Investigation on factors affecting drug delivery using polymers and phospholipids 2

CHAPTER General Introduction 1.1 Skin Structure The human skin is the largest and most easily accessible organ of the human body and its primary function is to control the loss of water and other body constituents and protect the body from the surrounding environment (Silva et al., 2006; Charalambopoulou et al., 2004; Cevc et al., 1996). histological layers: epidermis, dermis, hypodermis. The skin consists of three The hypodermis is a subcutaneous tissue consisting of fat and muscle and provides mechanical protection against physical shock. The dermis is about mm thick and contains blood and lymph capillaries, collagen, elastic fibers, nerves as well as hair follicles and sebaceous and sweat glands. The epidermis is avascular, therefore the essential substances are transported by passive diffusion from the underlaying dermis. The epidermis has a multilayered membrane reflecting different stages of differentiation of the skin cells (the keratinocytes) (Wiedersberg et al., 2008; Glombitza and Müller-Goymann 2002; Cevc et al., 1996). It is mainly constituted by keratinocytes, accounting for 95% of the epidermis. Other cell types in epidermis include; melanocytes, Langerhans cells, and Merkel cells. Epidermis, 100 to 150 μm thick, is biologically divided into four distinct layers: the stratum basale (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum corneum (SC) (Menon 2002). In the basal layer of the epidermis cells proliferate. After replication, cells leave the basal layer and start to differentiate and migrate upwards through the epidermis towards the skin surface where they are transformed into dead keratin filled cells (corneocytes) (Bouwstra and HoneywellNguyen 2002). Image of the epidermis is shown in Fig. 1.1. (a) (b) Fig. 1.1 (a) Binary image of the human epidermis and localization of green fluorescence, staining of cell nuclei with DAPI is shown as blue signal. Slice view of stratum granulosum is shown in red fluorescence. (b) Cross-section of human epidermis. The nucleated cells of the epidermis have been stained blue, unsaturated lipids, including fatty acids and esters have been stained red. Details on the method of sample preparation are mentioned in section 2.2.9 and 5.2.6. The SC, also known as the horny layer, consists of 10-15 cell layers of keratin-rich corneocytes embedded in a lipid matrix (Silva et al., 2006). The SC has been represented as a two-compartment mortar brick model proposed by Peter Elias (Elias 1981). In this model the keratinized cells represent the “bricks” which are imbedded in the hydrophobic crystalline lamellar lipid-rich matrix (mortar) (Menon 2002; Glombitza and Müller-Goymann 2002; Bouwstra and Honeywell-Nguyen 2002). The intercellular SC lipids (mortar) comprise mainly ceramides (~40% w/w), free fatty acids (~10% w/w) and cholesterol (~25% w/w). The distinct lipid composition of the skin is the major rate-limiting barrier for topical and transdermal drug delivery (Wiedersberg et al., 2008; Elias, 1983; Downing 1992; Fatouros et al., 2006; Kim et al., 2008; Glombitza and Müller-Goymann 2002). For any molecule applied to the skin, three pathways of skin permeation have been identified: (1) the intercellular lipid domains in SC; (2) transcellular pathway through the keratinocytes and (3) transappendageal permeation through the sweat glands and across the hair follicles (Godin and Touitou 2007; El Maghraby et al., 2008) 1.2 Topical and Transdermal Drug Delivery Topical drug delivery is widely used for the treatment of localized skin disease such as acne vulgaris as well as musculoskeletal disorders. However, over the past few decades there is an increasing emphasis on the transdermal delivery of drugs to treat systemic diseases. Both topical and transdermal delivery systems require the active ingredient to overcome the stratum corneum barrier. Topical drug delivery helps to achieve therapeutic concentrations in the soft tissues and peripheral nerves underlying the site of application while maintaining low serum concentrations. However, transdermal delivery is defined as delivery of the drug through intact skin so that it reaches the systemic circulation in sufficient quantity to achieve therapeutic action (Stanos 2007). Compared to conventional oral delivery methods, transdermal drug delivery (TDD) offers better patient compliance, improved bioavailability, reduced side-effects, elimination of first-pass effect, and easy termination of drug input. Additionally, sustained drug delivery via the skin would reduce the dosing frequency and eliminate peak plasma levels of the drug (Barry 2001; Schreier and Bouwstra 1994; Cappel and Kreuter 1991; Kim et al., 2008; Park et al., 2008; Kiptoo et al., 2008). Despite these advantages, the barrier nature of the skin presents a significant obstacle for molecules to be delivered through it; most drugs not cross skin at therapeutic rates and less than 20 drugs have been approved by FDA for transdermal delivery. Drugs that cross the stratum corneum barrier can generally diffuse to deeper capillaries for systemic distribution. Therefore, many techniques have been used to disrupt the highly organized crystalline lipid matrix of stratum corneum. Penetration enhancers help to facilitate the absorption of active compounds through the skin. These include chemical penetration enhancers, supersaturated drug delivery systems, iontophoresis, electroporation, sonophoresis and vesicle delivery systems (El Maghraby 2008). Most permeation enhancers act on the SC to increase drug solubility in the formulation, enhance drug partitioning within the SC layer, disrupt the crystalline lipid lamella and increase its fluidity, increase transepidermal water loss (TEWL) or cause skin lipid extraction (Barry 2001; Valenta et al., 2004; Vávrová et al., 2008; Bommannan et al., 1991). Some of the chemical enhancers that can temporarily disrupt the barrier properties of the skin include; water, Azone derivatives, fatty acids, fatty esters, sulphoxides, alcohols, pyrrolidones, glycols, surfactants, terpenes and phospholipids (Williams and Barry 2004; Kim et al., 2008; Barry 1987; Foldvari 2000). It is also possible that some chemicals will stabilize the skin lipids and retard the delivery of drugs across human skin (Hadgraft et al., 1996). Such compounds have potential uses in dermatological formulations containing sunscreens, insect repellants, or acne vulgaris-protecting agents. They can increase the accumulation of the active compound on the skin surface and minimize the adverse side effects which may be caused by systemic absorption (Asbill and Michnaik 2000). An ideal drug for the transdermal delivery should have a low molecular weight (< 500 Da) and sufficient lipophilicity to partition into the SC, but also be hydrophilic enough to penetrate deeper into the skin and reach the systemic circulation. Due to the barrier limiting properties of the SC layer, this delivery method is limited for the administration of potent drugs (Davidson et al., 2008; Kalia and Guy 2001; Guy and Hadgraft 1987). A number of physical techniques are used to gain information on the barrier function of the SC and to study the interaction of penetration enhancers with the skin. Some of these techniques include; differential thermal analysis (DSC), X-ray scattering, fourier transform infrared spectroscopy (FTIR) and confocal laser scanning microscopy (Boncheva et al., 2008; Alvarez-Román et al., 2004; ABüyüktimkin et al., 1996; Beastall et al., 1988). 1.3 Skin Permeation Models Bioavailability of topically or transdermally applied drugs may be determined using various techniques. The ideal method for assessment would be to study the systemic uptake in vivo by blood or urine sampling and measure the drug deposition on the skin layers by tape stripping. However due to compliance and ethic issues, these methods are not feasible during the initial development of a novel pharmaceutical dosage form, and in vivo testings are mainly carried out in animal models (Puglia et al., 2004; Godin and Touitous 2007). Numerous in vitro models, including diffusion cells are employed to assess the skin permeation profiles and kinetic parameters (Bender et al., 2008). The most relevant membrane for in vitro permeation studies is the human skin which is usually obtained from cosmetic surgery. However, human skin is often difficult to obtain therefore a wide range of animal models have been used as replacement for human skin. These include porcine, rabbit, mouse, rat, guinea pig and snake models (Godin and Touitous 2007; Bartek et al., 1972; Nicoli et al., 2006; Shin and Choi 2005). Other alternative substitutes include reconstructed skin models and artificial membranes (Pappinen et al., 2008; Iervolino et al., 2000; Nolan et al., 2003). 1.4 Recent Formulation Developments In the last two decades, colloidal drug delivery systems such as microemulsion, nanoemulsion, and lipid nanoparticles, have been employed to improve delivery of drug to the skin (Padamwar et al., 2006; Touitou et al., 1997; Verma et al., 2003; Puglia et al., 2004; Chen et al., 2007; Honeywell-Nguyen and Bouwstra 2005). These systems provide controlled release of the active ingredients, they can be used for both hydrophilic and hydrophobic drugs as well as have low production cost. The small particulate vehicles can ensure close contact with the SC which significantly promotes drug delivery into the skin (Fang et al., 2008). Ultra deformable vesicles have also been developed which can pass the stratum corneum as intact vesicular form before permeating deeper in to the skin layers thus improve drug diffusion into the skin (Cevc et al., 1998 and 2002). Another strategy for enhancing the percutaneous absorption of topically applied drugs is to use a vehicle or a carrier such as polymers which facilitate transport of drugs into and across the skin without affecting the physiochemical properties of stratum corneum (Tadicherla and Berman 2006; Asbill and Michniak 2000). Polymeric delivery systems can protect the active ingredient from degradation and prolong or modify the drug release pattern. Polymers such as chitosan, polyacrylic acid (Silva et al., 2008), cellulose (Bodhibukkana et al., 2006; taepaiboon et al., 2007), poloxymer (Nair and Panchagnula 2003), poly N-isopropylacrylamide (PNIPAM) (Lopez et al., 2004) poly (ε-caprolactone) (Jiménez et al., 2004; Shim et al., 2004), polylactide (Ga de Jalón et al., 2001a and b; Rolland et al., 1993; Tsujimoto et al., 2007), poly vinylalcohol (Kenawy et al., 2007; Tao and Shivkumar 2007) in forms of hydrogels, membranes, micro or nanoparticles, have been explored as promising carriers for controlled delivery of drugs to the skin. Recent research advances for the topical delivery of peptides, proteins, DNA and RNA to the skin include incorporation of lipid or polymer particles into devices such as microneedles (Prausnitz 2004; Tao and Desai 2003; Gill and Prausnitz 2007) and gene guns (Lee et al., 2008; Babiuk et al., 2000) to help target large protein molecules to Langerhans cells for vaccination purposes. 1.5 Hypothesis and Objective Over the past decades, there has been a general realization that the bioavailability of topically applied drugs is very low. Drugs in topical or transdermal delivery systems may not penetrate the skin in sufficient amounts to exert a therapeutic effect. In attempts to overcome the poor penetration of drugs and to optimize drug release characteristics, various strategies have been explored. Tables 1, and depict some of the recent developments in the field of topical and transdermal drug delivery. The potential of polymers and phospholipids for improving the topical and transdermal delivery of therapeutic agents and cosmetic ingredients is worth exploring. Factors that influence transdermal and topical drug delivery may be divided into three categories: 1) drug related, 2) vehicle related and 3) skin related factors. Drug related factors include; particle size, molecular weight, stability, partition coefficient (Kp), diffusion coefficient and solubility of the drug in the chosen vehicle. Factors related to the vehicle include; amount of drug release from the vehicle, physicochemical properties of the vehicle and the use of penetration enhancer. Skin related factors are; metabolism, barrier structure of the skin and biological variation pertaining to race, sex and age. This thesis attempts to explore some of these factors that influence transdermal drug delivery. Chapter and look into drug related factors, chapters 4-7 explore some vehicle related factors while Chapter looks at skin related factor. Lipid vesicles are being widely used as vehicles in transdermal delivery systems. A lot of work has been done regarding the effect of lipid concentration, vesicle content, polarity and size as well as method of production of these vesicles on the skin permeation of drug molecules. However the effect of mixture of surfactants and their hydrophilic-hydrophobic balance (HLB value) on the structure of the vesicles, their stability, drug solubility as well as their effect on the skin permeation is still in the initial stages. Therefore the objective of Chapter was to: 1) Explore the effect of two groups of non-ionic surfactants (Spans and Tweens) on drug solubility 2) Study the effect of surfactant mixtures on skin permeability of haloperidol 3) Investigate the HLB value and surface tension on the structural characteristics and stability of the vesicle as well as skin permeation of drug molecule. Properties of the drug molecule are the most important factor that influences transdermal delivery. As most drugs are solid in nature, therefore they are poorly absorbed through the skin. Hence a vehicle is needed to solublize and stabilize the drug and optimize its delivery through the skin. One approach to increase the solubility of low water soluble drugs is the use of cyclodextrins. These oligosaccharides can also help to decrease local irritation and reduce photosensitivity of the drug. The effect of cyclodextrin as a penetration enhancer is subject to controversies. While most authors agree that cyclodextrins may help increase the skin permeation of drugs, however the parabolic effect of cyclodextrin concentration on skin permeation of drug molecules is yet not clear. This chapter was the first attempt to explore the surface active effect of cyclodextrin derivatives and to relate the critical micelle concentration (CMC) of these surface active molecules to their penetration enhancing effect. This finding will help to explain the parabolic relationship between the cyclodextrin concentration and skin permeation of drug molecules. Objective of this chapter was to: 1) Explore the effect of cyclodextrin derivatives on the aqueous solubility of haloperidol and study the molecular complexation mode 2) Investigate the effect of ionization on the solubility and skin permeability of haloperidol 3) Study the surface active effect of cyclodextrin derivatives and to help explain the parabolic relationship regarding the cyclodextrin concentration and skin permeation of haloperidol. Recently much attention has been given to the role of polymers as potential carriers in transdermal delivery. Polymers in the forms of nanoparticles, microparticles, hydrogels, and conjugated forms with lipid vesicles have been widely explored. A 10 triplicates. Solubility enhancement ratios (ER) of HP were calculated using the equation below: ER  Co Cs (2-2) where Co is the HP solubility in the formulations with surfactants and Cs is the saturation solubility of HP in the control sample without surfactant. Surface tensions of solutions were measured by the Du Nouy ring method at room temperature (20 ± 2oC) using a digital tensiometer (Sigma 700 KSV Instruments, Helsinki, Finland). The precision of the force transducer of the tensiometer was 0.01 mN/m. Du Nouy ring method with a platinum–iridium ring having a mean diameter of 9.545 mm was employed. Following each measurement, the ring was washed with Milli-Q water and subjected to a high temperature flame to ensure complete removal of residual. Maximum surface tension values of each concentration and a total of three surface tension measurements for each solution were obtained. 2.2.4 Proniosome Formulations Proniosomes were prepared using a modified literature method (Alsarra et al., 2005; Fang et al., 2001). The compositions of different proniosomal formulations are listed in Table 2.1. HP mg, surfactant, cholesterol and lecithin, in the ratio of 9:9:1 were mixed with isopropyl alcohol. The jar was capped and warmed in a sonicator (120 rpm) water bath at 65 ± 3oC for 10 min. Then phosphate buffer saline was added and the mixture was further warmed in the water bath for min, so that a clear solution was obtained. Rhodamine-loaded proniosomes were used to study the depth of dye 31 Table 2.1 Composition and appearance of proniosomal formulations. Proniosomal formulations 1.8 6.7 10 16 16.7 Span 85 100 93.75 - Composition Surfactant (% of 225 mg) Span 60 Span 40 Tween 20 Tween 80 6.25 41 59 100 60 40 59 41 100 - C (mg) L (mg) B (ml) IPA (ml) Appearance 25 25 25 25 25 25 25 225 225 225 225 225 225 225 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.625 0.625 0.625 0.625 0.625 0.625 0.625 Translucent oil Clear oil Opaque amber oil Opaque amber oil Clear oil Clear oil Clear oil C = cholesterol, L = lecithin, B = pH 7.4 phosphate buffer saline, IPA = isopropyl alcohol. * All formulations contained mg/ml HP. 32 permeation with a confocal microscope. Samples were prepared using 0.03% w/v of rhodamine G. 2.2.5 Encapsulation Efficiency and Stability of Proniosomes HP-containing proniosomes were separated from the free un-entrapped drug by centrifuging the samples at 12 000 rpm for 30 at 20oC. The supernatant was recovered and assayed by an HPLC method for HP concentration. The percentage of drug encapsulation was calculated by Eq. 2.3, where Ct is the concentration of total HP, and Cf is the concentration of free un-entrapped HP. Encapsulation efficiency  Ct  C f Ct  100% Samples were stored at 4oC or 20oC for a 6-week period. (2-3) The encapsulation efficiencies of the samples were determined at day 0, weeks and after preparation. 2.2.6 Scanning Electron Microscopy (SEM) Proniosomal formulations were mounted on an aluminum stub using silver paint. The vesicles were sputter-coated with gold at 10 mA and 1.2 kV for min, using a vacuum evaporator (Jeol Fine Coat Ion Sputter JFC-1100, Japan). Electron micrographs were obtained with a scanning electron microscopy (Jeol JSM-T220A, Japan) equipped with a digital camera, operating at 15.0 kV alternating voltage. 33 2.2.7 Preparation of Human Epidermis The abdominal skin of an adult Chines female (with an average age of 52) was obtained with patient consent post plastic surgery. Full-thickness skin with epidermis facing downwards was immersed in water of 60oC for min, and then the epidermis was carefully peeled off and stored at -80oC. Prior to permeation studies, the epidermis was thawed and hydrated in an aqueous 0.9% w/v sodium chloride and 1% v/v antibiotic antimycotic solution under room conditions for h (Kligman and Christophers 1963). 2.2.8 In vitro Skin Permeation Studies The permeation of HP from proniosomal formulations was determined by flowthrough diffusion cell. The epidermis was mounted on the receptor compartment with the stratum corneum facing upwards. The exposed surface area for the drug permeation in the skin was 0.785 cm2. The receptor compartment was filled with isotonic phosphate-buffered saline (PBS). A ml of proniosomal formulation was placed in the donor compartment and covered with aluminum foil. Samples were collected at 4-h intervals for 36 h. The temperature was kept at 37 ± 0.5oC throughout the experiment. The samples were analyzed by the HPLC method. All experiments were performed in triplicate. The permeation of drug across the epidermis can be described by Fick’s 2nd law of diffusion (Crank 1975): D Q  AKLCo  t    L (1) n ( D / L2 ) n 2 2t  e   n 1 n   (2-4) 34 Permeation parameters are interpreted from the cumulative amount of released drug per unit skin area (Q/A) versus time (t) plot. The gradient and x-intercept of the linear portion of the plot yield steady-state flux (Jss) and lag time (tL) respectively. The drug permeability (KD/L) is derived as shown in Eq (2-5): KD J ss  L Co (2-5) where Co is the initial drug concentration in the donor cell. The diffusion parameter, D/L2 reflecting the mobility of the drug solute in the skin was calculated using the following equation: D  6t L L (2-6) The partition parameter, KL, reflecting the distribution of the drug between the skin and the donor solution is derived from: KL  KD / L D / L2 (2-7) The enhancement index, EI, a ratio of the drug permeability from the control and the proniosomal formulations, measures the enhancement in drug penetration (Vaddi et al., 2002). EI  ( KD / L) proniosome ( KD / L) control (2-8) 35 2.2.9 Confocal Laser Scanning Microscopy (CLSM) Skin penetration of rhodamine-loaded proniosomes was viewed using a CLSM. The CLSM was a Nikon A1R laser scanning confocal and digital camera from Japan. All samples were excited at 405, 488 and 561 nm and X-Z sectioning has been used to determine the depth of permeation using an objective of ×60. Following the in vitro skin permeation studies, the skin samples were placed on a glass slide and covered with a glass cover-slip. The slides were inverted and images were captured through the cover-slip side of the prepared samples. The full epidermis was scanned at different increments through the z-axis of the microscope. 2.2.10 Statistics The values are expressed as mean ± SD of (n = 3). Comparisons were made using one-way analysis of variance, ANOVA (Graph Pad Prism, Version 2) followed by Tukey’s multiple comparison post-test to determine the differences between treatment groups. The differences were considered statistically significant when p < 0.05. 2.3 Results and Discussion 2.3.1 Solubility and Surface Tension Studies The solubilities of HP in different surfactant mixtures and also the surfactant surface tensions in water: glycerol solutions are presented in Fig. 2.1. The drug has a relatively low solubility in the control solution. The addition of surfactants enhanced the solubility of HP significantly. The formulation with a surfactant mixture 36 corresponding to HLB 10 resulted in the highest drug solubility, almost 13-fold compared to that in the control solution. Surface tension measurements showed higher interfacial tensions for formulations with HLB values of 10, and formulations with HLB 1.8 and 16.7 had the lowest surface tension values. 4000 80 surface tension 3000 60 2000 40 1000 20 surface tension (mN/m) HP solubility (μg/ml) HP solubility control HLB 1.8 HLB HLB HLB 6.7 HLB 10 HLB 16 HLB 16.7 Fig. 2.1 Solubility and surface tension measurements of HP solutions (n=3). 2.3.2 Encapsulation Efficiency and Vesicle Stability As shown in Fig. 2.2 the encapsulation efficiencies were high and there was no significant difference amongst the formulations (p > 0.05). After the formulations were stored at room temperature (20oC) and fridge (4oC) for weeks, no statistical significance in drug encapsulation efficiencies was observed (p > 0.05). 37 Encapsulation efficiency (%) 120 100 Day Week (20ºC) Week (4ºC) Week (20ºC) Week (4ºC) 80 1.8 6.7 10 16 16.7 HLB value of Proniosomal formulations Fig. 2.2 Encapsulation efficiencies (%) of proniosomal formulations (n=3). 2.3.3 SEM Imaging The scanning electron microscopy images of the proniosomes are shown in Fig. 2.3. Proniosomes with lower HLB values seemed to be mostly spherical and discrete with sharp boundaries and smooth and rigid surfaces. However, vesicles made with surfactants of HLB 16 and 16.7, have deformable structures which may be attributed to the type of surfactant in the formulation. The main difference between deformable and rigid vesicles is due to the fluidity of the lipid bilayer of the deformable vesicles (Cevc et al., 2002). 38 (a) (b) (c) (e) (f) (g) (d) Fig. 2.3 Scanning electron microscopy images of proniosome formulations, (a) HLB 1.8, (b) HLB 2, (c) HLB 6, (d) HLB 6.7, (e) HLB 10, (f) HLB 16 and (g) HLB 16.7. 39 2.3.4 In vitro Skin Permeation Studies Fig. 2.4 shows the release profiles of HP over a 36-h period. There is a significant increase in the permeation of HP from formulations containing surfactants with HLB value 16.7 and the HP flux rate was almost 2.8-folds higher than that of the control (P < 0.05, Table 2.3). Formulations with single surfactants were found to increase the permeation of HP more than mixtures of surfactants. Thus these surfactants when used together in proniosomes did not show any synergism with regards to permeation of the drug through the skin. Although formulations with HLB 16 had high surface tension when compared to formulations with HLB 16.7, however formulation with HLB 16 is a mixture of two surfactants. The mechanism of surfactant mixture on skin permeability is not well understood. Mixture of surfactants result in complex structures, after mixing of the two surfactants a new type of mixed micelle is formed that could behave differently than the two single surfactants. Previous findings show that synergy in penetration enhancing effect is concentration-dependent and occurs when nearly equal fractions of the surfactants are present in the mixture (Karande and Mitragotri 2002; Karande et al., 2007 and 2006; Whitehead et al., 2008). In this work the concentration of the two surfactants were calculated to obtain the required HLB value and therefore are not equimolar. This could explain the non-synergistic effect of the two surfactants in the skin penetration of haloperidol. The lag time was increased probably due to the rate-limiting membrane barrier of the lipid bilayers and drug-reservoir characteristics of the proniosomes (Hu and Rhodes 40 2000; Alsarra et al., 2005). Nevertheless the cumulative drug amount permeated from all formulations was higher than that of the control. Fig. 2.4 Permeation profile of HP across human epidermis (n=3). Most investigators agreed that vesicle size did not influence the drug permeation profiles. Phospholipids not penetrate to the deeper layers of the skin but instead fuse with the skin and disrupt the lipid structure of the stratum corneum (Schreier and Bouwstra 1994; Ganesan et al., 1984; Gesztes and Mezei 1988; Honeywell-nguyen and Bouwstra 2005; Kirjavainen et al., 1996; Touitous et al., 1994). The concentrations of surfactants in these formulations are in excess of their critical micelle concentrations and micelles are too large to diffuse into the skin layers (Fang et al., 2001; Sarpotdar and Zatz 1986). Therefore direct contact and adherence of the vesicles with skin surface is important for the drug to penetrate and partition between the stratum corneum and the formulation (Wokovich et al., 2006). Surface tension values will determine the extent of the contact of the drug-loaded vesicle with the 41 skin surface. Formulations with Span 85 (HLB 1.8) did not change drug permeation significantly when compared to that of the control, indicating that there is an optimal balance between hydrophilicity and lipophilicity (Lo 2003). HLB 1.8 could be too hydrophobic to enhance the permeation rate of HP, a hydrophobic drug. Due to the high affinity of the drug to such vesicle, diffusion to the skin may be limited. Drug permeation may be correlated to the physicochemical properties of the surfactant. Table 2.2 demonstrates some characteristics of non-ionic surfactants including number of carbons in the alkyl chain (Dai et al., 1997; Al-Sabagh 2002). It can be seen that the shorter the chain length of the fatty alcohol, the higher the drug release rate (Devaraj et al., 2002). In our study, the rate of drug release from proniosomal formulations based on the carbon chain lengths can be ranked as: Tween 20 (C12) > Span 60 (C18) > Span 85 (C54). Therefore physicochemical characteristics of surfactants such as surface tension and hydrophobicity influence the rate of drug permeation from various proniosomal vesicles. To support the above hypothesis, confocal laser scanning microscopic studies were conducted. For this purpose proniosome formulations containing single surfactants with the highest and the lowest skin permeation rates were used. The extent of vesicular penetration was measured by CLS microscopy after the in vitro skin permeation studies of proniosome formulations with HLB 16.7 and HLB 1.8 each containing 0.03% rhodamine 6G. Proniosomes with HLB values of 16.7 resulted in increased permeation of rhodamine-label which corresponded to more rhodamine transport across the stratum corneum (SC) and into the deeper layers of the skin, 42 whereas rhodamine-labeled proniosomes of HLB 1.8 had limited penetration into the skin (Fig. 2.5). 43 a) HLB 1.8 HLB 16.7 b) HLB 1.8 HLB 16.7 Fig. 2.5 a) Images of the epidermis and localization of red fluorescence incorporated in to the proniosomes as a function of depth into the skin. b) For better visualization, skin samples were stained with fluorescein prior to skin permeation studies. The image depths (from left to right) are 0, 4, 8, 12, 16, 20 and 24 µm. 44 Table2.2 Properties of the surfactants incorporated in proniosomes. Surfactant trade name Chemical description Tween 20 Tween 80 Span 40 Span 60 Span 85 Polyoxyethylene (20) Sorbitan mono-laurate Polyoxyethylene (20) Sorbitan mono-oleate Sorbitan mono-palmatate Sorbitan mono-stearate Sorbitan trioleate Ethylene oxide units HLB Carbons in the alkyl chain 20 20 - 16.7 15 4.7 6.7 1.8 12 18 (unsaturate) 16 18 54 (unsaturate) Table 2.3 Permeation profiles of different proniosomal formulations, (n = 3). Proniosomal formulations Control 1.8 6.7 10 16 16.7 Jss (μg/cm .h) 8.792 ± 1.232 15.223 ± 1.441 10.406 ± 0.543 17.635 ± 1.953 22.392 ± 1.337 19.459 ± 0.868 10.337 ± 6.683 24.532 ± 0.961 KD/L (cm/h) 1.465 ± 0.205 2.537 ± 0.24 1.734 ± 0.091 2.939 ± 0.325 3.732 ± 0.223 3.243 ± 0.145 1.723 ± 1.114 4.089 ± 0.16 tL (h) 2.792 ± 0.46 4.336 ± 0.497 4.612 ± 0.376 4.28 ± 0.155 4.057 ± 0.207 3.155 ± 0.549 2.788 ± 0.978 4.299 ± 0.213 -1 D/L (h ) 0.061 ± 0.011 0.039 ± 0.005 0.036 ± 0.003 0.039 ± 0.001 0.041 ± 0.002 0.054 ± 0.01 0.066 ± 0.027 0.039 ± 0.002 KL 24.061 65.394 47.772 75.403 90.693 60.051 26.096 105.295 EI 1.732 1.184 2.006 2.547 2.213 1.176 2.79 45 2.4 Conclusion Proniosomal formulations with non-ionic surfactant were studied. The effect of hydrophilicity and hydrophobicity of one or two surfactants on drug solubility, proniosome surface structure and stability and skin permeation of haloperidol from different formulations were investigated. Haloperidol was entrapped in proniosomes with high efficiency for all formulations from 97 to 98%. Stability studies performed at 4oC and 25oC for a period of weeks did not reveal any significant drug leakage (p > 0.05). Formulations with single surfactants were found to increase the skin permeation of HP more than formulations containing two surfactants. The number of carbons in the alkyl chain of the non-ionic surfactant influenced the in vitro permeation of HP though the epidermis and the skin permeation was increased with increase in HLB value of the surfactant. Interfacial tension and surfactant hydrophobicity appeared to be useful for elucidating mechanism of skin permeation and for comparing drug fluxes from different proniosomal formulations. 46 [...]... Table 2. 1 Composition and appearance of proniosomal formulations Proniosomal formulations 1.8 2 6 6.7 10 16 16.7 Span 85 100 93.75 - Composition Surfactant (% of 22 5 mg) Span 60 Span 40 Tween 20 Tween 80 6 .25 41 59 100 60 40 59 41 100 - C (mg) L (mg) B (ml) IPA (ml) Appearance 25 25 25 25 25 25 25 22 5 22 5 22 5 22 5 22 5 22 5 22 5 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0. 625 0. 625 0. 625 0. 625 0. 625 0. 625 0. 625 Translucent... Sigurðardóttir and Loftsson 1995 Methyl β-CD, CM-β-CD, maltosyl- β-CD Hydrocortisone 21 Babu and Pandit 20 04 HP- β-CD, PM- β-CD 22 Sridevi and Diwan 20 02 23 24 Application Observation In vitro, Franz diffusion cell (mouse skin) In vitro, Franz diffusion cell (mouse skin) - CD complexation, pH adjustments and addition of oleic acid helped increase drug permeation through the skin - CD concentration had a... index, EI, a ratio of the drug permeability from the control and the proniosomal formulations, measures the enhancement in drug penetration (Vaddi et al., 20 02) EI  ( KD / L) proniosome ( KD / L) control (2- 8) 35 2. 2.9 Confocal Laser Scanning Microscopy (CLSM) Skin penetration of rhodamine-loaded proniosomes was viewed using a CLSM The CLSM was a Nikon A1R laser scanning confocal and digital camera from... systems and prevented drug crystallization, and thus increased the permeation rate 28 Lopez et al., 20 00 HP- β-CD, β-CD Dexamethasone In vitro, Franz diffusion cell (mouse skin) - Drug- CD complexation protected the drug against skin metabolism and increased the skin permeation (by increasing the drug availability on the skin surface) 23 Table 1 .2 An overview of the drug- cyclodextrin inclusion research... formulations contained 2 mg/ml HP 32 permeation with a confocal microscope Samples were prepared using 0.03% w/v of rhodamine G 2. 2.5 Encapsulation Efficiency and Stability of Proniosomes HP-containing proniosomes were separated from the free un-entrapped drug by centrifuging the samples at 12 000 rpm for 30 min at 20 oC The supernatant was recovered and assayed by an HPLC method for HP concentration The... (2- 5): KD J ss  L Co (2- 5) where Co is the initial drug concentration in the donor cell The diffusion parameter, D/L2 reflecting the mobility of the drug solute in the skin was calculated using the following equation: D 1  2 6t L L (2- 6) The partition parameter, KL, reflecting the distribution of the drug between the skin and the donor solution is derived from: KL  KD / L D / L2 (2- 7) The enhancement... hydrophobic drug, HP Single or blends of non-ionic surfactants were used and their effects on drug solubility, vesicle stability, surface tension, and skin permeation of HP from proniosome formulations were studied 2. 2 Materials and Methods 2. 2.1 Materials Span 40, 60, 85, Tween 80, sodium phosphate monobasic monohydrate, phosphatebuffered saline, cholesterol and haloperidol, fluorescein and rhodamin... al., 1998; Hu and Rhodes 20 00; Alsarra et al., 20 05; Yoshioka et al., 1994) Numerous studies have been conducted on the effects of drug concentration, the type of surfactant, alcohol and lipids on the in vitro permeation across the skin (Vora et al., 1998; Alsarra et al., 20 05; Varshosaz et al., 20 05) Formulations containing lecithin increased the drug encapsulation compared to formulations containing... Table 1 .2 An overview of the drug- cyclodextrin inclusion research in transdermal drug delivery No Reference Formulations Drug Application Drug- Cyclodextrin Inclusions 15 Ammar et al., 20 06 DM--CD, -CD, HP--CD, HP--CD Glipizide In vitro, Franz diffusion cell (rat skin) In vivo (rat) 16 Sridevi et al., 20 02 HP- β-CD Ketoprofen 17 Felton et al., 20 02 HP- β-CD Oxybenzone In vitro, Franz diffusion cell... increased the skin permeation of oxybenzone, however a parabolic relationship was observed between the CD concentration and drug permeation which was found to be due to the formation of a drug reservoir on the skin surface - Drug- CD inclusion decreased the skin permeation - Skin permeation observed from S-9977-CD solution is due to the lack of complex formation - Combination of oleic acid and RM- β-CD synergistically . (Menon 20 02; Glombitza and Müller-Goymann 20 02; Bouwstra and Honeywell-Nguyen 20 02) . The intercellular SC lipids (mortar) comprise mainly ceramides (~40% w/w), free fatty acids (~10% w/w) and. penetration enhancers, supersaturated drug delivery systems, iontophoresis, electroporation, sonophoresis and vesicle delivery systems (El Maghraby 20 08). Most permeation enhancers act on the SC. the skin. 20 Table 1 .2 An overview of the drug- cyclodextrin inclusion research in transdermal drug delivery. No. Reference Formulations Drug Application Observation 8 Maitre et al., 20 07 HP-ß-CD