Investigation on factors affecting drug delivery using polymers and phospholipids 4

Gold facial masks have been used at beauty clinics and saloons.They are deemed to improve blood circulation and skin elasticity and to rejuvenatethe skin and reduce the formation of wrin

CHAPTER 5 Development of a Nutrient-Rich Facial Mask for the Topical

Delivery of L-Ascorbic Acid and Retinoic Acid

5.1 Introduction

Many marketing strategies include the incorporation of antioxidants and other skinnutrients into cosmetic products L-ascorbic acid, (AA) has been widely used incosmetic and dermatological products because of its photoprotective effect and theability to scavenge free radicals and destroy oxidizing agents It can also inducecollagen synthesis and suppress the pigmentation of the skin while reducing signs ofphotoaging It is chemically unstable and it can easily be oxidized, therefore its stablederivatives of AA such as ascorbyl palmitate, ascorbyl tetraisopalmitate andmagnesium ascorbyl phosphate are widely used in the pharmaceutical industry(Segall and Moyano 2008; Campos et al., 2008; Gaspar and Campo 2007) Thesederivatives can easily be converted to the active compound, AA, after ingestion.However topical applications of these derivatives are not able to efficiently increasethe skin levels of this antioxidant (Pinnell and Madey 1998) A formulation strategy

to improve the stability of ascorbic acid is to incorporate in in emulsions The oilphase may partially protect AA from oxidative degradation caused in aqueoussolutions (Farahmand et al., 2006; Rozman and Gašperlin 2007; Kogan and Garti2006)

Retinoic acid (RA) enhances the repair of UV-damaged skin and reduces wrinklescaused by photoaging It can also be used for the treatment of acne (Watson et al.,2008; Cao et al., 2007; Kang and Voorhees, 1998; Fisher et al., 2002;Thielitz et al.,

2008) Due to its lipophilic structure it is practically insoluble in aqueous solutionwhich decreases its bioavailability (Lin et al., 2000; Montassier et al., 1997; Hu et al.,2005)

Gold nanoparticles have been studied as potential vaccine carriers and in transdermaldelivery systems (Sonavane et al., 2008; Menon et al., 2007; Mulholland et al., 2006;Dean et al., 2007) Gold facial masks have been used at beauty clinics and saloons.They are deemed to improve blood circulation and skin elasticity and to rejuvenatethe skin and reduce the formation of wrinkles (http://beauty.indobase.com/skin-

the use of gold facial masks

To improve the bioavailability of cosmetic products, there is a need to address thestability and solubility issues of these vitamins The aim of the present work is todevelop nutrient-rich electrospun nanofiber facial mask sheets for cosmetic purposes

AA, RA, gold and collagen-loaded electrospun facial masks of PVA and RM β-CDwere developed and characterized using FESEM and X-ray elemental analysis Invitro skin permeation studies of the vitamins were carried out across humanepidermis

5.2 Materials and Methods

5.2.1 Materials

L-ascorbic acid, tetrachloroauric acid, poly vinyl alcohol, trisodium citrate, collagenwere obtained from Sigma, Singapore 13-cis retinoic acid was obtained fromToronto Research Chemicals RM β-CD (degree of substitution of about 1.8) was agift from Wacker (Burghausen, Germany)

5.2.2 Electrospinning

Gold nanoparticles were prepared by trisodium citrate reduction of tetrachloroauricacid in 10 % w/v poly vinyl alcohol (PVA) 30 % v/v ethanol solution (Bai et al.,2007; Wang et al., 2007) Briefly, 2 ml of 2 % w/v trisodium citrate was added to thePVA solution mixture and stirred at 95 - 100 oC, and then 0.7 ml chloroauric acidaqueous solution was added to this mixture where the colour of the solution changed

to blood red indicating the formation of the gold nanoparticles The solution wascooled to room temperature before the other components were added RM β-CD wasadded to the solution to make a 20 % w/v concentration Characteristics of theelectrostatic spinning equipment and the conditions are mentioned in section 4.2.2.Details of the formulation are shown in Table 5.1

The control used is a commercially available cotton mask which was pre-moistenedwith the same concentration of ascorbic acid/ and or retinoic acid

Table 5.1 The composition of the face mask formulations.

IngredientFormulations

Ascorbic acid(1 % w/v)

Cis-retinoic acid(0.1 % w/v)

Collagen(0.01 % w/v)

Gold(0.1 % w/v)

AA and RA concentrations in the formulation were used based on the concentrations

of these vitamins in cosmetic products available in the market Collagen was studied

in various concentrations however it was found that higher concentrations influencedthe morphological structure of the fibers of the face mask and resulted in semispherical defects on the mat The amount of gold used was in accordance to previouspapers, PVA can help to stabilize gold nanoparticles (Khanna et al., 2005)

5.2.3 FESEM and Energy Dispersive X-Ray Spectroscopy (EDS) Analysis of the Fiber Mat

The surface topography of the electrospun fibers was assessed using the FESEM(described previously in section 4.2.3) The diameter distribution of the electrospunfibers was derived from a random sample of at least 20 fibers

EDS measurements were carried out by means of a FESEM equipped with an energydispersive X-ray source to identify the presence of gold in the fiber After coatingwith platinum, samples were analyzed at 15 kv voltage The area to be analyzed wasselected before the electron beam scanned and identified the intensity of characteristic

5.2.5 In vitro Skin Permeation Studies

Skin samples were prepared according to the method described in section 2.2.7.Flow-through diffusion cell was used for the skin permeation experiments asmentioned in section 2.2.8 The donor compartment was filled with 25 mg of vitamin-loaded mat and was hydrated with 300 µl of water The receptor compartment wasphosphate buffer saline with pH adjusted to 5.5 Control samples were cotton sheetpre-moistened with vitamin solutions Samples from the receptor compartment werecollected at predetermined time points over a 4-h period, and the amounts of AA or

RA permeated were analyzed by UV spectrometer

5.2.6 Skin Histology

Skin samples used in diffusion studies were processed for light microscopy Sampleswere soaked overnight in 85 ml of 80% v/v ethanol, 10 ml formaldehyde and 5 mlacetic acid (Gaspar and Campos 2007) After a series of dehydration, they wereembedded in paraffin and semi-thin sections were cut and stained with hematoxylinand eosin prior being examined with a light microscope (Leica EC 3, USA)

5.2.7 Statistical Analysis

Statistical analysis was made using, Student t-test or one-way analysis of variance,ANOVA mentioned in section 2.2.10

5.3 Results

5.3.1 Fiber Morphology and EDS Analysis

Continuous fibers without beads or sputtering of the solution were obtained withdiameter ranging from ~ 100 nm to 2 µm (Fig 5.1) There was a decrease in fiberdiameters from electrospun solutions containing gold

AA AA- Au

Fig 5.1 FESEM morphology of the electrospun fiber mats and micrographic image of the

nanofiber mat.

Fig 5.2 X-ray energy spectra of nanofiber face masks, demonstrating the presence of the gold element signals using area analysis and

spot analysis C and O represent the backbone of the nanofiber mat; presence of Na is due to the sodium citrate added for gold reduction.

C-K O-K

Na-K Au-M Au-M

This may be due to the increase in the charge density of the solution due to thepresence of gold element (Bai et al., 2008) Fig 5.2 illustrates the spectra of goldpresent on the fibers Spot analysis indicates that Au was only present on thefiber.

5.3.3 In vitro Skin Permeation Studies

Results of skin permeation are shown in Fig 5.3 It can be seen that nanofibersloaded with vitamins increased the skin permeation of AA and for RA whencompared to the vitamin-loaded cotton face mask (p > 0.05) Presence of goldincreases the permeation of ascorbic acid (p > 0.05) Although there was nosignificant increase in the skin permeation rate of the vitamis from the nanofibermask, however the permeation profiles were clearly different as compared to thecontrol samples and addition of a penetration enhacer to the facial mask may help

to further enhance the skin permeation rate These facial masks have high surfacearea-to-volume ratios when compared to cotton facial masks which can increasethe contact areas of the masks with the skin surface The dry nature of the productincreases the stability of its constituents and minimizes the oxidation of theantioxidants The presence of RM β-CD can increase the solubility of RA andaccelerate the dissolution rate of the fiber mat The concentration of RM β-CDwas able to provide a fiber mat with a disintegration time of less than one hour

After placing the mask on the face and hydrating it with water, the mask willgradually dissolve and release the entire active ingredient, ensuring maximal skinpenetration These electrospun fiber mats can be formulated to accommodatevarious skin nutrients and vitamins needed for a healthy skin These properties ofthe electrospun mats make them a promising alternative to facial cotton masks

Fig 5.3 Cumulative AA and RA permeation across human epidermis (n=3).

Microscopic appearance of the skin before and after treatment with goldnanoparticle-loaded fiber mat is shown in Fig 5.4 In the control, a clearly defined

SC could be seen, but after treatment, slight detachment of the SC layer occurred.The SC layer was fragmented and enlargement of inter-keratinocyte spaces wasobserved while the other epidermis layers became more compact

Before After

Fig 5.4 Morphology of human epidermis after skin permeation studies, (×400) The

nucleated cells of the epidermis have been stained blue, unsaturated lipids, including fatty acids and esters have been stained red.

5.4 Conclusion

L-ascorbic acid has been widely used in cosmetic and dermatological productsbecause of its ability to scavenge free radicals and destroy oxidizing agents.However, it is chemically unstable and can easily be oxidized The currentcosmetic facial masks available in the market are pre-moistened which means thatthe aqueous fluid content of the mask may oxidize some of the unstable activeingredients This work presents an anti-wrinkle nanofiber face mask containingascorbic acid, retinoic acid, gold nanoparticles and collagen This novel facemask will only be wetted when applied to the skin, thus enhancing productstability Once moistened, the content of the mask will gradually dissolve andrelease the entire active ingredient and ensure maximum skin penetration Thehigh surface area-to-volume ratio of the nanofiber mask will ensure maximumcontact with the skin surface and help to enhance the skin permeation to restoreskins healthy appearance

CHAPTER 6 Development of a Thermoresponsive Nanofiber Mat for Sustained Topical Delivery of Levothyrxine

6.1 Introduction

Poly (N-isopropylacrylamide) (PNIPAM) is a thermally reversible hydrogel with alower critical solution temperature (LCST) of around 32oC in water The cross-linked gel of this material swells and shrinks at temperatures below and above theLCST respectively, therefore a PNIPAM delivery system can provide sustainedtherapeutic levels of a drug by responding to the physiological signals of the body.Poly (N-isopropylacrylamide) (PNIPAM) nanoparticles (Wei et al., 2007; Shin etal., 2001), hydrogels (Zhang et al., 2004; Don et al., 2008) and liposomes coatedwith PNIPAM have been extensively studied as controlled drug delivery systems(Han et al., 2006; Wang et al., 2003; Kim and Kim 2002; Kono et al., 1999)

Levothyroxine (T4), a model drug, is a synthetic hormone administered orally forthe treatment of hypothyroidism and goiter (Patel et al., 2003; Volpato et al.,2004) Topical administrations of T4have been used to reduce deposits of adiposetissue on skin (Arduino and Eandi 1989; Sanntini et al., 2003) Presence of highconcentration of T4 in cosmetic creams may cause systemic effect Usingradioactive marker, radioactivity was found in the plasma after skin application of

T4 (James and Wepierre 1974) However recent in vivo studies using liposomalformulations were not able to detect any systemic effect (Santini et al., 2003)

Topical application of dimethyl-β-cyclodextrin (DM β-CD) was able to retain T4

on the skin without significant transdermal permeation (Padula et al., 2008)

This is an investigation to determine if polymeric nanofibers can sustain the skinpenetration of levothyroxine (T4) and maintain the effective concentration in theskin layers Electrospun nanofiber mats of PVA, PNIPAM and PVA- PNIPAMcomplex were developed as carriers for sustained release of T4across human skin

6.2 Materials and Methods

6.2.1 Materials

Poly vinylalcohol (Mw 70000-100000), poly (N-isopropylacrylamide) (Mw 2000025000), levothyroxine, fluorescein and 4’, 6-diamidino-2-phenylindole (DAPI)were purchased from Sigma (Singapore) All other reagents were of analyticalgrade

9 min Standard solutions of T4 (0.05 - 2 μg/ml) in 40% v/v ethanol wereprepared

6.2.3 Electrospinning of PVA/PNIPAM Nanofibers

Polymeric solutions were obtained by dissolving the drug and polymer in water(for PVA and PVA-PNIPAM) or in ethanol (for PNIPAM) Fluorescein-loadedPNIPAM solutions were developed for confocal imaging and studying the depth

of the penetration of the drug from these fibers into the skin Details of theelectrospinning process are mentioned in section 4.2.2 Polymeric solutions wereelectrospun at a voltage of 15kV with a flow rate of 1ml/h The non-wovenelectrostatically spun fabric was removed from the collector and was dried undervacuum for a week at room temperature to remove residual solvent prior to usage.Details of each formulation are shown in Table 6.1

Table 6.1 Nanofiber formulations.

Polymer Concentration (% w/v) Formulation

* All formulations contain 2 mg/ml of T4.

6.2.4 FTIR Studies of Nanofibers

Interaction between polymers and their functional groups were studied usingFTIR Spectra of the fibers were obtained using the method mentioned in section4.2.4

6.2.5 FESEM and Fluorescence Microscopy of the Nanofiber

The surface topography of the electrospun fibers was assessed using a FESEM(details are mentioned in 4.2.3) The diameter distribution of the electrospunfibers were derived from a random sample of at least 20 fibers Fluorescein-loadedPNIPAM fibers were viewed using a Nikon fluorescence microscope (EclipseTE2000-U, Japan)

6.2.6 In vitro Drug Release Studies

Total immersion method was used to study the cumulative release profiles of T4

from drug-loaded fiber mats A known amount of the fibers (5 mg) wassuspended in 10 ml of PBS and was placed in a shaking incubator at 37oC or 20oC.Samples of 1 ml were taken from the medium periodically and the released drugwas determined using HPLC The volume of the release medium was keptconstant by replacement with same volume of fresh medium All drug releasedata were averaged from three measurements

6.2.7 In vitro Skin Permeation Studies

Skin samples were prepared according to the method in section 2.2.7 Permeationstudies of drug-loaded 10% PNIPAM and 10% PVA nanofibers were performed(see section 2.2.8) The donor compartment was filled with 25 mg of fiber mat or

250 mg of control solution, all having an equal amount of drug The amount of T4

permeated was analyzed by HPLC Experiments were carried out in triplicates