Olympus BX50 microscope (Olympus, Middlesex, UK). Selected slides were subsequently stained using haematoxylin and eosin (H&E) to visualize the skin architecture.

Figure 6. Cryostat FSE used for sectioning

Figure 7. Human skin ready for performing injections 2.2.4 Preparation of frozen human skin for OCT data acquisition

Excised human skin was removed from – 20ºC storage and allowed to defrost for approximately for 1 h. Sub-cutaneous adipose tissue was removed by blunt dissection and the skin was pinned, with the D side down, onto a cork dissection board to facilitate injection. The treated skin and nearby areas were visualised immediately after MN injections, using the VivoSight System (Michelson Diagnostics, UK).

Figure 8. OCT equipment

2.3 Mouse skin sampling

Three mice were obtained post-sacrifice and stored at – 20ºC prior to the study. All skin samples were checked to be free from damage, scarring, moles, disease or other imperfections.

2.3.1 Preparation of frozen mouse for histological data acquisition

The mice were removed from storage at – 20ºC and allowed to defrost approximately for 3 h. Prior to skin treatment and to facilitate MN injections, all the mice were shaved with a hair clipper (Wahl, USA). After MN injections being performed, the mice were peeled off. Then the areas of injection were cut with a blender, put straight onto 10%

(v/v) formaldehyde solution and stored overnight at 4ºC. In the following day, the skin samples were washed from formaldehyde by putting it onto PBS solution and changing it 3 times, 30 minutes each. After the last PBS solution changing, the skin samples were put into cassettes embedded with OCT media, D side down, and stored overnight at – 80 ºC. In the next day, skin samples embedded in OCT blocks were sectioned using a Cryostat FSE (Thermo Scientific Shandon, UK). Skin sections (10 µm thickness) were collected onto Superfrost Plus microcope slides and observed with an Olympus BX50 microscope (Olympus, Middlesex, UK). Selected slides were subsequently stained using H&E to visualize the skin architecture.

2.3.2 Preparation of frozen mouse for OCT data acquisition

The mice were removed from – 20ºC storage and allowed to defrost for approximately 1 h. Prior to skin treatment and to facilitate MN injections, all mice were shaved. The treated skin and nearby areas were visualised immediately after MN injections, using the VivoSight System (Michelson Diagnostics, UK).

2.4 Data processing

Image processing was performed using ImageJ software (National Institute of Health, USA).

3 Results

3.1 Control

3.1.1 Human

The obtained histological picture (Figure 9) shows the two main layers of the human skin tissue, subjected to H&E staining. Accordingly, the darker superficial layer is the EP, which contacts with the exterior, while the SC is the outermost constituent. Below the EP there is the D, composed of the papillary and reticular parts. The PD is the thinner one, right below the EP. The top layer of the D consists of connective tissue, blood vessels and some lymphatic vessels, as shown in Figure 9. It is easy to distinguish the papillary from the reticular layer, as the latter is much thicker and constituted by dense irregular connective tissue. Despite the presence of various disruptions at RD seem to be full of disruptions, these are not considered to be structural. In fact, those are artefacts that occurred much probably due to the histological technique applied. This aspect was therefore taken in account when analysing all the histological data acquired.

Figure 9. Human control skin sample stained with H&E. Legend: SC – stratum corneum; EP – epidermis; PD – papillary dermis; RD – reticular dermis; LV –

lymphatic vessel

Figure 10 was obtained from a sample submitted to MB injection, but it clearly shows a transversal section of a hair follicle structure in detail. It is possible to observe the tubular structure of the hair follicle. At the base, one can see the hair bulb enclosing the

hair papilla. The cortex, a highly keratinised layer, surrounds the innermost layer of the follicle, the medulla. Attached to the hair bulb is the arrector pili muscle, a small smooth muscle, as well as sebaceous glands. The association between the hair follicle and the attached arrector pili muscle and sebaceous gland is called pilosebaceous unit. Finally, it is possible to observe the most superficial part of the hair follicle, the infundibulum.

Figure 10. Structure of a human hair follicle. Blue staining due to the MB injection. Legend: HF – hair follicle; INF – infundibulum; CX – cortex; MED –

medulla; MU – arrector pili muscle; SG – sebaceous gland; HP – hair papilla;

CT  perifollicular connective tissue sheath

The Figure 11, which was acquired using the OCT, highlights the main structural layers of the human skin, supporting the previous histological data, as well as its features. It is important to take in account that the uppermost initial bright brand at the skin’s surface is an optic effect, being thus non-structural (Annex A1). The top layer, located straight below the hyperreflective band, is the EP. It is characterized for having a heterogeneous granular texture, which is mainly due to its structural keratinocytes with different intracellular content in keratin. In this figure, it is also noticeable a drastic contrast change between EP and PD. For that reason, it is also possible to identify the dermo-epidermal junction. By its turn, the PD appears as a hyperreflective and mottled structure compared to the previous described layer, as a result of its high content in well packed collagen bundles and very few vessels. One of those is indicated in Figure 11,

appearing as hypo-reflectives and with a conical tapering shape. Under those circumstances, there were some hyperreflective areas composed of dense collagen (indicated with triangles), which were small collagen bundles closely packed together.

Finally, the innermost and lowest visible hypo reflective layer, the RD, is characterized for having, similarly to the other layer of the D, collagen bundles, despite they not being so well packed. Although being characterized for having some vessels, they are not visible in this picture.

Moreover, Figure 11 shows a hair protruding from epidermal surface, as well as its respective follicle (among other hair follicles, indicated with green arrows). The hair follicles are hypo-reflective, mainly due to the shadowing optical effects, which are caused by the presence of keratin in their constitution (Annex A1). They also are associated with sebaceous glands, hypo reflective and ovoid structures (indicated with orange arrows). These glands presenting a granular structure are surrounded by a darker hypo reflective border, in relation to surrounding dermal collagen.

Figure 11. OCT picture of human skin - control. Legend: EP – epidermis; DP – papillary dermis; DR – reticulary dermis; DEJ – dermo-epidermal junction; V – vessel; H – hair; Triangles – areas of dense collagen; Orange arrows – sebaceous

glands; Green arrows – hair follicles

These last pictures (Figures 12-A and 12-B) were obtained from OCT data using Fiji software. They provide an accurate visualization of the 3-dimensional structure of human’s skin, being easy to identify the two main layers, EP and D, together with the two sub-layers of the last one, papillary and RD, respectively.

3.1.2 Mouse

This histological picture (Figure 13) shows the different layers that mouse skin is consisted of, stained with H&E. The outermost part and also the thinnest one, is the EP, which is made of epithelial cells. The D is a lower, thick layer of loose connective tissue. It is possible to distinguish some structures on this layer, like hair follicles and sebaceous glands, both of them associated to the pilosebaceous unit. As it is possible to see in the Figure 13, the structures on the left were identified as being sebaceous glands, due to their clear inner, that can correspond to a group of cytoplasmic vacuoles filled with lipid. On the other hand, the hair follicles structures represented in the same figure are darker and very noticeable due to their hair bulb. These structures and associated adnexal glands and muscle could be seen in the D, as well as extending into the hypodermis, which is largely composed of brown fat (not seen in this picture). The aspect of the hair blub is highly dependent on the phase of the hair cycle. The panniculus

A

B

Figure 12. 3D human skin pictures obtained by OCT scanning treatment. Legend:

EP – epidermis; DP – pappilary dermis; DR – reticulary dermis; DEJ – dermo- epidermal junction; H - hair

carnosus (PC) is a constituent of the hypodermis, characterized for being a sheet of striated muscle that makes the boundary between the skin and the underlying adventitia (ADV), which consists of a loose and irregular collagenous connective tissue that allows for the flexibility of the skin. As the D and the PC are not joined in the histological pictures, which are in fact an artefact of them, it will not be considered as a big disruption henceforth.

Figure 13. Mouse control skin sample stained with H&E. Legend: EP - epidermis; D - dermis; HP – hypodermis; ADV: adventitia; PC – panniculus

carnosus; HF – hair follicle; SG – sebaceous gland

Figure 14 was acquired using the OCT, being the Figure 15 obtained from the treatment of the same data using the Image J software.

Figure 14. Mouse control skin picture obtained from an OCT scan. Legend: EP – epidermis; D – dermis; HP – hypodermis; PC – panniculus carnosus; H – hair

In both figures it is possible to distinguish the main layers of the mouse skin. At the top of the skin there are some hyperreflective structures, the hairs of the mouse, which are very abundant. They are hyperreflective due to the keratin. The outermost layer of the skin, the EP, because of its few thickness, is not easily recognized and identified. This area is located right below the band of hyperreflectivity, the same optical effect described earlier for human skin OCT data. By its turn, the D is easily seen as a granular layer, not so hyperreflective as the hairs and EP, but still brighter than the hypodermis, mainly because its connective fibre composition, like dense collagen fibrils. The hypodermis appears as being hypo-reflective compared with those last layers described, as a result of its high content in lipids. Its constituent layer, the PC, is particularly noticed by its three sheets of hyper and hypo reflective nature. They have different reflective behaviour due to the presence of some lipid containing cells present within the layers composed of smaller fibres, as myofibers.

3.2 MN injections 3.3 Human

The Video 2 shows a MB injection on human skin. The technique was performed following the manufacturer instructions (Annex A3). After this procedure, it is noticeable a MB blur spreading from the injection site, with approximately a round Figure 15. 3D mouse skin pictures obtained by OCT scanning treatment. Legend: EP –

epidermis; D – dermis; HP – hypodermis; PC – panniculus carnosus; H - hair

regular shape, which does not modify nor spreads more than it was immediately after the injection.

Video 2. MN injection on human skin

Figure 16 was obtained during a MN injection, which shows the MN device approaching, as well as the skin retracting following the pressure resultant from the impact of the device.

Figure 16. MJ approaching human skin right before injection obtained by OCT scanning. Legend: MD - MN device; blue arrows: hollow microneedles The Video 3 shows an injection with an interesting result: instead of making a round blue shape, it happened as if the MB solution was spreading inside a branch of pipes.

In fact, while the solution in the first injection flowed among all the skin structure in every direction, in the last one, the needles reached a group of vessels (most probably the lymphatic ones), from where the MB solution spread along (Figure 17).

Video 3. MN injection reaching the skin vessels

Figure 17. Human skin after being injected with 200 µl of 2% MB solution Figure 17 represents the outcome of the two injections described above, allowing for the direct comparison of the different patterns of spreading and diffusion of the MB solution, following its injection in the human skin.

3.4 Mouse

The Figure 18-A shows the aspect of the MB solution spreading right after the MN injection being performed, which has a regular round shape. The second one (Figure 18-B) shows the same mouse 5 minutes after the injection. Comparing the two pictures, it is noticeable the increment of the size of the blur spreading, despite maintaining the same regular round shape.

Figure 18. Spreading of MB on mouse skin after 200 µl injection. A – right after injection B – 5 minutes after injection

Even though it is not perceptible, a remarkable skin tissue expanding occurred temporarily in the adjacent area to the injection site right after the administrations, becoming larger or higher solution volumes. These phenomena known as “bleb”

disappeared with time, along with an increased blue spreading.

This histological picture obtained from a mouse skin in which it was performed a MB injection shows that some parts of the skin structure were stained by the solution injected (Figure 19). More importantly, it shows the injection point, which is characterized by the disruption of the outermost skin layer, the EP, allowing the formulation contained in the MN device to reach structures below the EP, by easily overcoming the SC barrier in this way.

Figure 19. Histological picture of mouse skin after 2% MB injection. Legend: EP – epidermis; D – dermis; HP – hypodermis; PC – panniculus carnosus; IP –

injection point

Figure 20-A shows the top view of the mouse skin with three injection points made by the EP disruption with the three hollow MNs from the device, while in the Figure 20- B it is possible to visualize the same piece of skin, but in a different perspective, in which it is possible to see the aspect of the holes created by the MN injection inside the skin, within the injection site.

Figure 20. Three-dimensional view of injection sites on mouse skin A – Top view B – side view

3.4 50 µl injection

3.4.1 Human

This histological picture taken from a sample subjected to a 50 µl injection of a 2%

(w/v) MB solution (Figure 21) permits to observe a big disruption of the tissue (indicated by a red square), in the PD. There also seem to be another smaller disruptions in this layer of the D (not indicated), in addition to others that appear to be in the EP region. Despite the RD showing many other disruptions, they most probably were not caused by the injection (Figure 9). Although the spreading of MB was uniform, it is also possible to observe a more intense staining in the EP and in the fibre bundles constituents of the RD.

Figure 21. Human skin after 50 µl MB injection. The red square indicates a disruption area

In Figure 22, it is possible to see an injection site, which is characterized by a disruption of the SC and, subsequently, the EP. It can be seen because the area of the injection site did not have the hyperreflective top layer of the surface, the SC, as well as the EP, which is represented by a hypo reflective colour, as described previously. There is only an areflective area, which suggests the absence of any structure in that space. A vessel is also recognizable in the upper RD (hypo reflective area). It is also possible to see a small tissue disruption (red arrow) in the upper part of the PD caused by the injection of the MB solution. This injection solution volume, despite being considered as a standard one, is very small, and thus it only can form very small blebs largely spaced between them. There are normally no tissue blebs associated, being all rather located in the PD.

Figure 22. Picture of human skin after 50 µl MB injection taken from an OCT scan. Legend: Yellow bold arrow – injection site; Red arrow – disruption; V – vessel The following picture (Figure 23) is part of the same section, but now in 3D. It shows small disruptions, most of them differing in size, but being all localized at the PD. The injection site is visible, too.

Figure 23. Three-dimensional model of the human skin after 50 µl MB injection.

Legend: Red arrows - disruption; Yellow arrow – injection site

The following Video 4 shows the scan OCT made right after the injection of 50 µl of MB 2% (w/v) solution, in which the area of the three injection points corresponds to the three MNs of the MJ600®. It can also be seen some small tissue disruptions.

Video 4. 50 µl MB injection on human skin 3.4.2 Mouse

Figure 24 shows the mouse skin after the 50 ul injection. The blue staining is very well localized in some skin structures, like the EP, hair follicles, some D content and the PC.

There are no visible disruptions of the tissue.

Figure 24. Mouse skin after 50 µl MB injection

In the following OCT data it is possible to visualize some small changes in the skin structure, as if these were very small disruptions or being only reminiscences of a previous solution flow inside the skin. There is no blebbing formation near the injection site and the skin structure remains equal to the control (Figures 25 and 26).

Figure 25. Picture of mouse skin after 50 µl MB injection taken from an OCT scan. Legend:

yellow arrow – injection site; red arrows – small disruptions

Some changes in skin strucutre are clarified in Video 5, since they appear darker as small hyporreflective areas like disruptions.

Video 5. 50 µl MB injection on mouse skin

3.5 100 µl injection

3.5.1 Human

Similarly to the histological data acquired for the volume of 50 µl, the human tissue also shows some disruptions in the papillary dermis (PD) (Figure 28). Some of them are more and larger, which could be explained by the injection of a larger volume. There is also an uniform spreading of the MB. The EP shows a more intense staining, while the D only shows a similar staining only in its fibre bundles.

Figure 26. 3-D picture of mouse skin after 50 µl MB injection. Legend: red arrows – small disruptions

Figure 27. Human skin after 100 µl MB injection. The red squares indicate tissue disruptions

In Figure 28 is possible to observe some disruptions, all taking place at the PD.

Figure 28 Picture of human skin after 100 µl MB injection taken from an OCT scan. Legend: red arrows – disruptions

It is also important to take into account that the disruptions do not have the same aspect every time. The following OCT 3-dimensional models (Figures 29-A and B) show disruptions with different appearance, which could occur after an 100 µl injection of the MB solution. In the left picture (Figure 29-A) there are some isolated disruptions, relatively close to each other in the PD. On the other hand, the second picture (Figure 29-B) shows a whole big disruption of the tissue.

Figure 29 A and B. Three-dimensional models of human skin after 100 µl MB injection. Legend: red arrows - disruptions

In the Video 6, larger disruptions are observed, also in a higher number than those obtained with a 50µl injection.

Video 6. 100 µl MB injection on human skin 3.5.2 Mouse

The histological data in the Figure 30 does not differ considerably from the one acquired using 50 μl of volume, despite the staining appearing more intense in latter, being possible to visualize in the EP, hair follicles and PC. Here, the D is also slightly blue, but there are some points more intensely stained than others, which could be due to some tissue fibres, as well as, cells nuclei.

Figure 30. Mouse skin after 100 µl MB injection

In the following OCT data (Figures 31 and 32) it is very pronounced the formation of a blebbing area, in which the skin near the injection site seems to be expanding, and then acquiring a “mountain” shape. There are no observable hyporreflective areas that could correspond to disruptions, neither other changes in the skin structure apart from the blebbing phenomenon (Video 7).

Figure 31. Picture of mouse skin after 100 µl MB injection taken from an OCT scan. Legend: AB – area of blebbing

Figure 32. Three-dimensional model of mouse skin after 100 µl MB injection.

Legend: AB – area of blebbing

Video 7. 100 µl MB injection on mouse skin

3.6 200 µl

3.6.1 Human

After an injection with 200 µl of MB, almost all skin tissue seems to be disrupted in the PD area. Figure 33 is a histological picture showing the aspect of the tissue with a great number of disruptions, despite their small dimensions. Similarly to previous results (Figures 21 and 27), the solution spreading among the skin tissue seems to be uniform, being the staining more intense in the region of EP and in some pools along all D, which are fibre bundles.

Figure 33. Human skin after 200 µl MB injection. The red squares indicate disruption areas

Figure 33 shows bigger disruptions in the skin tissue, coupled with smaller ones in the whole PD region. Moreover, it also evidences larger disruptions in the RD, in contrast to what we have observed for the last two lower injection solution volumes, 50 and 100 µl, respectively.

The following OCT picture (Figure 34) shows a high number of disruptions associated to the injection of 200 µl MB solution volume. Despite not being so large, they appear in a higher number among the entire PD layer. On the other hand, the last two OCT pictures show larger disruptions in the tissue, in addition to other smaller ones, but in lower number (Figure 33). Similarly to the third human skin histological picture, these OCT also permit to visualize some large disruptions in the RD region.

Figure 34. Picture of human skin after 200 µl MB injection taken from an OCT scan. Legend: red arrows – disruptions

This 3-D OCT modelling acquired immediately after the administration of 200 µl of the MB solution shows two injection sites, as well as some small disruptions all over the PD (Figure 35 and Video 8).

Figure 35. 3-D model of human skin after 100 µl MB injection. Legend: red arrows – disruptions; Yellow arrows – injection sites

Video 8. 200 µl MB injection on human skin

3.6.2 Mouse

In Figure 36 it is possible to observe a big and uniform MB spreading through all skin, being more concentrated in the EP and PC layers, as well as in the hair follicles and in some cell nuclei. The OCT data acquired during a scan of the skin region that was injected with 200 µl of MB is characterized by a large blebbing area (Figures 37 and 38), being more emphasized in Figure 38, and clearly visualized in the video. Despite very small, it is possible to visualize some disruptions in Video 9. It is possible to observe some hyporreflective small points in the blebbing area too.

Figure 37. Picture of mouse skin after 200 µl MB injection taken from an OCT scan. Legend: AB – area of blebbing; arrow – injection site

Figure 36. Mouse skin after 200 µl MB injection

Figure 38. 3-D model of human skin after 100 µl MB injection. Legend: AB – area of blebbing

Video 9. 200 µl MB injection on mouse skin

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.

6 Bibliography

(1) Akhthar N. Microneedles: an innovative approach to transdermal delivery - a review. International Journal of Pharmacy and Pharmaceutical Sciences. 2014, 6(4): 8- 25.

(2) Lambers H, Piessens S, Bloem A, Pronk H, Finkel P. Natural skin surface pH is on average below 5, which is beneficial for its resident flora. International Journal of Cosmetic Science. 5 Oct 2006; 28 (5): 359-370.

(3) Chapter 9: Skin. In: Young B, O’Dowd G, Woodford Phillip. Wheater's Functional Histology. 6^th edition. London, United Kingdom: Elsevier Health Sciences; 2006. p.

159-179.

(4) Amaral J, Pinto V, Costa T, Gaspar J, Ferreira R, Paz E, Cardoso S, Freitas P.

Integration of TMR Sensors in Silicon Microneedles for Magnetic Measurements of Neurons. IEEE Transactions on Magnetics. 2013; 49 (7): 3512-3515.

(5) Birchall J, Clemo R, Anstey A, John D. Microneedles in Clinical Practice–An Exploratory Study Into the Opinions of Healthcare Professionals and the Public.

Pharmaceutical Research. 2010; 28 (1): 95-106.

(6) Coulman S, Barrow D, Anstey A, Gateley C, Morrissey A, Wilke N, Allender C, Brain K, Birchall J. Minimally Invasive Cutaneous Delivery of Macromolecules and Plasmid DNA Via Microneedles. Current Drug Delivery. 2006; 3 (1): 65-75.

(7) Dul M, Stefanidou M, Porta P, Serve J, O’Mahony C, Malissen B, Henri S, Levin Y, Kochba E, Wong FS, Dayan C, Coulman SA, Birchall JC. Hydrodynamic gene delivery in human skin using a hollow microneedle device. Journal of Controlled Release [S0168-3659(17)30087-1]. 2017 Feb 28 [cited 2017 Apr 7]. Available from:

https://doi.org/10.1016/j.jconrel.2017.02.028

(8) Groves RB, Coulman SA, Birchall JC, Evans SL. An anisotropic, hyperelastic model for skin: Experimental measurements, finite element modelling and identification of parameters for human and murine skin. Journal of the Mechanical Behavior of Biomedical Materials. 2013, 18: 167-180

(9) Welzel J. Optical coherence tomography in dermatology: a review. Skin Research and Technology. 2001; 7: 1-9