Investigation on factors affecting drug delivery using polymers and phospholipids 5

PLGA microparticles were able to increase drug retention in the epidermis and decrease the drug permeation through the skin Ga de Jalón et al., 2001a and b; Rolland et al., 1993; Tsujimo

et al., 2007; Makino et al., 2001; Gutowska et al., 1992; Zhang et al., 2002; Guo et al., 2008).

PNIPAM microgels have been studied as transdermal carriers, however results did not show penetration enhancement across human epidermis (Lopez et al., 2005) PLGA microparticles were able to increase drug retention in the epidermis and decrease the drug permeation through the skin (Ga de Jalón et al., 2001a and b; Rolland et al., 1993; Tsujimoto et al., 2007) Several studies have shown sustained and controlled release of drugs from transdermal patches which contained EC (Mutalik and Udupa 2005; Mukherjee et al., 2005; Rama Rao et al., 2006; Rama Rao 2003; Mayorga et al., 1997 and 1996; Rama Rao and Diwan 1998; Amnuaikit et al., 2005).

These polymers have been studied individually as transdermal carriers in previous works Variation on the effect of partition coefficient and skin permeability was studied using PNIPAM microgels (Lopez et al., 2005) Formulation variation of PLGA microparticles was also investigated (Santoyo et al., 2002; Tsujimoto et al., 2007) Formulation strategies have also been studied using EC polymer as a transdermal vehicle ((Mutalik and Udupa 2005; Mukherjee et al 2005; Rama Rao et

al 2006) Here we wanted to compare and see the effect of polymer hydrophobicity, polymer transition temperature on the skin permeation.

The aim of this work is to determine if topical application of T4can produce systemic

7.2 Materials and Methods

7.2.1 Materials

L-levothyroxine, poly vinyl alcohol (MW 31,000), poly (N-isopropylacrylamide) (MW 20,000-25,000), ethyl cellulose and phosphate buffer saline tablets were purchased from Sigma, Singapore PLA (R 203H) and PLGA 48/52 (RG 503H) were gifts from Boehringer Ingelheim (Germany) The density of PLA and PLGA were 0.34 dl/g and 0.52 dl/g respectively.

7.2.3 Encapsulation Efficacy and Stability Studies

The drug-loaded microparticles were centrifuged at 17 000 rpm for 45 min at 20oC The free levothyroxine in the supernatant was determined by HPLC method and the encapsulation efficacy (EE %) was calculated using Eq 2-3.

Encapsulation efficacy was investigated after 14-week storage at room temperature (20oC) and in the fridge (4oC).

7.2.4 Microparticle Characterization

The surface morphology and appearance of microparticles were examined using FESEM mentioned in section 4.2.3.

7.2.5 HPLC Analysis

T4concentration was analyzed using the method mentioned in section 6.2.6.

7.2.6 In vitro Drug Release Studies

In vitro drug release from the drug-loaded beads was studied in phosphate buffer saline (PBS; pH 7.4) at 37oC in a horizontal shaker At specific intervals, 1-ml samples were taken and the microparticulate dispersions were centrifuged to remove impurities before being assayed for drug content by HPLC method An equal volume

of fresh PBS was immediately added to the receptor cell after each sampling.

7.2.7 In vitro Skin Permeation Studies

Permeation studies of drug-loaded microparticles were performed using a through diffusion cell apparatus (described in section 2.2.8.) The donor compartment was filled with 1 ml of aqueous polymeric microparticle solution and the receptor compartment was phosphate buffer saline pH 7.4 Samples from the receptor

flow-7.2.8 FTIR of Skin Sample

FTIR spectra of the skin samples treated with polymeric particles were obtained with Perkin Elmer Spectrum 100 (USA) After treating the epidermis with each formulation for 24 h, the samples were washed 3 times with PBS and vacuum-dried at room temperature Samples were then subjected to FTIR measurements Details are mentioned in section 4.2.4.

7.2.9 Confocal Studies of the Treated Skin

To study the effect of polymeric microparticles on the extent of skin penetration, confocal study was carried out Skin samples were treated with polymeric microparticles and then aquous solution of 0.03% w/v fluorescein dye was applied and its skin penetration was viewed using a CLSM described in section 2.2.9.

7.3 Results and Discussion

7.3.1 FESEM Characterization of Microparticles

FESEM images of the microparticles are shown in Fig 7.1 It can be seen that the appearance of the microparticles clearly varied with the polymer type Ethyl cellulose microspheres had a uniform microporous and sponge-like structure No considerable difference was observed between the microstructures of PLA and PLGA microparticles Cracks in the surface of PLGA microparticles were probably artifacts due to the high energy of the electron beam at high magnifications PLGA has a low glass transition temperature (Tg), therefore the polymer transforms from a glassy to a

rubbery state which is more susceptible to the vacuum pressure of FESEM (Wischke

et al., 2006) PNIPAM microcapsules were fragmented but not deformed.

The rate of solvent evaporation, polymer precipitation and stability of the inner aqueous phase play a major role in microcapsule morphology (Crotts and Park 1995) Surface tension of the solution greatly affects the microparticle structure Reduction

in surface tension of the solution will lead to fast and rapid solvent evaporation which will result in fewer pores on the particle surface (Niwa et al., 1993) In our study, parameters such as compositions of solvent system and aqueous phase were kept constant, therefore any difference in the morphology or structure of the particles is likely to be related to the intrinsic properties of the polymers.

(a) (b)

Fig 7.1 FESEM images of T 4 -loaded (a) PLA, (b) PLGA, (c) EC and (d) PNIPAM microparticles.

7.3.2 Determination of Encapsulation Efficacy

The encapsulation efficiencies of T4-loaded microparticles consisting of different polymers and their physical stability over a 14-week period are displayed in Fig 7.2.

It was found that ethyl cellulose microparticles exhibited the highest drug encapsulation of 85.98 ± 8.84% PLA and PLGA resulted in similar encapsulation efficacy of 75.54 ± 12.11% and 76.47 ± 17.88%, respectively PNIPAM had the lowest T4encapsulation of 67.59 ± 1.81%.

Fig 7.2 Stability of lipid suspensions: Encapsulation efficacy of the vesicles over time at

room temperature (20 o C) and fridge temperature (4 o C), n=3.

The in vitro degradation behavior of polymeric microparticles was investigated at

20oC and 4oC (Fig 7.2) It was found that irrespective of the storage temperature, ethyl cellulose microparticles remained stable during the 14-week storage period without significant drug leakage (p > 0.05) The degradation rate of PNIPAM microparticles was faster than PLA and PLGA microparticles PLGA microparticles stored at 4oC did not show any significant drug loss over the study period (p > 0.05) however, storage at 20oC resulted in significant drug leakage (p < 0.05) After 14 weeks at 20oC and 4oC, the T4 contents of PLA and PNIPAM microparticles were significantly lower than the original (p < 0.05).

7.3.3 In vitro Release of Levothyroxine

irrespective of polymer type The drug release from microparticles seems to occur in two phases: an initial rapid release followed by a slow release The initial burst effect

is probably due to the adsorption of the drug onto the wall of the microparticles which would be immediately released After which, the drug release profile displayed a delayed release that may be attributed to diffusion of the drug entrapped within the core of the microparticles.

Fig 7.3 In vitro release profile of T 4 from microparticles in phosphate buffer (pH 7.4) at the body temperature (37 o C), n = 3.

7.3.4 In vitro Skin Permeation Studies

In vitro skin permeation studies were performed to evaluate the skin absorption of T4from these preparations Fig 7.4 depicts the permeation profile of T4 from the polymeric particles The systems with PLA, PLGA and PNIPAM did not provide any

T4 penetration, however EC microparticles showed some drug penetration across the

skin This work showed that the use of the T4-loaded PLA, PLGA and PNIPAM microparticles increased drug retention in the epidermis and decreased drug permeation through the skin Consequently, these polymeric microparticles represent

a good delivery system to retard the release rate of drugs into the skin and improve topical therapy.

Fig 7.4 Permeation profile of T 4 across human epidermis (n=6-8).

Amorphous polymers exhibit glass transition temperature (Tg) Below this temperature, polymer is in a glass-like state Above this temperature, the polymer passes from a glassy to a rubber-like state which may cause coalescence and precipitation of the polymer network on the surface (Wischke et al., 2006; Kangarlou

Tipton 2000; Packhaeuser and Kissel 2007; Passerini and Craig 2001; Henry et al., 2005; Royall et al., 2001) As compared to polyesters, EC has a high Tg of >100oC (Tarvainen et al., 2003; Frohoff-Hülsmann et al., 1999; Hyppölä et al., 1996; Rowe et al., 1984) PNIPAM is a synthetic thermosensitive polymer that responds to internal

or external stimuli Aqueous PNIPAM solutions exhibit a LCST of 32oC At temperatures below the LCST, PNIPAM is hydrophilic and exists in a random coil form; however, above the LCST, it becomes insoluble and precipitates out from the aqueous solution Furthermore, this precipitation could delay the drug release by acting as an additional diffusion barrier (Choi et al., 2006; Geever et al., 2006).

It is possible to hypothesize that the low Tg of PLA and PLGA, and low LCST of PNIPAM may cause precipitation of the rubber-like, insoluble polymer on the skin surface This could create an impermeable barrier and prevent drug penetration across the epidermis T4skin penetration observed for EC microparticles may be due

to its high Tgtherefore polymer remains in the glassy state which is soluble and does not precipitate.

Luengo and coworkers studied the effect of PLGA nanoparticles on the skin permeation of flufenamic acid At shorter incubation times there was no significant differences in the permeated amount of drug, however after long incubation time due

to the degradation of the polymer to lactic and glycolic acid and the reduction of the

pH of the donor compartment, skin permeation was enhanced (Luengo et al., 2006) The authors could not explain the non-enhancing effect of nanoparticles at shorter

incubation time, however this phenomenon might be due to the change in the physical state of the polymer with regards to its low transition temperature.

An alternative view is that the presence of oxygen atoms in the polymer molecule could facilitate the formation of hydrogen bonds with the skin lipids This could have stabilized the rigidity of the solid-lipid state of the skin structure by increasing the skin Tg and therefore retarding the skin penetration (Hadgraft et al., 1996; El Maghraby et al., 2005; Asbill and Michnaik et al., 2000).

Confocal images of the skin samples treated with the polymeric microparticles are shown in Fig 7.5 It can be seen that the fluorescein dye easily penetrated through the control skin samples Skin samples treated with EC microparticles show some skin penetration of flourescein However it can be seen that the fluorescein dye could not penetrate the skin samples treated with polyesters and PNIPAM microparticles and the dye was mainly focused on the outer layer of the skin This may be due to the impermeable barrier of the polymer on the skin surface which prevents the penetration of the fluorescein.

Control EC PNIPAM

Fig 7.5 Binary image of the epidermis and localization of green fluorescence on the skin after treatment with the polymeric

particles For better visualization cell nuclei were counter stained with DAPI.

7.3.5 FTIR of Human Skin Samples

Fig 7.6 presents polymer-induced changes in the skin structure monitored through FTIR Spectra of the skin samples were recorded at the end of the in vitro permeation study The CH2 asymmetric and symmetric stretching vibrations were observed at

2920 and 2851 cm-1 respectively These peaks indicate that the majority of the stratum corneum lipids are in the solid-gel state The shift of these peaks to a higher wavenumber after skin treatment would suggest increased lipid fluidity of the skin structure Peaks typical of SC proteins were those occurring in the region of 1500-

1700 cm-1 The carbonyl stretching observed at 1743 cm-1 is due to C-O stretching Amide I band at 1650 cm-1 is associated with α-helix conformation of the protein backbone (Bernard et al., 2007; Tanojo et al., 1997; Goates and Knutson 1993, 1994) After the exposure of the skin samples to microparticle solutions, no peak shift in the

CH stretching area and the protein domain was observed These polymers did not alter the lipid fluidity of the SC, and did not interact with the SC proteins.

Fig 7.6 FTIR spectrum of (a) untreated human epidermis, and skin treated with (b) 10% w/v

PVA mat, (c) 10% w/v PNIPAM mat and (d) PBS.

7.4 Conclusion

Formulations containing polymeric microparticles suitable for topical and transdermal delivery systems were studied using four different polymers, poly D,L lactide (PLA), poly D,L lactide co glycoside (PLGA), poly (N-isopropylacrylamide) (PNIPAM) and ethyl cellulose (EC) It was found that ethyl cellulose microparticles had the highest drug encapsulation and minimal drug leakage during the 14-week storage period PNIPAM microparticles had the lowest drug encapsulation efficiency and a fast degradation rate PLGA microparticles exhibited a temperature dependent drug leakage It was observed that transition temperature (Tg) may influence the skin permeation rate of the drug from these microparticles Polyesters (PLA and PLGA) and PNIPAM acted as skin penetration retardant These microparticles have potential

use in skin formulations containing sunscreens and other active ingredients that are meant to be concentrated on the skin surface However skin permeation was observed from EC microparticles, therefore such polymers may be used as carriers in transdermal formulations and can help achieve therapeutic concentrations of the drug

in the plasma

Vesicular lipid systems are used as carriers for topical and transdermal drug delivery However, it is generally agreed that conventional liposomes have little or no effect on the penetration of drugs through the skin and are chemically and physically unstable (Desai and Finlay 2002; Hashizume et al., 2003) Niosome vesicles from nonionic surfactants, were thought to be an improvement over the conventional liposomes (Hofland et al., 1991; Hao et al., 2002) Alternatively, polyethyleneglycol (PEG) containing niosomes (Liu et al., 2007; Hua and Liu et al., 2007) and other formulations such as proniosomes, containing cholesterol and non-ionic surfactants, were developed (Alsarra et al., 2005; Fang et al., 2001; Hu and Rhodes 1999) Ethanol, a skin permeation enhancer, was incorporated into liposomes and termed ethosomes These vesicles have the ability to permeate through the human skin and effect intracellular delivery (Touitou et al., 2000 and 2001; Paolinoa et al., 2005).

Transferosomes, another form of lipid carriers, are regarded as deformable liposomes These ultra-deformable carriers contain an edge activator and have high elasticity which enables them to squeeze through intercellular regions of stratum corneum (SC) (Cevc et al., 1998; Mishra et al., 2007; Elsayed et al., 2006) Recently, liposomes with similar composition to that of the SC, cerosomes, have been formulated and used

to enhance the skin delivery of drugs (Hatziantoniou et al., 2007; Contreras et al., 2005).

The aim of this work was to identify the most effective formulation for delivering a hydrophilic drug in terms of drug encapsulation efficiency, stability and skin permeation properties The vesicles of conventional liposomes, ethosomes, transferosomes, proniosomes, niosomes and polyethyleneglycol-block- polypropyleneglycol-block-polyethyleneglycol (PEG-PPG-PEG) niosomes were formulated and compared to SC liposomes (cerosomes) for their ability to increase skin permeation of diclofenac.

Although the lipid vesicles have different names but actually they only vary in some

of the ingredients The effect of various compositions of the lipid vesicles on the skin permeation of diclofenac was studied Alterations of the biophysical structure of the

SC in the presence of these vesicles were determined using fourier transform infrared spectroscopy.

8.2 Materials and Methods

8.2.1 Materials

Lipoid E 80 (Phosphatidylcholine from egg yolk lecithin) and Cerosome 9005 were gifts from Lipoid GmbH (Ludwigshafen, Germany) Diclofenac sodium, cholesterol, sodium phosphate monobasic monohydrate, PEG-PPG-PEG, Span 85 and Tween 20 were purchased from Sigma, Singapore Tween 80 was purchased from Bio-Rad Laboratories (Singapore).

8.2.2 Preparation of Diclofenac Sodium-Loaded Vesicles

The compositions of different vesicle formulations are listed in Table 8.1 Diclofenac-loaded conventional liposomes were prepared by cast film method as reported previously with slight modification(Dubey et al., 2007) Briefly, Lipoid E 80 was dissolved in ethanol in a clean and dry round bottom flask followed by removal

of the organic solvents using rotary vacuum evaporator above the lipid transition temperature to form a thin film on the wall of the flask After removal of solvent traces, thin lipid film was hydrated with phosphate buffer saline (PBS) pH 7.4 containing diclofenac by magnetic stirring (1000 rpm, 10 min) at the corresponding temperature.

Ethosome colloidal suspensions, PEG-PPG-PEG niosomes, transferosomes, niosomes were prepared as reported elsewhere (Paolinoa et al., 2005; Liu et al., 2007; Elsayed

et al., 2006; Devaraj et al., 2002) Ingredients were solubilized in absolute ethanol.

PBS containing diclofenac sodium was added gradually while mixing at 1000 rpm with a magnetic stirrer.

Proniosomes were prepared as described previously with slight modification (Alsarra

et al., 2005) Briefly, Tween 20, Lipoid E 80, and cholesterol (9:9:2) were mixed with absolute ethanol and warmed in a water-bath sonicator at 65oC for 5 min Then PBS containing diclofenac sodium was added and the mixture was further warmed in the water bath for about 2 min until a clear solution was obtained The mixture was allowed to cool at room temperature to form the proniosomal suspension.

All formulations were finely homogenized for 1 min at amplitude 30, and at pulser of

2 by means of an ultrasonic processor (ITS Science).

Cerosomes (containing 6.6% v/v SC lipids) were used as obtained without further modification Diclofenac sodium was physically blended with this readily made cream and stirred to make a homogenous formulation, therefore vesicle size and encapsulation efficacy was not calculated for this formulation A PBS solution of diclofenac was used as control All formulations contained a total of 5 mg/ml diclofenac sodium.

Table 8.1 Composition of lipid suspensions.

8.2.3 Determination of Encapsulation Efficiency

The unentrapped diclofenac was removed following centrifugation at 17 000 rpm for

45 min at 20oC and analysed by HPLC method The encapsulation efficiency (EE %) was calculated using Eq 2.3.

8.2.4 Storage Stability of Vesicles

Storage stability studies are important in the development of pharmaceutically acceptable product The ability of vesicles to retain the drug was assessed by keeping the formulation suspensions at 4 ± 2oC (fridge) and 20 ± 2oC (room temperature) for a period of 60 days Drug leakage was observed by measuring encapsulation efficacy

of the vesicular formulations The diameters of the particles were measured on days

30 and 60 at two different temperatures The vesicular suspensions were kept in sealed vials Mean vesicle sizes of drug-loaded liposomes were determined using Zetasizer 300HSA (Malvern Instruments, Malvern, UK) Analysis (n = 3) was carried out at room temperature and an angle of detection of 90o.

8.2.5 In vitro Skin Permeation Studies

The permeation profiles of drug from drug-loaded ethosomes, niosomes,

medium was PBS Samples were withdrawn at specific time intervals for a period of

48 h Diclofenac was quantified using HPLC method.

8.2.6 FTIR Studies of the Human Skin

After treating the epidermis with each formulation for 48 h, the samples were washed

3 times with PBS and vacuum-dried at room temperature The samples were then subjected to FTIR spectroscopy mentioned in section 4.2.4.

8.2.7 HPLC Assay of Diclofenac Sodium

The quantitative determination of diclofenac was performed by HPLC using acetonitrile/pH 3 buffer solutions (35:65 v/v) delivered at a flow rate of 1 ml/ min A sample of 20 μl was eluted from the Agilent hypersil column, C18, 5 µm, 4.6 × 250

mm Drug peaks, detected at wavelength 290 nm, were separated at 5.2 min.

8.3 Results and Discussion

8.3.1 Vesicle Size Measurment

Fig 8.1 presents the sizes of the vesicles over an 8-week storage period The smallest vesicles were transferosomes with a mean vesicle size of 118.9 ± 9.8 nm Proniosomes had the largest vesicles of 710.8 ± 135.3 nm This may be due to the high concentrations of cholesterol and lecithin incorporated in the vesicles.

The size of the PEG-PPG-PEG niosomes increased upon 8-week storage compared to 1-h after preparation The highest flocculation rate was observed for formulations

stored at room temperature Aggregation was not found to be temperature-dependent for the rest of the formulations as it was similarly observed during storage at room temperature and in the fridge Lack of net electrical charge of conventional liposomes could have caused the aggregation of the vesicles This effect might have resulted from storage of the liposomal formulation in PBS or other aqueous phase containing polyvalent ion (Grit and Crommelin 1993; Fransen et al., 1986; Lau et al., 2005) However, vesicles of ethosomes, transferosomes, niosomes and proniosomes decreases in size after 8 weeks as compared to 1-h after preparation.

8.3.2 Determination of Encapsulation Efficiency

Fig 8.2 represents the encapsulation efficiency of the vesicles There was no significant difference in the amount of drug encapsulated in all formulations (p > 0.05) Proniosomes and conventional liposomes entrapped the most drug of 63.43 ± 9.31% and 67.15 ± 5.84%, respectively (p> 0.05) The preparation methods required these vesicles to be hydrated at above 60oC while the rest of the formulations were hydrated at room temperature The hydration temperature was thought to influence the extent of drug encapsulation, therefore less drug was entrapped at temperatures below the lipid transition point (Hao et al., 2002) This could explain the low drug encapsulation observed for other vesicle Lipid vesicles were thought to effectively entrap both hydrophobic and hydrophilic drugs However, studies have also demonstrated that the entrapment of hydrophilic molecules could be less efficient than that of hydrophobic molecules (Touitou et al., 2001; López-Pinto et al., 2005) The latter supports our findings as relatively small amounts of diclofenac sodium were encapsulated in the vesicles when compared to those of hydrophobic drugs in the literature Encapsulation efficiencies of the drug in our formulated systems after 12-week storage at 20oC and 4oC showed no difference from that of a freshly prepared sample These formulations were relatively stable with minimal drug leakage (p > 0.05).

8.3.3 In vitro Skin Permeation Studies

The effects of 6 vesicular formulations on the in vitro percutaneous permeation of diclofenac sodium through human epidermis are shown in Fig 8.3.

Cerosomes significantly increased drug penetration compared to the other formulations (p < 0.01) and this could be attributed to the penetration enhancement of the fatty acids in these formulations (Sintov and Botner 2006; Hua and Liu 2007; Niazy 1991; Fang et al., 2003; Chi et al., 1995) Transferosomes gave a slight increase in drug permeation relative to aqueous solution (p > 0.05) Small vesicle size and the flexibility of the lipid membrane could have enhanced the skin permeation (Boinpally et al., 2003; Cevc et al., 1998; Cevc and Blume 2001; Jain et al., 2005; Honeywell-Nguyen et al., 2002 and 2003; El Maghraby et al., 1999; 2001).

Cerosome Control

Fig 8.3 Cumulative concentrations of diclofenac sodium across human epidermis (n=3).

Interestingly, drug penetration across human epidermis was not significantly different

in other vesicles types and this was also reported in previous findings (Bhattachar et al., 1992) A few possible mechanisms could explain this effect It is known that the degree of hydration of the SC influences skin permeability Skin hydration loosens the packing of the tight cell junctions which makes the skin more permeable (Brown

et al., 2001) Presence of ethanol in the formulations would reduce the

thermodynamic activity of the drug and result in skin dehydration, hampering drug permeation from these vesicles (Levang et al., 1999; Gao and Singh 1998; Babita et al., 2006) Researchers ascribed the structural similarity of vesicles and components

of the skin; vehicles introduce another lipid barrier by creating a drug reservoir which eventually retards diffusion of the drug This may result in prolonged drug release and retention by the skin (Fang et al., 2006) Literature reports support the lack of correlation between entrapment efficiency and permeation of a drug across the skin (Elsayed et al., 2006; Barry 2001) Greater drug permeation from cerosomes could be due to free uncapsulated diclofenac sodium, thus favoring drug uptake into skin Drug solubility in SC lipids and affinity of the drug to the vesicles may influence the skin penetration of diclofenac The low skin permeability of diclofenac could be explained by the limited solubility of the drug in the skin lipids and the low partition coefficient of the drug from the vesicle formulation into the SC layer (Elsayed et al., 2006; El Maghraby et al., 2001; Verma et al., 2003; Dreher et al., 1997; Okumura et al., 1989; Barry 1988; Honeywell-Nguyen and Bouwstra 2003).

8.3.4 FTIR Studies of the Human Skin

Vesicle-induced changes in the skin structure were monitored by FTIR Spectra of the skin samples were taken after the in vitro permeation study The vesicle-induced

from 2955 to 2958 cm-1 Carbonyl (C=O) stretching at wave number 1740 cm-1corresponds to the esterified ester lipids This peak became negligible after skin treatment with vesicles which indicates fusion of the vesicles with the epidermal layer and alteration of SC protein conformation (Babita et al., 2006; Goates and Knutson 1994; Narishetty and Panchagnula 2005; Tanojo et al., 1997).

Protein secondary conformation is characterized by Amide I bond which corresponds

to different component bands, each representing a different state of peptide secondary structure, β-helix protein (1620-1635, 1670, and 1692-1697 cm-1) and α-sheet protein structure (1649-1656 cm-1) In the presence of vesicles, both low (1634, 1640, 1645

cm-1) and high (1660, 1667, 1671 cm-1) wavenumber shoulders were observed within the Amide I region This pattern is diagnostic for formation of β-sheet structure and indicates change in the conformation of protein secondary structure in the presence of these vesicles (Goates and Knutson 1994; Zhang et al., 2007) The extent of the changes in Amide I band position varied for different formulations, however the change was almost negligible for skin samples treated with ethosome This is in accordance with our permeation results where ethosome yielded the lowest drug permeation It is important to consider the influence of vehicle in order to delineate the effect of lipids or non-ionic surfactants on the skin structure and FTIR spectra Amide I band observed for ethosomes was comparable to that produced by aqueous mixture alone, inferring that the branching effect of amide I area observed for the other formulations is not due to the presence of lecithin or ethanol Non-ionic surfactants, cholesterol, ceramides or fatty acids seem to play an important role in the branching effect of amide I region Also the intensity of this effect appears to be

concentration dependent and influenced by the type of components used in each formulation Transferosomes which contained 1% v/v Tween 20 demonstrated less effect as compared to PEG-PPG-PEG niosomes with higher concentration of the surfactant.

sodium from these vesicles may not be affected by the fluidity of the SC lipid layer as all formulations exerted similar effects on the SC but only cerosomes produced significantly higher drug permeation Hydrophilic drugs such as diclofenac sodium were thought to permeate the skin mainly via the polar transcellular pathway which is

a protein rich domain whereas alteration in the lipid structure of the skin is thought to

be responsible for the non-polar passage of hydrophobic drugs (Yokomizo 1996) Results of the permeation study suggest that the solubility of the drug in SC lipids and the partitioning of the drug molecule from the vesicles to skin may be key factors that influenced the permeation of diclofenac sodium across the skin.

Fig 8.4 Representative FTIR spectra of (a) untreated human epidermis, and skin in the

presence of (b) PBS, (c) Ethosome, (d) transferosome, (e) Proniosome, (f) niosome, (g) PPG-PEG niosome and (h) cerosome.

PEG-8.4 Conclusion

Various lipid suspensions including conventional liposomes, ethosomes, transferosomes, proniosomes, niosomes, PEG-PPG-PEG niosomes and stratum corneum liposomes (cerosomes) were developed Stability profile of the preparations over 12 weeks did not show any significant drug leakage from the studied vesicles (p

> 0.05) FTIR observations indicate that the vesicles increased stratum corneum (SC) lipid fluidization and altered protein conformation Skin permeability experiments showed that the free unencapsulated drug in the cerosome formulations caused significant increase in the drug permeation across the skin (p < 0.01) The low skin permeability of the drug from the other vesicles could be due to entrapment of the drug in these vesicles which decreased the solubility of hydrophilic drug in the skin lipids and reduced the partition coefficient of the drug from these vesicles into the SC.