Investigation on factors affecting drug delivery using polymers and phospholipids 4

CHAPTER 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 skin nutrients into cosmetic products. L-ascorbic acid, (AA) has been widely used in cosmetic and dermatological products because of its photoprotective effect and the ability to scavenge free radicals and destroy oxidizing agents. It can also induce collagen synthesis and suppress the pigmentation of the skin while reducing signs of photoaging. It is chemically unstable and it can easily be oxidized, therefore its stable derivatives of AA such as ascorbyl palmitate, ascorbyl tetraisopalmitate and magnesium ascorbyl phosphate are widely used in the pharmaceutical industry (Segall and Moyano 2008; Campos et al., 2008; Gaspar and Campo 2007). These derivatives can easily be converted to the active compound, AA, after ingestion. However topical applications of these derivatives are not able to efficiently increase the 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 oil phase may partially protect AA from oxidative degradation caused in aqueous solutions (Farahmand et al., 2006; Rozman and Gašperlin 2007; Kogan and Garti 2006). 79 Retinoic acid (RA) enhances the repair of UV-damaged skin and reduces wrinkles caused 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 solution which 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 transdermal delivery 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 rejuvenate the skin and reduce the formation of wrinkles (http://beauty.indobase.com/skincare/facial-skin-care.html), however there is no published scientific evidence about the use of gold facial masks. To improve the bioavailability of cosmetic products, there is a need to address the stability and solubility issues of these vitamins. The aim of the present work is to develop nutrient-rich electrospun nanofiber facial mask sheets for cosmetic purposes. AA, RA, gold and collagen-loaded electrospun facial masks of PVA and RM β-CD were developed and characterized using FESEM and X-ray elemental analysis. In vitro skin permeation studies of the vitamins were carried out across human epidermis. 80 5.2 Materials and Methods 5.2.1 Materials L-ascorbic acid, tetrachloroauric acid, poly vinyl alcohol, trisodium citrate, collagen were obtained from Sigma, Singapore. 13-cis retinoic acid was obtained from Toronto Research Chemicals. RM β-CD (degree of substitution of about 1.8) was a gift from Wacker (Burghausen, Germany). 5.2.2 Electrospinning Gold nanoparticles were prepared by trisodium citrate reduction of tetrachloroauric acid in 10 % w/v poly vinyl alcohol (PVA) 30 % v/v ethanol solution (Bai et al., 2007; Wang et al., 2007). Briefly, ml of % w/v trisodium citrate was added to the PVA solution mixture and stirred at 95 - 100 oC, and then 0.7 ml chloroauric acid aqueous 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 was cooled to room temperature before the other components were added. RM β-CD was added to the solution to make a 20 % w/v concentration. Characteristics of the electrostatic 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-moistened with the same concentration of ascorbic acid/ and or retinoic acid. 81 Table 5.1 The composition of the face mask formulations. Formulations A B C D E Ascorbic acid (1 % w/v) √ √ Ingredient Cis-retinoic acid Collagen (0.1 % w/v) (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 influenced the morphological structure of the fibers of the face mask and resulted in semi spherical defects on the mat. The amount of gold used was in accordance to previous papers, 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 electrospun fibers was derived from a random sample of at least 20 fibers. EDS measurements were carried out by means of a FESEM equipped with an energy dispersive X-ray source to identify the presence of gold in the fiber. After coating with platinum, samples were analyzed at 15 kv voltage. The area to be analyzed was selected before the electron beam scanned and identified the intensity of characteristic X-ray energies of specific elements. 82 5.2.4 UV Spectroscopy UV absorption measurements were carried out at room temperature on a UV-VIS spectrophotometer (U-1800 spectrophotometer, Hitachi, Japan) at 261 and 349 nm for AA and RA respectively. Standard solutions of AA and RA (0.05 to 2.00 μg/ml) were prepared in water and water ethanol solutions respectively. 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 as mentioned in section 2.2.8. The donor compartment was filled with 25 mg of vitaminloaded mat and was hydrated with 300 µl of water. The receptor compartment was phosphate buffer saline with pH adjusted to 5.5. Control samples were cotton sheet pre-moistened with vitamin solutions. Samples from the receptor compartment were collected 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. Samples were soaked overnight in 85 ml of 80% v/v ethanol, 10 ml formaldehyde and ml acetic acid (Gaspar and Campos 2007). After a series of dehydration, they were embedded in paraffin and semi-thin sections were cut and stained with hematoxylin and eosin prior being examined with a light microscope (Leica EC 3, USA). 83 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 with diameter ranging from ~ 100 nm to µm (Fig. 5.1). There was a decrease in fiber diameters from electrospun solutions containing gold. 84 AA AA- Au RA AA-RA-collagen AA-RA-collagen-Au Nanofiber mat Fig. 5.1 FESEM morphology of the electrospun fiber mats and micrographic image of the nanofiber mat. 85 005 006 001 30 30 µm µm 1000 C-K C o u n ts C o u n ts O-K 4000 3000 2000 Au-M Au-M Na-K Au-M 400 300 Au-L Au-L 1000 800 C-K 700 600 500 200 Au-M Au-M Na-K Au-M keV 0.00 600 500 400 300 Au-L Au-L 100 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 006 900 800 C-K 700 O-K 6000 5000 1000 900 8000 7000 10 10 µm µm 10 10 µm µm 005 C o u n ts 9000 001 200 Au-M Na-K Au-M Au-M O-K Au-L Au-L 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 keV 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 keV 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. 86 This may be due to the increase in the charge density of the solution due to the presence of gold element (Bai et al., 2008). Fig. 5.2 illustrates the spectra of gold present on the fibers. Spot analysis indicates that Au was only present on the fiber. 5.3.3 In vitro Skin Permeation Studies Results of skin permeation are shown in Fig. 5.3. It can be seen that nanofibers loaded with vitamins increased the skin permeation of AA and for RA when compared to the vitamin-loaded cotton face mask (p > 0.05). Presence of gold increases the permeation of ascorbic acid (p > 0.05). Although there was no significant increase in the skin permeation rate of the vitamis from the nanofiber mask, however the permeation profiles were clearly different as compared to the control 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 surface area-to-volume ratios when compared to cotton facial masks which can increase the contact areas of the masks with the skin surface. The dry nature of the product increases the stability of its constituents and minimizes the oxidation of the antioxidants. The presence of RM β-CD can increase the solubility of RA and accelerate the dissolution rate of the fiber mat. The concentration of RM β-CD was 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 will gradually dissolve and release the entire active ingredient, ensuring maximal skin penetration. These electrospun fiber mats can be formulated to accommodate various skin nutrients and vitamins needed for a healthy skin. These properties of the electrospun mats make them a promising alternative to facial cotton masks. 87 Cumulative RA (μg/cm 2) 12 Control Electrospun mat-RA 0 50 100 150 200 250 200 250 Time (mins) Cumulative AA (µg/cm 2) 50 Electrospun mat-AA 40 Electrospun mat-Au-AA 30 Control 20 10 0 50 100 150 Time (mins) Fig. 5.3 Cumulative AA and RA permeation across human epidermis (n=3). Microscopic appearance of the skin before and after treatment with gold nanoparticle-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 was observed while the other epidermis layers became more compact. 88 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 products because of its ability to scavenge free radicals and destroy oxidizing agents. However, it is chemically unstable and can easily be oxidized. The current cosmetic facial masks available in the market are pre-moistened which means that the aqueous fluid content of the mask may oxidize some of the unstable active ingredients. This work presents an anti-wrinkle nanofiber face mask containing ascorbic acid, retinoic acid, gold nanoparticles and collagen. This novel face mask will only be wetted when applied to the skin, thus enhancing product stability. Once moistened, the content of the mask will gradually dissolve and release the entire active ingredient and ensure maximum skin penetration. The high surface area-to-volume ratio of the nanofiber mask will ensure maximum contact with the skin surface and help to enhance the skin permeation to restore skins healthy appearance. 89 CHAPTER 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 a lower critical solution temperature (LCST) of around 32oC in water. The crosslinked gel of this material swells and shrinks at temperatures below and above the LCST respectively, therefore a PNIPAM delivery system can provide sustained therapeutic levels of a drug by responding to the physiological signals of the body. Poly (N-isopropylacrylamide) (PNIPAM) nanoparticles (Wei et al., 2007; Shin et al., 2001), hydrogels (Zhang et al., 2004; Don et al., 2008) and liposomes coated with 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 for the treatment of hypothyroidism and goiter (Patel et al., 2003; Volpato et al., 2004). Topical administrations of T4 have been used to reduce deposits of adipose tissue on skin (Arduino and Eandi 1989; Sanntini et al., 2003). Presence of high concentration of T4 in cosmetic creams may cause systemic effect. Using radioactive marker, radioactivity was found in the plasma after skin application of T4 (James and Wepierre 1974). However recent in vivo studies using liposomal formulations were not able to detect any systemic effect (Santini et al., 2003). 90 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 skin penetration of levothyroxine (T4) and maintain the effective concentration in the skin layers. Electrospun nanofiber mats of PVA, PNIPAM and PVA- PNIPAM complex were developed as carriers for sustained release of T4 across human skin. 6.2 Materials and Methods 6.2.1 Materials Poly vinylalcohol (Mw 70000-100000), poly (N-isopropylacrylamide) (Mw 20000 25000), levothyroxine, fluorescein and 4’, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (Singapore). All other reagents were of analytical grade. 6.2.2 HPLC Analysis Concentration of T4 was determined by HPLC from Agilent HP 1100 Series (USA). The analysis was carried out using an X-bridge column (3.5 µm, 4.6 mm × 100 mm; USA). Mobile phase (70:30 acetonitrile and 0.05 M phosphate buffer adjusted to pH using phosphoric acid) was delivered at a rate of 0.6 ml/min. UV detection at wavelength 220 nm, injection volume 100 μL gave a retention time of min. Standard solutions of T4 (0.05 - μg/ml) in 40% v/v ethanol were prepared. 91 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-loaded PNIPAM solutions were developed for confocal imaging and studying the depth of the penetration of the drug from these fibers into the skin. Details of the electrospinning process are mentioned in section 4.2.2. Polymeric solutions were electrospun at a voltage of 15kV with a flow rate of 1ml/h. The non-woven electrostatically spun fabric was removed from the collector and was dried under vacuum 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. Formulation A B C D E Polymer Concentration (% w/v) PVA PNIPAM 10 10 10 10 10 10 Solvent Water Ethanol water Water Water * All formulations contain mg/ml of T4. 6.2.4 FTIR Studies of Nanofibers Interaction between polymers and their functional groups were studied using FTIR. Spectra of the fibers were obtained using the method mentioned in section 4.2.4. 92 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 electrospun fibers were derived from a random sample of at least 20 fibers. Fluorescein-loaded PNIPAM fibers were viewed using a Nikon fluorescence microscope (Eclipse TE2000-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) was suspended in 10 ml of PBS and was placed in a shaking incubator at 37oC or 20oC. Samples of ml were taken from the medium periodically and the released drug was determined using HPLC. The volume of the release medium was kept constant by replacement with same volume of fresh medium. All drug release data 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. Permeation studies 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. 93 6.2.9 Confocal Laser Scanning Microscopy (CLSM) Skin penetration of fluorescein loaded PNIPAM nanofibers were viewed using a CLSM described in section 2.2.9. 6.3 Results and Discussion 6.3.1 FTIR Measurements of the Drug-Loaded Nanofibers The interactions between the polymers and T4 were analyzed by FTIR (Fig. 6.1). For pure PVA (Fig. 6.1a), a broad band around 3336 cm-1 is attributed to the O–H stretching vibration of the hydroxyl group. The vibrational bands at 2942 and 1438 cm-1 represent the –CH stretching. The sharp peak band at 1095 cm-1 corresponds to C–O–C symmetrical stretching present in the PVA backbone (Fig. 6.1a). There was a decline in the intensity of the –OH band when PVA was mixed with T4 (Fig. 6.1b). It is clear that hydrogen abstraction occurred from PVA molecule in the presence of T4 suggesting the formation of hydrogen bond between PVA and T4 molecules (Şanlı et al., 2007; Hong et al., 2007; Arndt et al., 1999). The bands at 2971, 2932 and 2875 cm-1 are associated with the –CH stretching vibration of PNIPAM fiber in 100% v/v ethanol (Fig. 6.1f). The positions of these three peaks are sensitive to changes in the conformation of the hydrocarbon chain and they shift towards lower frequencies when placed in aqueous solutions. This could be related to the interaction of the alkyl chain with water causing a decrease in the degree of freedom of the PNIPAM molecules (Liu et al., 2005; Maeda et al., 2001a and b). 94 Fig. 6.1 FTIR spectra of nanofibers of (a) 10% w/v PVA - No drug, (b) 10% w/v PVA, (c) 10%w/v PVA - 5% w/v PNIPAM, (d) 10% w/v PVA - 10% w/v PNIPAM, (e) 10% w/v PNIPAM and (f) 10% w/v PNIPAM - No drug. All formulations contain drug unless otherwise mentioned. FTIR spectra of the polymer blends show the presence of both PVA and PNIPAM in the nanofibrous networks. The interactions between the two polymers could be due to hydrogen bonding between the hydroxyl group in PVA and amide group in PNIPAM. 6.3.2 FESEM and Florescence Imaging of Nanofibers Morphological structures of electrospun PVA, PNIPAM and polymer mixtures are shown in Fig. 6.2. In our study, the electrospinning parameters such as voltage, flow rate and distance between the injector and collector were kept constant, therefore any difference in the morphology or structure of the fibers is probably related to the intrinsic properties of the polymeric solution. PVA fibers loaded with mg/ml of T4 were obtained using drug/polymer solutions at a concentration of 10% w/v polymer in water. Formulation A exhibits uniform fibers with 95 diameters ranging from ~ 100 to 200 nm (Fig. 6.2a). Continuous fibers of formulation B without beads or sputtering of the solution were obtained with fiber diameters ranging from ~ 30 to 300 nm. High viscosity and lower surface tension of the ethanolic solution favor the formation of continuous nanofibers as observed with formulation B (Fong et al., 1999; Xu et al., 2007; Verrecka et al., 2003). Formulation C resulted in spindle-like defects therefore it was not used in the other experiments (Fig. 6.2c). Nevertheless continuous nanofibers were obtained from the electrospinning of formulations D and E, however some elongated and semi-spherical defects were formed along these fibers (Fig. 6.2d, e). The mean diameters of the fibers correlated with viscosities of the solutions. Fiber diameter increases (about 100 to 1000 nm) when blends of PVA and PNIPAM were used. This could be due to the hydrogel formed when PNIPAM was added into aqueous solutions resulting in higher surface tension values. FESEM showed that levothyroxine crystals were not formed on the surface of the fibers indicating that levothyroxine has been completely embedded in the fibers. Fig. 6.2f shows that fluorescein was homogenously distributed cross the PNIPAM fiber net. 96 (a) (c) (e) (b) (d) (f) Fig. 6.2 FESEM images of T4-loaded nanofibers of (a) 10% w/v PVA, (b) 10% w/v PNIPAM in ethanol, (c) 10% w/v PNIPAM in water, (d) 10% w/v PVA - 5% w/v PNIPAM, (e) 10% w/v PVA - 10% w/v PNIPAM, (f) fluorescein -loaded 10% w/v PNIPAM. 97 6.3.3 In vitro Drug Release Studies The release of T4 from formulations A, B, D and E was investigated in phosphate buffer solution of pH 7.4 both at body temperature, 37oC, (Fig. 6.3) and room temperature, 20oC (Fig. 6.4). The release of T4 from the polymer mat is speculated to be by drug diffusion, polymer erosion (degradation) or both mechanisms. At body temperature, 37oC, formulation B released approximately 97% of its drug content whereas formulation A released only 65% of its total drug content. The release of T4 from the mixed polymer mat was found to be a function of PNIPAM concentration used, therefore more drug was released from formulation E compared to that of formulation D. This could be explained by the high water solubility of PNIPAM which dissolved almost immediately leading to a rapid release of T4. 100 Cumulative T4 release (%) 80 60 40 PVA 10% PNIPAM 10% 20 PVA 10%- PNIPAM 5% PVA 10%- PNIPAM 10% 0 15 30 45 60 75 90 105 120 135 Time (min) Fig. 6.3 In vitro release profile of T4 from electrospun mat in phosphate buffer (pH 7.4) at body temperature (37oC), n=3. 98 Drug release rates from formulations containing PVA were found to be lower than PNIPAM due to the slow degradation of PVA. Therefore PNIPAM and PVA blends prolonged drug release with lower risk of toxicity compared to that of PNIPAM fibers. The drug release at 20oC from formulation B was relatively lower than at 37oC (>LCST). Considering that PNIPAM is a thermosensitive polymer, it can be used for regulating drug release via response to temperature change. At temperatures below LCST, the polymer is stable and the drug release is slow, however at higher temperatures the polymer collapses thereby enhancing drug release (Kato et al., 2000). Drug release from PVA fibers was not affectd by temperature. 100 Cumulative T4 release (%) 80 60 40 PNIPAM 10 % PVA 10% 20 PVA 10%-PNIPAM 10% PVA 10%-PNIPAM 5% 0 15 30 45 60 75 90 105 120 135 Time (min) Fig. 6.4 In vitro release profile of T4 from electrospun mat in phosphate buffer (pH 7.4) at room temperature (20oC), n=3. 99 6.3.4 In vitro Skin Permeation Studies Fig. 6.5 represents cumulative T4 for 10% w/v PVA, 10% w/v PNIPAM and control as a function of time. It can be seen that T4 permeation across the skin was gradual (p > 0.05) and there is evidence of drug accumulation in the skin as shown by the small amounts of drug permeated. The recommended therapeutic dose of T4 is 50-100 μg/day, therefore the cumulative T4 skin penetration obtained in this study would not be sufficient to produce a systemic effect in vivo. This improves therapeutic efficiency and helps to accumulate drug on the skin to obtain a prolonged delivery (Padula et al., 2008). Fig. 6. Cumulative T4 permeation across human epidermis (n=3). To support the above hypothesis, CLS microscopic studies were conducted. FITC-labelled PNIPAM nanofibers were located primarily in the stratum corneum 100 (SC) layers of the skin (Fig 6.6). Fluorescein was detected in the lower layers of the epidermis from aqueous solution that did not contain any polymer. Skin was counterstained with DAPI to visualize cell nuclei. The binary images (Fig. 6.6b) indicate the localization of green fluorescein on the outer layer of the skin for the PNIPAM formulations as compared with the control. Such delivery systems may have potential use in skin formulations containing sunscreens and other active ingredients that are meant to be concentrated on the skin surface. Due to the lower dosing frequency and simpler dosage regimes, patient compliance may be improved. 101 a) PNIPAM nanofiber mat Control b) PNIPAM nanofiber mat Control Fig. 6.6 (a) Image of the epidermis and localization of green fluorescence incorporated in to the PNIPAM nanofibers as a function of depth into the skin. The image depths (from left to right) are 0, 8, 16 and 24 µm. (b) Binary image of the skin after the flow through diffusion studies. Staining of cell nuclei with DAPI is shown as blue signal. 6.4 Conclusion A series of nanofibrous membranes were electrospun into blends of poly vinyl alcohol (PVA) and poly-N-isopropylacrylamide (PNIPAM) to develop a sustained topical delivery of T4. The polymeric nanofiber mats were characterized by field emission scanning electron microscopy (FESEM) and fourier transform infrared (FTIR) spectroscopy. In vitro permeation of the drug from the polymeric nanofibers was studied using excised human skin and the permeation mechanism 102 investigated using confocal microscopy. It was observed that polymeric nanofibers were able to sustain the penetration of T4 to the skin and help maintain the effective drug concentration in the skin layers for longer period of time. These formulations may have potential uses in topical skin products and can help to increase the accumulation of the active compound on the skin surface thus minimize the adverse side effects which may be caused by systemic absorption. This may result in great improvement in consumer compliance, avoid frequent dosing and enhance the therapeutic effectiveness. 103 [...]... mg/ml of T4 6.2 .4 FTIR Studies of Nanofibers Interaction between polymers and their functional groups were studied using FTIR Spectra of the fibers were obtained using the method mentioned in section 4. 2 .4 92 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... 20oC (Fig 6 .4) The release of T4 from the polymer mat is speculated to be by drug diffusion, polymer erosion (degradation) or both mechanisms At body temperature, 37oC, formulation B released approximately 97% of its drug content whereas formulation A released only 65% of its total drug content The release of T4 from the mixed polymer mat was found to be a function of PNIPAM concentration used, therefore... Results and Discussion 6.3.1 FTIR Measurements of the Drug- Loaded Nanofibers The interactions between the polymers and T4 were analyzed by FTIR (Fig 6.1) For pure PVA (Fig 6.1a), a broad band around 3336 cm-1 is attributed to the O–H stretching vibration of the hydroxyl group The vibrational bands at 2 942 and 143 8 cm-1 represent the –CH stretching The sharp peak band at 1095 cm-1 corresponds to C–O–C symmetrical... al., 20 04; Don et al., 2008) and liposomes coated with 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 for the treatment of hypothyroidism and goiter (Patel et al., 2003; Volpato et al., 20 04) Topical administrations of T4 have... 30 45 60 75 90 105 120 135 Time (min) Fig 6 .4 In vitro release profile of T4 from electrospun mat in phosphate buffer (pH 7 .4) at room temperature (20oC), n=3 99 6.3 .4 In vitro Skin Permeation Studies Fig 6.5 represents cumulative T4 for 10% w/v PVA, 10% w/v PNIPAM and control as a function of time It can be seen that T4 permeation across the skin was gradual (p > 0.05) and there is evidence of drug. .. been used to reduce deposits of adipose tissue on skin (Arduino and Eandi 1989; Sanntini et al., 2003) Presence of high concentration of T4 in cosmetic creams may cause systemic effect Using radioactive marker, radioactivity was found in the plasma after skin application of T4 (James and Wepierre 19 74) However recent in vivo studies using liposomal formulations were not able to detect any systemic effect... Nanofibers Polymeric solutions were obtained by dissolving the drug and polymer in water (for PVA and PVA-PNIPAM) or in ethanol (for PNIPAM) Fluorescein-loaded PNIPAM solutions were developed for confocal imaging and studying the depth of the penetration of the drug from these fibers into the skin Details of the electrospinning process are mentioned in section 4. 2.2 Polymeric solutions were electrospun at... (PVA) and poly-N-isopropylacrylamide (PNIPAM) to develop a sustained topical delivery of T4 The polymeric nanofiber mats were characterized by field emission scanning electron microscopy (FESEM) and fourier transform infrared (FTIR) spectroscopy In vitro permeation of the drug from the polymeric nanofibers was studied using excised human skin and the permeation mechanism 102 investigated using confocal... penetration of T4 to the skin and help maintain the effective drug concentration in the skin layers for longer period of time These formulations may have potential uses in topical skin products and can help to increase the accumulation of the active compound on the skin surface thus minimize the adverse side effects which may be caused by systemic absorption This may result in great improvement in consumer... tension of the ethanolic solution favor the formation of continuous nanofibers as observed with formulation B (Fong et al., 1999; Xu et al., 2007; Verrecka et al., 2003) Formulation C resulted in spindle-like defects therefore it was not used in the other experiments (Fig 6.2c) Nevertheless continuous nanofibers were obtained from the electrospinning of formulations D and E, however some elongated and . formulations contain 2 mg/ml of T 4 . 6.2 .4 FTIR Studies of Nanofibers Interaction between polymers and their functional groups were studied using FTIR. Spectra of the fibers were obtained using. 97% of its drug content whereas formulation A released only 65% of its total drug content. The release of T 4 from the mixed polymer mat was found to be a function of PNIPAM concentration used,. 261 and 349 nm for AA and RA respectively. Standard solutions of AA and RA (0.05 to 2.00 μg/ml) were prepared in water and water ethanol solutions respectively. 5.2.5 In vitro Skin Permeation