Skin Models for Screening of Topical Delivery

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STOCKHOLM SWEDEN 2020,

Skin Models for Screening of Topical Delivery

JOHANNA CARLSSON

KTH ROYAL INSTITUTE OF TECHNOLOGY

SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

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1

Abstract

The interest of drug delivery via the skin is progressively increasing due to its convenience and affordability. However, the skin has an effective barrier function which hinders drugs to penetrate. This can be overcome, using topical formulations, which contain vehicles optimized for penetration into skin or permeation through skin into underlying tissues or blood stream.

In development of transdermal drugs, ex-vivo skin models are frequently used for permeability studies.

However, these models may suffer from limited reproducibility, due to their biological variability, and are related to ethical issues. This has led to development of in-vitro skin models. These new skin models need to be verified by comparison to the existing models before applied in drug studies.

In this project, penetration and permeation of proteins and small molecules was studied using an ex- vivo pig skin model. The effect of topical formulations and penetration enhancers was also investigated.

The results showed a significant increase in penetration and permeation in the absence of stratum corneum, proving its barrier effect. While no significant differences could be seen regarding molecule size in the presence of stratum corneum (intact skin model), there was significantly less permeation measured for large protein molecules tested than for smaller tested compounds in the absence of the stratum corneum (damaged skin model). The permeation and penetration were slightly increased in presence of the penetration enhancing limonene oil.

Key words: Transdermal delivery, biological actives, topical formulations, penetration enhancers, skin models, permeation through skin, penetration into skin

Abstrakt

Intresset för läkemedel som tas upp genom huden ökar ständigt eftersom administreringen är både bekväm och lättillgänglig. Däremot har huden en effektiv barriärfunktion, vilket hindrar penetration av läkemedlen. För att lösa detta problem kan läkemedlen presenteras i olika formuleringar, vilka innehåller transportmedel anpassade för att öka penetrationen in i huden eller permeationen genom huden, till underliggande vävnad samt blodomloppet.

Vid utvecklandet av läkemedel som ska tas upp genom huden används ofta ex-vivo hudmodeller i penetrations- och permeationsstudier. Dessa modeller kan emellertid ha otillräcklig reproducerbarhet, på grund av biologisk variation, samt är kopplade till etiska problem. Detta har lett till utvecklandet av hudmodeller av in-vitro typ. Dessa nya hudmodeller måste verifieras genom jämförelse med existerande modeller, innan de används i läkemedelsstudier.

I det här projektet studerades penetration och permeation av proteiner och små molekyler i en ex-vivo hudmodell från gris. Effekten av olika formuleringar och penetrationsförstärkare undersöktes också.

Resultaten visade en signifikant ökning av penetration och permeation vid avsaknad av stratum corneum, vilket påvisar dess barriärfunktion. Samtidigt som ingen märkbar skillnad gällande molekylstorlek kunde urskiljas vid närvaro av stratum corneum (intakt hudmodell), var permeationen av större molekyler tydligt minskad i jämförelse med små molekyler vid avsaknad av stratum corneum (skadad hudmodell). Både penetrationen och permeationen var svagt förhöjda vid närvaro av penetrationsförstärkaren limonen.

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Table of contents

Introduction... 3

Topical formulations ... 3

Topical formulations of biologically active substances ... 3

Types of penetration enhancers for topical delivery through skin ... 4

Skin structure ... 4

Assessment of the key aspects of transdermal delivery analysis... 6

Skin models for permeation and penetration studies ... 7

Ex-vivo skin models ... 7

In-vitro skin models ... 7

Objectives ... 9

Materials and methods ... 10

Materials ... 10

Methods ... 10

Collecting ex-vivo skin samples... 10

Labelling of proteins ... 11

Solvent exchange ... 11

Penetration experiments by Franz cells ... 12

Cryosectioning by microtome ... 14

Fluorescence microscopy ... 14

Results ... 15

The effect of stratum corneum ... 15

Permeation of actives through intact and damaged skin ... 15

Penetration of actives into intact and damaged skin ... 18

Limonene as a penetration enhancer ... 19

Permeation through intact skin using limonene as a penetration enhancer ... 19

Penetration into intact skin using limonene as a penetration enhancer... 21

Permeation through damaged skin using limonene as a penetration enhancer ... 22

Penetration into damaged skin using limonene as a penetration enhancer ... 24

Discussion ... 25

Future perspectives ... 26

References... 27

Tables and figures ... 28

Appendices ... 29

Appendix 1. Protocol for size exclusion chromatography... 29

Appendix 2. Protocol for using Amicon® Ultra-0.5 centrifugal filters ... 29

Appendix 3. Protocol for penetration experiments using Franz cells ... 30

Appendix 4. Protocol for cryosectioning by microtome ... 31

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3

Introduction

The interest of drug delivery via the skin is progressively increasing due to its convenience and affordability [1]. The non-invasive route of drug administration has advantages such as decreased side-effects and prolonged therapeutic effect [2].

The skin is an ideal site for drug administration as the drugs can be delivered both locally and systematically [1]. However, the skin has an effective barrier function to protect us from chemical and physical assaults and invasion of pathogens, as well as regulating levels of water and solutes. The low permeability of the skin also hinders drugs to penetrate [3]. This drawback can be overcome using topical formulations, which contain vehicles optimized for penetration through skin [4].

In development of transdermal drugs, ex-vivo skin models (e.g. excised human skin or animal skin) are frequently used for permeability studies of transdermal drugs. However, these models often suffer from limited reproducibility, due to their biological variability, and are also hampered by some legal and ethical issues. These issues have led to development of in-vitro 3D skin models, hoping a better reproducibility could be provided by these models [5].

For transdermal delivery of biological actives (i.e. biological substances with the ability to activate cellular response) through or into the skin, delivery vehicles need to be developed and optimized. This requires development of reliable analytical methods and skin models for evaluation of vehicle efficiency. Testing of some of the possible methods and models was the core of this work.

Topical formulations

Topical formulations of biologically active substances

Topical formulations are applied to a limited part of the body with interface sites such as skin (e.g. lotions, creams and dermal patches) or mucus membranes (e.g. nasal spray and eye drops).

They consist of active substances and vehicles optimized for penetration through or adhesion to a particular site of the body [4]. Topical formulations of biologically active substances active substances for delivery through or into the skin was at focus in this project.

Biological actives are commonly used as therapeutic drugs due to their ability to activate cellular response. The actives, which are usually large proteins, could for example be growth factors, promoting wound healing, or agents to promoting immune response [6, 7]. However, the therapeutic proteins, especially antibodies, are often too large for transcellular diffusion, too hydrophilic for passive diffusion through the lipid rich uppermost layer of the skin (stratum corneum), and the required doses are often higher than what can be delivered through diffusion via hair follicles and sweat glands [6].

To overcome the penetration problem of biological actives such as proteins into the skin, specific vehicles need to be formulated and optimized. The vehicles for skin delivery are designed to enhance skin penetration or to moisturize the skin and may consist of water, alcohol, propylene glycol or oil mixed with emulsifiers, absorption/penetration promoters and often also preservatives and fragrances [4].

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4 Types of penetration enhancers for topical delivery through skin

Chemical penetration enhancers are chemical substances which aim to reversibly disrupt the structure of stratum corneum, making the skin more permeable. One example of chemical penetration enhancers is lipophilic penetration enhancers, e.g. hydrophobic solvents, which can be used for transportation of substances through the hydrophobic layer. However, they may cause aggregation or denaturation of the proteins reducing the protein functions. Non-ionic surfactants can therefore be added as they have the ability to protect hydrophilic proteins in a hydrophobic phase, while simultaneously enhancing penetrating through the lipid barrier stratum corneum part of the skin. This have successfully been used for insulin with no significant loss of activity. Lipophilic terpene-based penetration enhancers are also commonly used as they increase the fluidity of the lipid extracellular matrix by association with the lipids in stratum corneum [6].

Peptide-based penetration enhancers are short amino acid sequences which damage cell membranes by entering the cells without the use of specific receptors, creating pore-like routes.

These are of particular interest for topical delivery of proteins as they may reduce toxicity compared to chemical penetration enhancers, and have been successfully used to topically deliver elastin, fluorescein and anti-scarring agents [6].

Another class is physical penetration enhancers. An example of those are microneedle arrays, which have grown in popularity in recent years. It is a minimally invasive procedure where thin needles pierce the stratum corneum, reaching only into the underlying viable epidermis.

The assay can either be used to deliver drugs directly into the skin or to pre-treat the skin to improve the permeability for topical formulations [7]. Other types of physical penetration enhancers include ultrasound, which create small bubbles in the targeted tissue allowing fluid to pass through the stratum corneum as the cells collapse, and electroporation, where transient pores are formed in the barrier by applying high-voltage pulses across the skin [6]. It has also been shown that some physical penetration enhancers are able to deliver fibroblast growth factor, promoting repair and regeneration of tissue [7].

Skin structure

Skin is our largest organ, accounting for more than 10% of body mass with a surface area of about 2 m2. It works as an effective barrier, protecting us from chemical and physical assaults and invasion of pathogens, as well as regulating levels of water and solutes. Human skin is typically about 2-3mm thick and consists of three different layers: epidermis, dermis and hypodermis [3, 8].

Figure 1. Schematic picture of the skin structure [3]

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5 The primary barrier function comes from the stratum corneum, the uppermost layer of the epidermis, which has very high density and low hydration. The 10-15µm thick stratum corneum consists of around 15-25 layers of flattened, stacked and cornified cells (corneocytes).

However, the corneocytes (cells containing keratin bundles) make up only 5% of the stratum corneum weight. The main barrier function comes from extracellular matrix of lipids, arranged into bilayers in the intercellular spaces of the stratum corneum. These lipid bilayers consist of ceramides (40-50%), fatty acids (15-25%), cholesterol (20-25%) and cholesterol sulphate (5- 10%). The lipids are essential for maintaining skin structure and help to avoid trans-epidermal water loss [6, 7].

Figure 2. Simplified illustration of the stratum corneum [3]

The stratum corneum is fully renewed once every 2-3 weeks as corneocytes originating in the viable epidermis continuously collapse into a flattened shape. The epidermis therefore contains cells with varying levels of differentiation which further facilitates the barrier function of the stratum corneum [3].

The dermis is 0,1-0,5mm thick and consists mainly of collagenous and elastin fibers, promoting the elasticity of the skin. The dermis contains nerves, blood vessels, lymphatic vessels, sweat glands and hair follicles which contribute to skin nutrition and repair as well as thermal regulation and immune response. Another important property of the dermis is its ability to remove waste from the skin [3].

The hypodermis, which is the deepest layer of the skin, acts as a heat insulator, shock absorber and energy storage as well as connecting the skin to underlying muscle tissue. It consists mainly of fat cells [3].

The penetration routes of the skin through the stratum corneum include:

• The route through skin appendages, such as hair follicles and sweat glands which form pathways through the intact epidermis

• The transcellular route, crossing through the corneocytes

• The intercellular route in-between the corneocytes through intervening lipids.

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6 Transcellular diffusion is a fairly uncommon route for transdermal drugs, which are usually hydrophilic, as the route requires diffusion through lipophilic cell membranes. Intercellular diffusion however, is acceptable for transdermal diffusion as the interlamellar regions in the stratum corneum contain more flexible hydrophobic chains and less ordered lipids, creating non-planar spaces between the cells [6, 3].

Figure 3. Schematic representation of penetration routes throughout the stratum corneum [3].

Assessment of the key aspects of transdermal delivery analysis

Two of the key aspects to consider when evaluating a new transdermal drug is permeation and penetration, whereas the first indicates the rate at which the substance is transported through all skin layers and the latter the transportation of the substance into different skin layers. The skin varies in thickness over the body, resulting in different permeation, penetration and distribution in cells and tissue at different administration sites.

The permeation of actives through the skin is commonly evaluated by the use of Franz-type diffusion cells, where a skin sample is attached as a membrane between the donor (containing the transdermal drug) and receptor phase. Samples are collected from the receptor liquid several times throughout the experiment, so they can be analysed and compared with the added formulation in the donor compartment.

The penetration of actives into different skin layers can be evaluated by cross-sectioning of the skin. Cryosectioning by microtome is a popular approach for this, slicing thin pieces of the skin which can be evaluated in a microscope. A common approach is to label the molecule with a fluorescent dye to be able to evaluate the penetration with fluorescent microscopy.

Distribution in different skin cells and tissues is also an important aspect, as different actives have different assigned destinations. For example, wound healing can be promoted by absorption of growth factors in the skin, attaching to extracellular receptors, while other actives must enter the cells to cause effect.

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7 Skin models for permeation and penetration studies

Ex-vivo skin models

Skin penetration studies are essential when evaluating drugs for dermal or transdermal application. The choice of ex-vivo skin models is of big importance for these studies. Although the ideal model would be human skin, excised animal skin is more commonly used as it is more easily accessible due to ethical issues related to the use of human skin for scientific experiments. From a study performed at the Novartis Research Institute in Vienna, comparing the penetration properties of different ex-vivo skin models (human, pig and rat skin), using four different topical dermatological drugs, pig skin was found to be a suitable model for the human skin. The experiments using terbinafine and clotrimazole as active compounds gave almost identical values in permeation rate with human skin and pig skin, and for hydrocortisone and salicylic acid the difference was about twofold, with the human skin yielding higher values than pig skin. These differences are considered small in comparison to rat skin which had up to 55 times higher penetration rate than the human skin in the study [9].

The storage of skin samples has shown to influence the skin models’ properties. A study at School of Pharmaceutical Sciences of Ribierão Preto in Brazil was performed to evaluate the influence of freezing of skin samples prior to permeation studies. Analysis of fluorescein permeation through stored frozen porcine ear skin gave almost twofold higher values compared to fresh skin, however, the differences related to storage time in freezer (15 days and 30 days) were negligible. By analysis by fluorescent microscopy and photomicrographs it was possible to see some parts of the epidermis were thinner in the frozen skin, in comparison to fresh skin, resulting in higher permeation [10].

While not completely reproducing human skin in all aspects, the frozen ex-vivo pig skin was considered to be a sufficiently good model used of human skin for the purpose of this study and was used for relative comparison of skin penetration and permeation of different formulations and actives. Better ex-vivo or in-vitro models would be though needed for studies related to metabolism of viable skin cells.

In-vitro skin models

Instead of using excised animal skin as a replacement for human skin in skin penetration studies of candidate drugs, reconstructed human skin or epidermis equivalents can be used as alternatives, especially for studies related to metabolism of viable skin cells such as toxicity, growth and synthesis of biomarkers. The in-vitro models on the market can generally be categorized into reconstructed epidermal equivalents or reconstructed full-thickness models [5, 9, 2].

The reconstructed epidermal equivalents are manufactured by cultivating human epidermal keratinocytes on polycarbonate membranes or collagen matrices. This promotes cell differentiation, resulting in a reconstructed artificial epidermis with a functional stratum corneum. The models can differ in cultivation process and choice of membrane, resulting in slightly different models. Models with different characteristics resemble different properties of human skin, which make them suitable for different types of skin studies. For example, EpiDermTM is an excellent model for epidermal penetration studies as the model has a lipid content similar to the native human skin [5].

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Table 1. Commercially available reconstructed dermal equivalent models [5].

Skin model Company Epidermis eq.

thickness

Stratum corneum eq.

thickness EpiSkin^TM SkinEthic ~ 7 cell layers

38-48µm

60-100 cell layers 73-102µm SkinEthic^TM RHE SkinEthic 5-9 cell layers

23-59µm

14-24 cell layers 15-32µm

EpiDerm^TM MatTek 6-8 cell layers

28-43µm

16-25 cell layers 12-28µm

Reconstructed full-thickness models consist of epidermal keratinocytes and dermal fibroblasts, cultured to form a differentiated skin model. The existence of both epidermal and dermal components results in models with more complex barrier functions than the reconstructed epidermal equivalents-models. The NIKS® keratinocyte progenitors, used in the StrataTestTM

skin model, are exceptional at reproducing normal human skin tissue architecture and barrier function upon cultivation. The cells have the ability to undergo normal epidermal differentiation, generating a fully stratified epithelium, almost identical to native human skin [5].

Table 2. Commercially available reconstructed full-thickness models [5].

Skin model Company Special features

StrataTest^TM StrataTech Corporation Cultured NIKS^® keratinocyte progenitors

EpiDerm FT MatTek Presence of signaling proteins

EpiCS^TM CellSystems Cells cultivated on a polycarbonate membrane

A study performed at the Novartis Research Institute in Vienna compared two in-vitro skin models with human skin, whereas one of them was SkineticTM RHE. The two models had similar permeation in the experiments with the hydrophobic compounds (terbinafine, clotrimazole and hydrocortisone) but in comparison to human skin the permeation rates were over 28 times higher. For salicylic acid, however, the permeation rate for SkineticTM RHE was only seven times higher than for ex-vivo human skin [9].

The in-vitro models aspire to reproduce human skin, but despite their structural similarity their barrier function is still significantly weaker, making the models much more permeable than native skin. Further research and studies are essential to improve current models and develop new ones for the study of human skin penetration [5].

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9 Objectives

The objective of this thesis project was to develop reproducible methods with high sensitivity for studies of transdermal delivery of proteins.

In particular to:

• Develop a standardized methodology to fluorescently label proteins

• Study penetration and permeation of labelled proteins and small fluorescent molecules using ex-vivo pig skin model

• Compare penetration and permeation of the labelled large proteins with small molecules to study influence of molecule size on barrier function of the skin

The developed methods should to be used for later permeation and penetration studies for evaluation of penetration enhancers and transdermal formulations.

Collection of a data set for penetration and permeation of different molecules in pig skin was also an important aspect of this project. The collected data should serve as reference values for evaluation of performance of in-vitro skin models.

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Materials and methods

Materials

Below are the chemicals and other materials used throughout the project listed.

Table 3. Chemicals used throughout the project.

Chemical Company Product no. Properties

Alexa Fluor^TM 488 Invitrogen A20000 Mixed isomers

Mw: 643Da

Bovine Serum Albumin (BSA) Sigma-Aldrich A2153 Lyophilized powder Mw: 66,4kDa Dimethyl sulfoxide (DMSO) Sigma-Aldrich 41640

DL-Limonene Merck 814546 Mix of D- and L-form

(~1:1)

Fluorescamine (FA) Sigma-Aldrich F9015

Fluorescein sodium salt (NaFITC) Sigma-Aldrich 46960 Mw: 376Da Phosphate buffered saline tablets (PBS) Sigma-Aldrich P4417 pH: 7,4

Polysorbate 80 Sigma-Aldrich 59924

Sodium dodecyl sulfate (SDS) Sigma-Aldrich 75746 Pellets

Table 4. Other materials used throughout the project.

Material Company

Amicon® Ultra-0.5 Centrifugal filters Millipore

Liquid nitrogen Linde

PD-10 desalting columns GE Healthcare

Pig skin (from inner part of pig ears) Lövsta Kött AB

Methods

Collecting ex-vivo skin samples

Ex-vivo skin samples were collected from the inner part of pig ears. The ears were obtained as rest products after slaughter of pigs for human consumption from Lövsta Kött AB slaughterhouse (Funbo-Lövsta, Uppsala). The ears were received and processed the day after slaughter. 2-4 pieces (dimensions: ~2x6cm, thickness: 1-2mm) could be collected from each ear as illustrated in figure 4. The prepared skin pieces were kept frozen at -85ºC until used for permeation experiments.

Figure 4. Illustration of how skin pieces were collected (modified picture from dribbble.com [11])

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11 Labelling of proteins

The standardized procedure for protein labelling was set as adding 12,5µL Alexa (10mM in DMSO) to 2,77mL BSA (2mg/mL in PBS), corresponding to a labelling ratio of 1,5 (mol dye/mol protein). The labelling mixture was left on a rocking-board mixer in room temperature one hour and in cold room (4ºC) over night (~18 hours). The labelled mixture was stored in fridge (4ºC) and size exclusion chromatography was performed within 24 hours after termination of labelling procedure.

The labelled protein mixture was run twice through size exclusion PD-10 chromatography columns to eliminate any free Alexa from the sample, while each time a new column was used.

PD-10 desalting columns was used following the gravity protocol (see appendix 1), and PBS buffer, pH 7,4, was used as equilibration buffer. A loading volume of 2,5mL and an elution volume of 3,5mL resulted in a 1,96x dilution of the primary sample.

Evaluation of the labelling efficiency was executed after the two size exclusion procedures, by fluorescence measurements in a plate reader. Since BSA is not fluorescent, 20µL fluorescamine (4mg/mL in acetone) was added to each well of 180µL sample, to be able to evaluate the protein concentration. The excitation and emission wavelengths used for evaluation can be seen in table 5.

The concentration of dye and protein was estimated by comparison of measured fluorescence with the corresponding calibration curve (see figures 28 and 29 under Tables and figures), and the labelling ratio after SEC was 0,35 ± 0,1 (mol dye/mol protein). The protein concentration after two SEC was 0,70 ± 0,1mg/mL.

Table 5. Excitation and emission wavelengths used for evaluation in plate reader.

Substance to be evaluated Excitation wavelength Emission wavelength

BSA (+ fluorescamine) 390nm 465nm

Alexa / NaFITC 490nm 520nm

Solvent exchange

For the experiments with limonene, the original PBS solvent used for protein labelling was exchanged after the second size exclusion by concentrating the labelled proteins using Amicon® Ultra-0.5 centrifugal filters (with a cut-off of 30kDa) following the protocol in appendix 2, and then diluting the concentrate in the limonene buffer (10% limonene, 0,1%

PS80 in PBS). The protein concentration of the concentrate was evaluated in plate reader before dilution in the new buffer.

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12 Penetration experiments by Franz cells

Penetration studies were performed using the PermeGear 6-station stirrer (V6-CA-02) equipped with 6 jacketed Franz cells with penetration areas of 0,64 cm2 and receptor volumes of 5mL (4G-01-00-09-05).

The skin pieces were prepared by bisection and put in PBS buffer to avoid drying of skin pieces.

For the experiments with damaged skin, the stratum corneum was peeled off after soaked in 60°C water for 90 seconds (can be seen in picture 5).

The penetration experiments were carried out at 32ºC, following the protocol described in appendix 3, adding 400µL of test liquid in the donor compartments. The accepted concentration of each substance in the test liquids are stated in table 6.

Table 6. The accepted concentrations for Franz cells experiments.

Substance Accepted concentration

BSA (labelled with Alexa) 10,00 ± 1,5 µM

Alexa 3,33 ± 0,2 µM

NaFITC 10,00 ± 0,2 µM

Samples (~200µL) were collected from the receptor phase each hour the first six hours of the experiment as well as after 24 hours. To avoid excitation of the fluorescent substances, the equipment was wrapped in aluminum foil throughout the entire experiment.

At termination of the experiments, after 24 hours, 3 samples á 200µL was collected from each receptor cell, before removal of donor liquid and disassembling of cells. To prevent any further penetration during storage, the skin pieces were cleaned by addition and removal of PBS in the donor compartment. After disassembling, excessive liquid was removed from the skin pieces by pat-drying with a paper towel, before wrapping them up in parafilm and aluminum foil. In case there was no donor liquid left at termination, the skin piece was discarded. The skin pieces were stored in freezer (-18ºC) until processed by microtome (within up to four weeks).

The samples were run in plate reader within 5 days from sampling (stored in fringe until processed) and evaluation of permeation was done by comparison of fluorescence of the collected samples to a full penetration standard. The full penetration standard was created by dilution of the test liquid in PBS, proportional to 400µL diluted to a total volume of 5mL.

Figure 5. Peeling off the stratum corneum for the damaged skin-experiments.

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Figure 6. An illustration of the components of a Franz cell [12].

Figure 7. Set-up of six penetration experiments using Franz cells.

Figure 8. Skin piece after termination of experiment.

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14 Cryosectioning by microtome

Cryosectioning by microtome of the skin samples was performed after penetration experiments according to the protocol in appendix 4. Cross-sectioning of 50µm thick slices from the center of the penetration area at a temperature of -10ºC, using the microtome Leica-RM2265 equipped with Cryo-set LN22.

The slices were put on glass slides and stored in room temperature, in large petri dishes and wrapped in aluminum foil to avoid excitation of the fluorescent molecules. Imaging by fluorescent microscope was performed within 24 hours after cutting.

Fluorescence microscopy

Penetration of fluorescent molecules into the skin was evaluated using the fluorescence microscope Zeiss Axiovert 100 with reflector slider equipped with blue excitation filter (pass wavelengths 450-490nm) and green emission filter (pass wavelengths 510-560nm). Pictures were taken at 5x, 10x and 20x optical magnification with a 3,6-digital zoom of the camera. The camera (Canon Powershot S80) had the settings ISO 400, 1’’, F/8 in all pictures under results.

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Results

The effect of stratum corneum

Permeation of actives through intact and damaged skin

Results of skin permeation experiments obtained using Franz diffusion cells are shown in figures 9 and 10 for intact skin and in figure 11 and 12 for damaged skin. Clearly, the permeability of intact skin was lower than damaged skin for all three tested compounds, both for Alexa labelled Bovine Serum Albumine (BSA-Alexa) with molecular weight of about 66,4 kDa as well as for Sodium Fluorescein (NaFITC) and free-Alexa dye with molecular weights order of magnitude lower. When compared to the hypothetical full penetration, i.e. situation at which all added fluorescent molecules would permeate through the skin into receptor liquid of the Franz Cell, the average permeability of intact skin over 24 hours was very low: 0,12% for BSA-Alexa, 0,31% for NaFITC and 0,20% for Alexa (see figure 10). For damaged skin the permeability was substantially higher: 14,51% for BSA-Alexa, 41,98% for NaFITC and 42,79% for Alexa (se figure 12). Removal of the stratum corneum thereby increased the permeability by more than hundred times for all tested compounds.

In intact skin, all three molecules had very similar average permeation over 24 hours, 0,12%- 0,31% (see figure 10), regardless of size. Possible differences in permeability of the tested molecules are less than the experimental error related to quantification of low permeation through stratum corneum barrier.

After removal of the stratum corneum the smaller molecules were clearly more permeable than the larger protein, with average permeations of 41,98-42,79% compared to 14,51% (see figure 12).

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Figure 9. Permeability of intact skin with substances in PBS solutions.

[Based on average values from 2 experiments with BSA-Alexa and free Alexa, and 3 experiments with NaFITC]

Figure 10. Summary of the permeability of intact skin over 24h, with substances in PBS solutions.

[Based on average values from 2 experiments with BSA-Alexa and free Alexa, and 3 experiments with NaFITC]

0.0%

0.2%

0.4%

0.6%

0.8%

1.0%

0 5 1 0 1 5 2 0 2 5

PERMEABILITY

HOURS

P ER M EA B I LI T Y - I N TACT S KI N ( I N P B S )

BSA-Alexa NaFITC Alexa

0.12%

0.31%

0.20%

0.0%

0.2%

0.4%

0.6%

0.8%

1.0%

PERMEABILITY

P ER M EA B I LI T Y OVER 24H - I N TACT S KI N ( I N P B S )

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Figure 11. Permeability of intact skin with substances in PBS solutions.

[Based on average values from 4 experiments with BSA-Alexa, 3 experiments with NaFITC and 2 experiments with free Alexa]

Figure 12. Summary of the permeability of damaged skin over 24h, with substances in PBS solutions.

[Based on average values from 4 experiments with BSA-Alexa, 3 experiments with NaFITC and 2 experiments with free Alexa]

0%

10%

20%

30%

40%

50%

60%

0 5 1 0 1 5 2 0 2 5

PERMEABILITY

HOURS

P ER M EA B I LI T Y - DA M AG ED S KI N ( I N P B S )

14.51%

41.98% 42.79%

0%

10%

20%

30%

40%

50%

60%

PERMEABILITY

P ER M EA B I LI T Y OVER 24H - DA M AG ED S KI N ( I N P B S )

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18 Penetration of actives into intact and damaged skin

From the analysis performed with fluorescence microscope, differences in penetration between intact and damaged skin could be noted, whereas damaged skin was significantly more penetrated than intact skin. This can be seen in figures 13-15, where the fluorescence is much wider spread in damaged skin. For the intact skin, the fluorescence is mostly located within the uppermost part of the skin indicating a functional barrier function of stratum corneum.

For all three compounds, i.e. BSA-Alexa, NaFITC and free Alexa, the penetration is clearly hindered by the stratum corneum, in good agreement with low permeation values presented in the previous section.

When removing the stratum corneum (i.e. damaged skin samples) only minor differences in penetration could be seen between the compounds. The high fluorescence intensity in the uppermost part could be due to some accumulation of free Alexa (see figure 13B, 14B and 15B).

For the small molecules, the penetration of Alexa in damaged skin seems to be slightly higher than for NaFITC as a higher intensity of fluorescence can be noted (see figures 14B and 15B).

However, this could be a result of the different fluorescence intensities of the two compounds.

Figure 13. Penetration of BSA-Alexa (in PBS) through intact skin (A) and damaged skin (B).

[Photographed with identical camera settings. 18x and 36x indicate different magnifications, i.e. magnification of the objective used multiplied by the digital zoom used]

Figure 14.Penetration of NaFITC (in PBS) through intact skin (A) and damaged skin (B).

Figure 15. Penetration of Alexa (in PBS) through intact skin (A) and damaged skin (B).

A – 18x B – 18x

18X 18X

18X 36X 18X 36X

36X 36X

A – 18x

A – 36x

B – 36x B – 18x

B – 36x

B – 36x B – 18x

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19 Limonene as a penetration enhancer

Permeation through intact skin using limonene as a penetration enhancer

The permeability of BSA-Alexa through intact skin was slightly improved when administered in emulsion kind of formulation containing limonene oil (10% limonene, 0,1% polysorbate 80 in PBS buffer) in comparison to formulation in bare phosphate buffered saline (PBS). The permeation over 24 hours increased from 0,12% in bare PBS to 0,25% in presence of limonene (see figure 17). However, the experiments with NaFITC did not show the same trend. Instead the average permeation over 24 hours decreased from 0,31% to 0,08% in presence of limonene.

For both tested compounds, BSA-Alexa and NaFITC, the observed changes could still be within the range of the experimental error for low permeation, which could be as high as 0,2%

for NaFITC in PBS (see figure 10 and figure 19).

Figure 16. Permeability of BSA-Alexa through intact skin in two different solutions.

[Based on average values from 2 experiments with PBS and Limonene]

Figure 17. Summary of the permeability of BSA-Alexa through intact skin in two different solutions.

[Based on average values from 2 experiments with PBS and Limonene]

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Figure 18. Permeability of NaFITC through intact skin in two different solutions.

[Based on average values from 3 experiments with PBS and 1 experiment with Limonene]

Figure 19. Summary of the permeability of NaFITC through intact skin in two different solutions.

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21 Penetration into intact skin using limonene as a penetration enhancer

The analysis with fluorescence microscope showed that in presence of limonene, the stratum corneum still retained an efficient barrier function. This is indicated in the pictures 20B and 21B, by the dense fluorescence in the top layer of the intact skin. However, an increase in penetration could still be noted for both compounds as the fluorescence is slightly wider spread through the skin in the presence of limonene (see pictures 20 and 21).

When comparing the pictures of the different compounds, the penetration into intact skin with BSA-Alexa seemed to be slightly higher than with NaFITC, in the presence of limonene, as the fluorescence is more intense (see figures 20B and 21B) inside the skin for BSA-Alexa.

However, since the limonene experiment was not done in repeats, no conclusions should be drawn from this.

Figure 20. Penetration of BSA-Alexa into intact skin in bare PBS (A) and in presence of limonene (B).

Figure 21. Penetration of NaFITC into intact skin in bare PBS (A) and in presence of limonene (B).

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22 Permeation through damaged skin using limonene as a penetration enhancer

The permeability of damaged skin was slightly improved in the presence of limonene in comparison to actives formulated in bare phosphate buffered saline (PBS), for both BSA-Alexa and NaFITC. The average permeation over 24 hours was increased from 14,51% to 19,82% for the labelled protein BSA-Alexa (see figure 23), and from 41,98% to 50,08% for low molecular weight compound NaFITC (see figure 25). For both compounds, permeation measured in the presence of limonene improved beyond the experiments error of measured in bare buffer, however, more measurement in the presence of limonene would still be needed to confirm these results.

Figure 22. Permeability of BSA-Alexa through damaged skin in two different solutions.

Figure 23. Summary of the permeability of BSA-Alexa through damaged skin in two different solutions.

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Figure 24. Permeability of NaFITC through damaged skin in two different solutions.

Figure 25. Summary of the permeability of NaFITC through damaged skin in two different solutions.

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24 Penetration into damaged skin using limonene as a penetration enhancer

The analysis with fluorescence microscope showed an increase in penetration for damaged skin in the presence of limonene. This could be noted for both compounds as the fluorescence intensity was visibly higher in the skin that was exposed to formulations with limonene, compared to the same experiments using PBS (see pictures 26 and 27).

Figure 26. Penetration of BSA-Alexa into damaged skin in bare PBS (A) and in presence of limonene (B).

Figure 27. Penetration of NaFITC into damaged skin in bare PBS (A) and in presence of limonene (B).

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25

Discussion

Removal of the stratum corneum significantly increases skin permeability and penetration by actives, which proves the efficient barrier function of stratum corneum. The permeability increase by removal of stratum corneum was more than hundred-fold for all tested compounds, i.e. the large labelled protein BSA-Alexa and small compounds of NaFITC and free Alexa label.

A trend of smaller molecules being more permeable and more easily penetrated than proteins could be seen in both intact and damaged skin. The differences were highly noticeable in damaged skin, however small and within the range of experimental error in intact skin. No conclusions can therefore be made regarding the effect of molecule size in intact skin, but it clearly has an effect in damaged skin.

The minimal difference in permeation and penetration of intact skin between the small molecules and the protein could indicate that even the smaller molecules (the smallest molecule NaFITC, 376.27Da) are too large to pass through the stratum corneum. To evaluate this aspect, permeation and penetration of even smaller molecules would need to be tested. This observation is somewhat in disagreement with some references [13, 14] stating molecules smaller than 500Da can pass stratum corneum. Perhaps highly hydrophilic character (and poor interaction with the skin lipids) of the compounds tested in this study played a role and caused lower permeability than expected based on reports.

In the presence of limonene, an improvement of permeation and penetration could be seen, especially for the damaged skin. The experiments should be repeated in order to draw conclusions.

The improvements of permeation of intact skin in this study by using limonene, was barely two-fold at its highest, not even permeating 1% through the skin over 24 hours. Although a slight increase could be seen, the solvent cannot be considered effective enough for transdermal delivery of biological actives based on the results in this study. Further research on penetration enhancers would be needed and tested to find a suitable solvent. One approach might be to use higher concentration of limonene.

The Franz cells and plate reader, used for evaluating permeation, proved to be sensitive methods allowing for quantitative analysis. The cryo-sectioning and fluorescence microscope evaluation had lower sensitivity, whereas differences could be distinguished but not quantitatively analyzed. For further evaluation of penetration, a method must be optimized to achieve higher sensitivity.

As the permeation and penetration studies were carried out during this project, data sets were collected for different molecules in ex-vivo pig skin. However, some experiments would need to be repeated to have enough results to draw conclusions. The next step would be to compare the results from this skin model with in-vitro skin models.

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Future perspectives

Throughout this project, the methods for penetration and penetration have been tested, evaluated and (some of them) optimized. Permeation and penetration of small molecules and proteins have been compared and a set of data have been collected for ex-vivo pig skin, which were the objectives of this project.

For future studies, the same experiments can be carried out in in-vitro skin models to evaluate their barrier function. Since in-vitro skin models have no legal or ethical issues related them, as well as have high reproducibility, they would be preferred over ex-vivo skin models in evaluation of new transdermal drugs.

Another aspect of future studies is development of topical formulations to enhance penetration of biological actives through stratum corneum. Drug administration via the skin is of great interest as it is a very convenient route for the user and has the ability of both local and systematic drug delivery. Development of topical formulations is essential for administration of biological actives and for development of new transdermal drugs for topical delivery.

Biological actives are of great interest due to their ability to activate cellular response.

Chemical drugs could give similar effects, however, by activating cellular response the risk of immunological response of the unknown chemical substance, is eliminated.

Acknowledgements

I would primarily like to thank my external supervisor Maja Hellsing at RISE for giving me the opportunity to carry out this interesting skin project. Also, for her supervision during the whole process.

The co-supervisor Lubica Macakova have been of great help throughout the entire project, and to her I am eternally grateful. She has supported me through the toughest times, making me able to finish my thesis.

I would also like to thank my supervisor My Hedhammar for supervision and making sure I pass the KTH requirements.

My special thanks are also assigned the examiner Qi Zhou for being flexible and letting me finish my thesis outside the assigned timeframe, due to illness.

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References

[1] M. Sharadha, D. V. Gowda, N. Vishal Gupta and A. R. Akhila, "An overview on topical drug delivery system," International Journal of Research in Pharmaceutical Sciences, vol. 11, no. 1, pp.

368-385, 2020.

[2] D. Prakash, A. P. Singh, N. S. Katiyar, K. Pathak, D. Pathak and Arti, "Penetration enhancers:

Adjuvants in transdermal drug delivery system," World Journal of Pharmacy and Pharmaceutical Sciences, vol. 5, no. 5, pp. 353-376, 2016.

[3] C. L. Domínguez-Delgado, I. M. Rodríguez-Cruz and M. López-Cervantes, "The Skin: A Valuable Route for Administration of Drugs," in Current Technologies to Increase the Transdermal Delivery of Drugs, Chapter 1, Bentham Science Publishers Ltd., 2010, pp. 1-22.

[4] A. Oakley, "Topical formulations," DermNet NZ, February 2016. [Online]. Available:

https://www.dermnetnz.org/topics/topical-formulations/. [Accessed 8th June 2020].

[5] F. Rodrigues and M. B. P. Oliveira, "Cell-based in vitro models for dermal permeability studies," in Concepts and Models for Drug Permeability Studies, Elsevier Ltd., 2016, pp. 155-167.

[6] N. E. Stevens and A. J. Cowin, "Overcoming the challenges of topical antibody administration for improving healing outcomes: A review of recent laboratory and clinical approaches," Wound Practice and Research, vol. 25, no. 4, pp. 188-194, 2017.

[7] A. Z. Alkilani, M. T. McCrudden and R. F. Donnelly, "Transdermal Drug Delivery: Innovative Pharmaceutical Developments Based on Disruption of the Barrier Properties of the stratum corneum," Pharmaceutics, vol. 7, pp. 438-470, 2015.

[8] E. Proksch, J. M. Brandner and J.-M. Jensen, "The skin: An Indespensable Barrier," Experimental Dermatology, 17, p. 1063–1072, 2008.

[9] F. P. Schmook, J. G. Meingassner and A. Billich, "Comparison of human skin or epidermis models with human and animal skin in in-vitro percutaneous absortption," International Journal of Pharmaceutics, vol. 215, pp. 51-56, 2001.

[10] F. S. G. Praça, W. S. G. Medina, J. O. Eloy, R. Petrilli, P. M. Campos, A. Ascenso and M. V. L.

Bentley, "Evaluation of critical parameters for in vitro skin permeation and penetrationstudies using animal skin models," European Journal of Pharmaceutical Sciences, vol. 111, pp. 121-132, 2018.

[11] K. Oles, "Dribbble.com," 9 October 2018. [Online]. Available: https://dribbble.com/shots/5371635- Ears-and-snout. [Accessed 30 May 2020].

[12] PermeGear, "Franz Cell – The Original," 2019. [Online]. Available: https://permegear.com/franz- cells/. [Accessed 14 June 2020].

[13] D. Bhowmik, Chiranjib, M. Chandira, B. Jayakar and K. P. Sampath, "Recent advances in

transdermal drug delivery system," International Journal of PharmTech Research, vol. 2, no. 1, pp.

68-77, 2010.

[14] J. D. Bos and M. M. H. M. Meinardi, "The 500 Dalton rule for the skin penetrationof chemical compounds and drugs," Experimental Dermatology, vol. 9, pp. 165-169, 2000.

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Tables and figures

Figure 28. BSA calibration curve. Acquired by using fluorescamine. Excitation/emission wavelengths: 390/465nm.

Figure 29. Alexa calibration curve. Excitation/emission wavelengths: 490/520nm.

y = 1631.3x + 0.557

0 5 10 15 20 25 30 35 40

0 0.005 0.01 0.015 0.02 0.025

Fluorescence intensity (a.u.)

Concentration (mg/mL)

BSA calibration curve (390/465nm)

y = 1440.5x + 1.3528

0 200 400 600 800 1000 1200 1400 1600

0 0.2 0.4 0.6 0.8 1 1.2

Fluorescence intensity (a.u.)

Concentration (µg/mL)

Alexa calibration curve (490/520nm)