Label Free Spectromicroscopy on Drug and Drug Loaded Nanocarriers in Skin

Label free microscopy provides insights into nanocarrier and drug penetration characteristics in human and murine skin. Selective probing of drugs and nanocarriers in skin, using X-ray spectromicroscopy, was conducted to determine the drug and nanocarrier distribution, penetration pathways, and concentration in skin. A unique stimulated Raman microscopy setup is introduced. Results on the protein, lipid, and water distribution in human skin together with results from life science microscopy are reported

Review of Modern Techniques for the Assessment of Skin Hydration

Skin hydration is a complex process that influences the physical and mechanical properties of skin. Various technologies have emerged over the years to assess this parameter, with the current standard being electrical probe-based instruments. Nevertheless, their inability to provide detailed information has prompted the use of sophisticated spectroscopic and imaging methodologies, which are capable of in-depth skin analysis that includes structural and composition details. Modern imaging and spectroscopic techniques have transformed skin research in the dermatological and cosmetics disciplines, and are now commonly employed in conjunction with traditional methods for comprehensive assessment of both healthy and pathological skin. This article reviews current techniques employed in measuring skin hydration, and gives an account on their principle of operation and applications in skin-related research

Application of biophysics and bioengineering to the assessment of skin barrier function

Atopic dermatitis (AD) is one of the most common inflammatory skin diseases. The cause of AD is multifactoral and it is affected by both genetic and environmental factors. Of all the causes of potential barrier defects, the lowered amino-acid derived natural moisturizing factor (NMF) in the stratum corneum (SC), especially associated with a known filaggrin mutation, shows the strongest link to AD. As a result, quantification of NMF in the SC in both healthy and compromised SC is the principal aim of this thesis. Because tape stripping is a key technique used to harvest the SC, a novel imaging method to measure the amount of SC per tape strip was validated. This method offers rapid, simple and reproducible SC quantification. It shows good correlation with existing gravimetric and infrared absorption methods and may provide a better standard method in the future. The tape-stripping extraction of NMF showed an abundant SC ‘reservoir’ of the constituents in healthy skin. Iontophoretic extraction of NMF was highly dependant upon molecular properties, particularly charge and concentration. In general, charged NMF constituents were easily extracted by reverse iontophoresis, whereas iontophoresis only offered modest enhancement of zwitterionic species. Quantification of NMF at different body sites, specifically forehead and forearm, showed similar profiles. However, forehead SC was thinner, and in general contained a lower total amount of NMF and less-ordered lipids. Forehead SC may therefore be considered a less competent barrier. A 3-week application of 0.1% w/v sodium lauryl sulphate (SLS) to healthy volunteers was used to model damaged skin similar to that in AD and chronic irritant contact dermatitis. The SC barrier post-treatment showed significantly reduced NMF, substantial lipid disordering, and the presence of immature corneocytes. The methods employed were sufficiently sensitive to detect these changes. In particular, the NMF components present at high levels in the SC may be useful, potential markers for skin ‘health’ and for its resistance to irritant chemicals.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Photobiological Basics and Clinical Indications of Phototherapy for Skin Rejuvenation

Sunlight is essential to almost all forms of life for both light and heat. Plants need sunlight for photosynthesis, and man and animals alike need plants for many vital purposes. The sun featured many Millennia ago not only as a deity but also as a therapeutic source, so phototherapy is by no means a recent phenomenon. Niels Finsen’s therapeutic arc lamp system in the early 1900s replaced the sun as a therapeutic source. Since then, many light sources have been successfully applied for phototherapy, with laser diodes and light-emitting diodes the most efficient. This chapter will explore what phototherapy is, and examine its important role in the fast-developing indication of skin rejuvenation. Systems used in phototherapy will be discussed and compared. Photobiological basics and light/tissue interaction underlying the process will be examined, together with the importance of treatment parameters. The wound healing process, on which skin rejuvenation rests, will be dissected with a discussion of the optimum wavelengths to photoactivate the skin cells, leading to the clinical indications in photorejuvenation

Influences of skin barrier, a nanoparticle-based vehicle and solvents on cutaneous drug delivery

It is challenging to overcome the stratum corneum (SC) barrier to deliver drugs into the skin. Nanoparticle (NP)-based drug delivery systems show the advantages of cutaneous penetration improvement and controllable and targeted drug release. Besides, solvents, as a big group of penetration enhancers, provide another strategy, which is easily accessible, low-cost, and flexibly changeable in components. Apart from the skin penetration enhancement based on formulations, the disrupted barrier of diseased skin could change the skin penetration of drugs too. Thus, the present thesis investigated the influences of the SC barrier function, a pH-sensitive Eudragit® L00 nanoparticle, and the solvents of water and ethanol on cutaneous drug delivery. To determine the influence of the SC barrier on drug delivery, the SC thickness remaining on the skin after different numbers of tape stripping (TS) or cyanoacrylate stripping (CS) were quantified using two-photon microscopy, and the correlation of the SC reduction with the skin permeability changes was studied. The amount of SC removed by each tape decreased along with the skin depth, while a nearly constant SC thickness was removed by each CS. CS can remove the SC, viable skin layers and the hair follicle (HF), while TS can only remove the SC. Nevertheless, the removal of the entire human SC can be attained by both TS and CS, which were 4 times CS or 50 tape strips. The skin permeability to the model drug PCA linearly increased with the reduction of the SC thickness on the skin. These findings provide useful references to separating different skin layers for the quantification of drugs in the skin and establishing ex vivo barrier-disrupted skin models with different extents of barrier disruption. Especially, the barrier-disrupted skin, obtained by performing 30 tape strips on the intact porcine ear skin, could simulate atopic dermatitis (AD) skin to some extent. This ex vivo skin model could be used for evaluating dermal formulations in the initial development stage and reduce the number of animal and human studies. Next, the influences of the SC barrier on the skin penetration behavior of DxPCA-loaded pH-sensitive Eudragit® L100 NPs were investigated by EPR and CLSM using intact and barrier disrupted porcine skin. The pH-sensitive NP exhibited a triggered drug release in vitro at a pH above 5.9. When applied to the skin, the drug DxPCA was slowly released from the NPs in the case of the barrier-disrupted skin, whereas this was under the EPR detect limited for the intact skin. The disrupted SC barrier increased the exchange of the endogenous fluid of the skin with the external medium of the NP dispersion. Due to the exchange, the pH of the external medium of the NPs was increased, leading to the change in the NP structure and thus to the drug release. The improved drug release of the NPs is part of the reason for the higher drug penetration into the viable skin layers of the barrier-disrupted skin compared to the intact skin. These results indicate the feasibility of using this pH-sensitive NP to realize the targeted drug release and enhanced drug delivery into the viable skin layers of the AD skin lesions so that the side effects of the drug Dx could be reduced. Concerning the spatial localization of the NP, the EPR results indicate that the pH-sensitive NPs cannot pass through the disrupted SC of the skin, let alone the intact SC barrier. Besides, the accumulation of Nile red-loaded NPs in HFs and the transfolliclular penetration of Nile red was observed, which indicates that HFs can serve as a reservoir where drugs are sustained released from the NPs and provide a shortcut for drugs to penetrate across the HF into the deep viable skin layers. The drug release of the pH-sensitive NPs inside HFs may be due to the high sebum content and the high HF pH. Lastly, water and ethanol are omnipresent in topical formulations, serving as dispersion media for NPs, low-toxic dissolution media, and penetration enhancers. The solvent effects of ethanol, PBS and the cosolvent ethanol-PBS (1:1, V/V) on the penetration of the hydrophilic model drug PCA into the excised human skin and porcine ear skin were investigated by EPR. Absolute ethanol showed poor ability to deliver PCA into the skin due to the crystallization of PCA caused by ethanol evaporation. PBS and the cosolvent are superior to ethanol, delivering a similar high amount of PCA into the skin. Despite a similar total amount of PCA in the skin, the cosolvent delivered more drugs into the viable skin compared to PBS. This shows the solvents effects on the macroscopic localization of drugs in the skin. Nevertheless, more than 95% of the penetrated drugs accumulated in the SC regardless of the solvents, showing that the SC is a predominant barrier and the main reservoir for the skin penetration of hydrophilic PCA. Furthermore, the solvents influenced the microscopic localization of PCA in the SC. PCA distributed in both the intercellular skin lipids and corneocytes when using the three solvents. From PBS to ethanol, with more ethanol in the solvent, the fraction of PCA distributed in the intercellular lipids decreased from 74% to 37%. The reason may be that ethanol enhances the diffusion of PCA from the intercellular lipids into the corneocytes, implying the coexistence of intercellular and transcellular skin pathways for PCA. In conclusion, the studies conducted in this thesis i) provide correlation of the extent of the SC barrier disruption with the number of applied TS or CS and show the feasibility of using TS to establish an ex vivo barrier-disrupted skin model that mimics AD skin to some degree; ii) give insights of the influence of the SC barrier on the drug release of NPs on the skin and the following skin penetration of drugs, and show the promising application of the pH-sensitive NP in reducing the side effects of Dx for the treatment of AD; iii) expand the knowledge of solvent effects on the spatial localization of drugs in the SC and give a hint for the skin pathway of hydrophilic drugs

VIBRATIONAL SPECTROSCOPY FOR THE ASSESSMENT OF VULVAL DISEASE

Vibrational spectroscopic diagnostic techniques have significant potential to improve the care of women with benign, premalignant and malignant vulval diseases by reducing the reliance on traditional biopsy and histopathology. These techniques also have the potential to augment clinicians’ ability to differentiate different types of vulval disease at the time of surgery for neoplastic vulval disease. In addition, vibrational spectroscopic techniques offer the opportunity to assess molecular changes associated with the development of vulval cancer that are not apparent on routine histopathological assessment. The work outlined in this thesis evaluates the role of emerging techniques in vibrational spectroscopy to address this need within three key themes: 1. Developmentofavibrationalspectroscopicdiagnostictechniquetoreducethe reliance on traditional biopsy and histopathological diagnosis. 2. Developmentofavibrationalspectroscopicdiagnostictechniqueforimproving the delineation of disease margins at the time of surgery for pre-malignant and malignant vulval conditions. 3. Evaluation of a vibrational spectroscopic tool for augmenting and automating aspects of vulval histopathology. Raman spectroscopic mapping of 91 fresh frozen vulval tissue sections combined with multivariate spectral analysis was used to demonstrate that malignant vulval disease could be differentiated from non-neoplastic and premalignant vulval disease with a sensitivity of 97% and specificity of 78%. The technique was then tested in experimental conditions closer to in-vivo application, measuring spectra from 91 whole fresh frozen tissue blocks using microscope and probe Raman systems. This demonstrated the technique could differentiate malignant from non-neoplastic and premalignant vulval disease with sensitivities of 84% to 92% and specificities of 84% to 64% respectively. In a separate investigation vulval tissue blocks from 27 women with suspected lichen sclerosus underwent Raman spectroscopic point measurements. Multivariate analysis demonstrated Raman spectroscopy could be used to differentiate lichen sclerosus from other vulval disorders with a similar clinical appearance with a sensitivity of sensitivity of 91% and specificity of 80%. Fourier transform infrared (FTIR) spectroscopic mapping of 93 fixed paraffin embedded tissue sections was used to demonstrate that malignant vulval disease could be differentiated from non-neoplastic and premalignant with vulval disease with an approximate sensitivity of 100% and specificity of 79%. In addition FTIR spectroscopy was used to differentiate molecular changes in vulval intraepithelial neoplasia (VIN) and lichen sclerosus (LS) found in association with vulval squamous cell carcinoma (SCC). Analysis of FTIR spectroscopic tissue maps from 48 patients demonstrated the technique could differentiate LS associated with SCC with a sensitivity of approximately 100% and specificity of 84% and VIN associated with SCC with a sensitivity of approximately 100% and specificity 58%. This thesis demonstrates the considerable potential of vibrational spectroscopy in this clinical setting. The research has made significant progress in each of the three themes outlined above and indicates that further work is warranted to develop the techniques towards routine clinical application

Funduse sinine ja lähi-infrapuna autofluorestsentsuuring autosoom-retsessiivse Stardgardti tõve, koroidereemia, PROM1-maakuli düstroofia ja okulaarse albinismi patsientidel

Väitekirja elektrooniline versioon ei sisalda publikatsiooneFunduse sinine ja lähi-infrapuna autofluorestsentsuuring autosoom-retsessiivse Stardgardti tõve, koroidereemia, PROM1-maakuli düstroofia ja okulaarse albinismi patsientidel Pärilikud võrkkestahaigused on juhtivaks nägemiskaotuse põhjuseks tööealise elanikkonna seas arenenud riikides. Tegemist on kliiniliselt ja geneetiliselt väga heterogeense haiguste grupiga, mistõttu diagnostika ja haiguse patogeneesi uurimine on olnud vaevarikas. Võrkkesta piltdiagnostika on oluline mitte-invasiivne meetod haiguste diagnoosimiseks ja uurimiseks. Konfokaalne skanneeriv laseroftalmoskoop valgustab võrkkesta erineva lainepikkusega laserkiirega ning salvestab tagasikiirgavat valgust luues silmapõhjast pildi. Funduse autofluorestsents (AF) uuringul kasutatakse ära silmapõhja enda naturaalseid fluorofoore. Lipofustsiini ergastamiseks kasutatakse sinise spektri laserkiirt (sinine AF) ja melaniini jaoks lähipuna laserkiirt (lähipuna AF). Nende fluorofooride jaotus ja kogus silmapõhjas muutub erinevate haigusprotsesside mõjul ning need muutused on tuvastatavad AF uuringul. Antud doktoritöös uurisime sinise ja lähipuna AF uuringu pilte autosoom-retsesiivse Stargardti tõve (STGD1), koroidereemia, PROM1-maakuli düstroofia ning okulaarse albinismi patsientidel. Töö eesmärgiks oli paremini mõista sinise ja lähipuna AF signaali allikaid erinevate haigusseisundite korral, kus võrkkesta fluorofooride jaotus ning kogused on muutunud. Lisaks kvalitatiivsele piltide hindamisele kasutamise kvantitatiivset AF signaali tugevuse mõõtmist hindamaks lipofustsiini ja melaniini taset. Uurimustöös näitasime, et melaniin on lähipuna AF signaali peamiseks allikaks. Lisaks näitasime, et melanin võib kaudselt moduleerida lipofustsiinist tuleneva sinise AF signaali, sest okulaarse albinismi kandjate hüpopigmenteeritud võrkkesta alade sinise AF signal oli tavapärasest kõrgem. AF signaali tugevuse mõõtmisel leidsime, et lipofustsiini kuhjumine võrkkestas põhjustab lisaks sinise AF signaali tõusule ka lähipuna AF signaali tõusu STGD1 patsientidel. Kvantitatiivsel analüüsil näitasime ka, et PROM1-maakuli düstroofia patsientide sinise AF signaal oli võrreldav terve silmapõhja signaali tugevusega, eristades seda fenotüübiliselt sarnasest STGD1 haigusest ning viidates ka sellele, et lipofustsiini üleliigne kuhjumine ei ole antud haigusele omane mehhanism. Koroidereemia ja STGD1 haigete uurimisel leidsime, et pigmentepiteeli rakkude kärbumine on nähtav AF signaali hääbumisena, samas lähipuna AF uuringaitab tuvastada varasemaid muutusi kui sinine AF uuring. Lipofustsiin ja melanin on mõlemad olulised võrkkesta rakkude seisundi biomarkerid, mida on võimalik mitte-invasiivsel moel AF uuringu abil analüüsida ning hinnata haiguse progressiooni.Inherited retinal diseases are the leading cause of visual impairment among the working age-group in the developed countries. Because of genetic and phenotypical heterogeneity, diagnosis and understanding pathogenesis of inherited retinal disease has been challenging. Retinal imaging studies which are noninvasive, are an invaluable source of information. Fundus autofluorescence (FAF) utilizes natural fluorophores to create an image of the retina. Lipofuscin is the primary source for short-wavelength autofluorescence (SW-AF) and melanin for near-infrared autofluorescence (NIR-AF). The amount and distribution of these fluorophores changes in the different disease processes and is detectable in FAF images. In this study we analyzed SW-AF and NIR-AF images in cases of genetically confirmed recessive Stargardt disease (STGD1), choroideremia, PROM1-macular disease and ocular albinism. The aim was to qualitatively describe FAF in conditions with varying levels of lipofuscin or melanin as well as to quantify FAF signal intensities. We also aimed at finding new clinical implications for autofluorescence imaging in evaluating inherited retinal disease. We confirmed that melanin is the major source of NIR-AF signal by analyzing ocular albinism carriers and mice models with varying fundus pigmentation, but we also found that presence of melanin can modulate SW-AF signal strength. As a novel finding we confirmed that lipofuscin contributes to NIR-AF signal intensity in cases with excessive bisretinoid lipofuscin levels like seen in STGD1. The analysis of choroideremia and STGD1 patients showed that retinal pigment epithelium atrophy causes loss of signal in both SW-AF and NIR-AF, but NIR-AF could be more sensitive in detecting early cell degeneration. Quantifying the autofluorescence signal intensity helps to further understand disease processes as it is an indirect measure for levels of retinal fluorophores. We showed PROM1-macular dystrophy does not present with elevated levels of SW-AF indicating that excessive lipofuscin accumulation is likely not part of its disease mechanism. That knowledge is valuable in differentiating it from phenotypically similar STGD1 or when developing therapeutic approaches. Lipofuscin and melanin are both valuable retinal biomarkers for evaluating retinal health by using non-invasive autofluorescence imaging.https://www.ester.ee/record=b555738