Effect of colloidal carriers on ascorbyl palmitate stability

European Journal of Pharmaceutical Sciences 19 (2003) 181–189 www.elsevier.com / locate / ejps

Effect of colloidal carriers on ascorbyl palmitate stability a, b ˇ *, Marjeta Sentjurc ˇ Julijana Kristl a , Breda Volk a , Mirjana Gasperlin , Polona Jurkovicˇ a a

ˇ ˇ 7, 1000 Ljubljana, Slovenia Faculty of Pharmacy, University of Ljubljana, Askerceva b ˇ Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Jozef

Received 14 October 2002; received in revised form 2 April 2003; accepted 3 April 2003

Abstract Active compounds can be protected against degradation by incorporation into colloidal carrier systems. The stabilizing effect of carrier systems for ascorbyl palmitate (AP) was investigated using microemulsions (ME), liposomes and solid lipid nanoparticles (SLN). Analysis of chemical stability by HPLC showed that AP is most resistant against oxidation in non-hydrogenated soybean lecithin liposomes, followed by SLN, w / o and o / w ME, and hydrogenated soybean lecithin liposomes. The molecular environment of the AP-like nitroxide probe (C 16 -Tempo) in colloidal carriers was characterized using electron paramagnetic resonance (EPR) spectroscopy. We have found that the nitroxide groups are located in environments with different polarity and mobility. The hydrophilic part of AP is the reactive moiety, and high stability is obtained in systems in which this part is exposed to a less polar environment. Additionally, the determined accessibility of nitroxide groups to reduction correlated well with the chemical stability of AP. It is more deeply immersed in the interface when entrapped in a liquid-state carrier than when applied in gel-state particles. Encapsulation of AP in SLN core leads to greater stability. We conclude that the location of the sensitive group of the drug-molecule in a carrier system is crucial for its stability.  2003 Elsevier B.V. All rights reserved. Keywords: Stability; Ascorbyl palmitate; Microemulsion; Liposomes; Solid lipid nanoparticles; Electron paramagnetic resonance (EPR)

1. Introduction Vitamin C ( L-ascorbic acid) is a strong and powerful water-soluble non-enzymatic antioxidant that efficiently protects biological molecules against oxidative degradation. It shows synergistic effect in conjunction with other antioxidants such as tocopherols and b-carotenoids, by establishing a peculiar recycling system (Keller and Fenske, 1998). As an antioxidant it can scavenge and destroy reactive oxidizing agents and free radicals, which are important in the processes of skin ageing (Colven and Pinnell, 1996). Vitamin C also improves the elasticity of the skin and reduces wrinkles by stimulating collagen synthesis (Philips et al., 1994). Since it suppresses pigmentation and decomposes melanin, it is used as a skinwhitening agent (Kameyama et al., 1996; Zhai and Maibach, 2001). Because of these favourable effects, vitamin C has long

*Corresponding author. Tel.: 1386-1-476-9644; fax: 1386-1-4258031. ˇ E-mail address: [email protected] (M. Gasperlin). 0928-0987 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00104-0

been used in pharmaceutical and cosmetic preparations (Colven and Pinnell, 1996). However, its low stability is a serious limitation. It is easily oxidized, especially under aerobic conditions and light exposure, being degraded, firstly in a reversible step to dehydroascorbic acid and secondly, irreversibly to oxalic acid (Austria et al., 1997). Chemical modification of ascorbic acid has led to more stable derivatives such as ascorbyl esters with C 6 to C 18 fatty acids or ascorbyl phosphate salts. Among the lipophilic derivatives, ascorbyl palmitate (AP) is often used in topical preparations against oxidative changes of biological components of the skin, and as an antioxidant to protect lipophilic ingredients in formulations (Reynolds, 1996; Kibbe, 2000; Silva and Campos, 2000). Colloidal carriers show great potential as multipurpose delivery systems, especially in targeting, controlling release, and increasing drug stability (Lasic, 1998; Lawrence ¨ and Rees, 2000; Muller et al., 2000). Because ascorbyl palmitate is almost insoluble in water at room temperature, carrier systems such as microemulsions (ME), with high solubilization capacity, are of special interest (Lawrence and Rees, 2000; Bagwe et al., 2001). Our earlier stability studies on ascorbyl palmitate in microemulsions showed that its long-term stability in microemulsions is still not

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

182

ˇ adequate (Spiclin et al., 2001). This led us to further investigation of ascorbyl palmitate stability in colloidal carriers such as liposomes and solid lipid nanoparticles (SLN). Because the stability of ascorbyl palmitate is oxygen-dependent, the composition of carriers and the accessibility of the cyclic ring to oxidation play an important role. In this study AP was selected as an active compound against oxidative changes in the skin. It served as a good test compound because of its high lipophilicity, which makes it an excellent candidate for particulate encapsulation and skin penetration. The aim of this work was to formulate ascorbyl palmitate entrapped in colloidal carrier systems such as microemulsions, liposomes and lipid nanoparticles, and to investigate the influence of these carriers on ascorbyl palmitate stability. The location of ascorbyl palmitate in the different carriers was assessed using electron paramagnetic resonance (EPR) in particular, in order to study the effect of the carrier nanoenvironment on ascorbyl palmitate stability. Comparison of these findings with the results of chemical stability in liposomes, lipid nanoparticles and microemulsions, has enabled us to elucidate the influence of a particular colloidal carrier system on the stability of ascorbyl palmitate.

2. Materials and methods

2.1. Materials Components of carrier systems were: caprylic / capric ¨ triglyceride, Miglyol 812 (Huls, Germany); PEG-8 caprylic / capric glycerides, Labrasol (Gattefosse, France); polyglyceryl-6 dioleate, Plurol oleique (Gattefosse, France); non-hydrogenated soybean lecithin (NSL), Phospholipon 80 (Natterman, Germany); hydrogenated soybean lecithin (HSL), Emulmetik 320 (Lucas Mayer, Germany); cholesterol (Sigma, Germany); glyceryl behenate, Compritol 888 ATO (Gattefosse, France); and poloxamer 188, Pluronic F68 (BASF, Germany). Water was purified by

reverse osmosis. The quantitative composition of the dispersions is shown in Table 1. Other chemicals were sodium ascorbate (ASC; Pliva, Croatia) and ascorbyl palmitate (AP; Hoffmann La Roche, Switzerland). Spin probe (SP) 1-oxyl-2,2,6,6-tetramethyl4-hexadecanoyloxypiperidine (tempyl palmitate or C 16 Tempo) (Fig. 1) was synthesized at the Faculty of Pharmacy, Ljubljana, Slovenia.

2.2. Methods The composition of colloidal dispersions without the active component is shown in Table 1. The amount of ascorbyl palmitate in all dispersions was 1 or 2% (w / w) with respect to the final dispersion. For EPR measurements C 16 -Tempo concentration was 1310 24 M in the final dispersion.

2.2.1. Preparation of microemulsions AP was dissolved in Labrasol, and the other three components were then added. Microemulsions were formed spontaneously after gentle hand mixing. 2.2.2. Preparation of liposomes Liposomes were prepared by the thin film method. AP was added to the lipids and the mixture dissolved in chloroform and dried in a rotary evaporator. The dry lipid film was hydrated with water above the phase transition temperature of phospholipids; i.e. at room temperature for NSL and at 85 8C for HSL. The dispersions of liposomes were extruded through a LiposoFast extruder (Avestin, Canada) using polycarbonate membranes. 2.2.3. Preparation of solid lipid nanoparticles SLN were prepared by the melt emulsification method using a Lab-Tek rotor-stator homogeniser (Omni, USA). AP was dissolved in the melted lipid phase before dispersing in stabilizer solution. The water–lipid dispersion was stirred for 10 min at 20 000 rpm at 85 8C. After emulsification the dispersion was cooled for 10 min at 5000 rpm at room temperature and nanoparticles, consisting of a solid

Table 1 Composition of colloidal carrier systems (in w / w %) Microemulsion

Lipid phase Surfactants

Water phase

Compritol 888 ATO Miglyol 812 Phospholipon 80 Emulmetic 320 Cholesterol Labrasol Plurol oleique Pluronic F68 Water

Liposomes

o/w

w/o

NSL

– 7.4

– 24.8

–

– – – 38.0 9.5 – 45.1

– – – 47.5 11.9 – 15.8

–

HSL – –

1.4 0.6 – – – 98.0

Lipid nanoparticles

– 1.4 0.6 – – – 98.0

5.1 – 2.0 – – – – 0.5 92.4

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

183

vern, UK). The particle charge was quantified as zeta potential. The electrophoretic mobility, m, was determined by laser Doppler anemometry using a Zetasizer 3000 (Malvern Instruments). Measurements were performed at field strength of 20 V/ cm in distilled water. The zeta potential was calculated by the Helmholtz-Smoluchowski equation. The samples were diluted with purified water (1:50; v / v) prior to pH, zeta potential and size determination. pH of the samples was 6.1–6.4.

2.2.5. HPLC assay All samples containing AP were stored in tightly-closed glass flasks at room temperature (2261 8C) in the dark. The amount of non-degraded AP was determined quantitatively by HPLC analysis at the beginning of storage and subsequently at predetermined time intervals. The HPLC apparatus consisted of HPLC pump K-501, sample injector A0258 with a 20-ml-sample loop (Knauer, Germany), and a 2151 variable wavelength detector (LKB Bromma, Sweden). To determine AP in microemulsions and liposomes, the stationary phase was a 12034-mm I.D. column packed with 5-mm Nucleosil C 18 , and the mobile phase a mixture of methanol–acetonitrile–0.02 M phosphate buffer, pH 2.5 (75:10:15). The flow rate was 1.5 ml min 21 and UV detection was at 254 nm. A 100-ml sample of microemulsion or liposome dispersion was diluted 1:100 (v / v) with methanol prior to HPLC assay. For determination of AP in lipid nanoparticles the stationary phase and UV detection were the same, but the mobile phase was a mixture of methanol–acetonitrile–0.02 M phosphate buffer, pH 2.5 (40:40:20) and the flow rate was 2.0 ml min 21 . For HPLC assay, 100 ml lipid nanoparticles dispersion was dissolved in 5 ml of dichloromethane and then further diluted with methanol to a final dilution of 1:100 (v / v). All analyses were performed in triplicate at ambient temperature.

Fig. 1. Chemical structure (a) and space formulae (b) of ascorbyl palmitate (A) and C 16 -Tempo (B). Due to keto-enol tautomerization two possible conformations of ascorbyl palmitate occur. The slight differences in orientation and hydrophilicity of the cyclic rings of AP and C 16 -Tempo are seen in the space formulae.

lipid core surrounded by a phospholipid layer and stearic stabilizer, were formed (Ahlin et al., 1998).

2.2.4. Characterization of colloidal carriers The diameter and the polydispersity index of liposomes and SLN were determined using photon correlation spectroscopy on a Zetasizer 3000 (Malvern Instruments, Mal-

2.2.6. EPR measurements For the EPR studies the colloidal carriers were labelled with the nitroxide spin probe, C 16 -Tempo. Then 0.01 M aliquots of spin probe (SP) dissolved in 95% (v / v) ethanol were placed in glass tubes and dried before adding the lipid phase for preparation of colloidal dispersions. Further procedures of sample preparation were as described above. Samples were stored at 15 8C to prevent SP degradation. For EPR measurements samples were drawn into a 40-ml standard quartz capillary and spectra were recorded on a Bruker ESP 300 X-band spectrometer at room temperature. Settings were: modulation frequency, 100 kHz; modulation amplitude, 0.15 mT; sweep width, 10 mT (337–347 mT); microwave power, 10 mW; and microwave frequency, 9.6–9.8 GHz. 2.2.6.1. Parameters obtained directly from the EPR spectra To determine the localization of spin probe in colloidal

184

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

carriers, EPR spectra were measured and hyperfine splitting constant, a N , correlation time tC , and reduction with ascorbate determined. The a N , as the distance between the low and middle field line was measured, and tC , was calculated according to Eq. (1):

tC 5 kDH0 [(h 0 /h 21 )1 / 2 2 1)]

(1)

where DH0 is the line-width of the middle line of the EPR spectra, h 0 and h 21 are amplitudes of the middle and high-field lines, and k is a constant typical for the SP used (k value for C 16 -Tempo is 1.93310 26 s / T; Marsh, 1981). The formula is valid for fast isotropic motion in not well-ordered domains with tC ,5 ns.

Table 2 Degradation of ascorbyl palmitate (AP) after 4 weeks in different colloidal carrier systems at an initial concentration of 1% w / w (mean6S.D.) Carrier system

Amount of non-degraded AP (%)

w / o ME o / w ME NSL liposomes HSL liposomes SLN

1961 1361 2662 764 25612

2.2.6.2. The reduction of SP with sodium ascorbate To estimate the accessibility of the SP nitroxide groups to reducing agent, the sample was mixed with 1 mM sodium ascorbate (ASC); the amount of ASC added was 10 times higher than the amount of the nitroxide. SP reduction was followed by EPR spectral decrease with time. The intensity was calculated from the amplitude of the low-field line (first peak) of the EPR spectra, as this line-shape is not influenced by ascorbate (it has absorption signal in the area of the middle-field line of the nitroxides). The intensity (I) after adding ascorbate is calculated from Eq. (2): I 5 h 1 DH 21

(2)

where h 1 is the amplitude and DH1 width of the peak-topeak low-field EPR spectral line.

2.2.7. Statistical analysis The results are expressed as the mean6S.D. Statistical analysis was carried out using Student’s t-test. Significant differences were determined at P,0.05.

Fig. 2. Influence of carrier systems on the rate of degradation of AP at an initial concentration of 1% with time (HPLC determination).

3. Results and discussion

3.1. Stability of AP in different colloidal carrier systems Five different colloidal carrier systems were selected to incorporate AP, and their influence on AP stability was monitored over a period of 4 weeks. Two of them were microemulsions, w / o and o / w type (compositions were chosen on the basis of the phase diagram prepared by titration method), two were liposomes, composed of hydrogenated (HSL) and non-hydrogenated (NSL) soybean lecithin, and the fifth comprised solid lipid nanoparticles (SLN). The observed instability of AP is a consequence of its oxidative degradation. Fractions of non-degraded AP remaining after 4 weeks in the different colloidal carrier systems are shown in Table 2. AP was found to be most stable in NSL liposomes, followed by SLN, w / o and o / w microemulsions, and HSL liposomes. Its degradation

Fig. 3. The effect of deairing (flooding with argon) on the rate of degradation of AP at an initial concentration of 2% in w / o and o / w microemulsions, as determined by HPLC; dea, deaired.

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

ranged from 93 to 75% (w / w), depending on the type of carrier system. The degradation of AP entrapped in liposomes and in SLN is shown in Fig. 2 and in ME in Fig. 3. Statistical comparison using Student’s t-test at P,0.05, showed that the type of microemulsion and liposome composition significantly influences AP stability. The active compound was significantly more stable in NSL than in HSL liposomes and in w / o type ME than in o / w. Further, the extent of AP degradation was significantly higher in SLN than in HSL liposomes, and in ME of both types, but there was no statistically significant difference between NSL liposomes and SLN. In all systems, differences became significant after 1 week. Degradation profiles of AP were evaluated by fitting experimental data to different order kinetics. The calculated Pearson values are listed in Table 3, bold print indicating the best fits. For both types of ME and NSL liposomes we found second order kinetics assuming equal importance for both concentration-active component and dissolved oxygen. For HSL liposomes the degradation followed pseudo first order kinetics indicating that oxygen was abundant compared to AP. In the case of nanoparticles pseudo first order or zero order kinetics were found possible. Lower Pearson values are a consequence of higher standard deviations. It is obvious that different kinetics of AP degradation is a consequence of different local environments, also reflecting the different observed stabilities. In a previous publication, the higher stability of AP in w / o ME was explained in terms of its different partition ˇ patterns (Spiclin et al., 2001). Due to the amphiphilic structure of AP, it is envisaged as being located at the interface, with the palmitic residue in the lipophilic phase and the cyclic ring in the aqueous phase. Only the cyclic ring is expected to be sensitive to oxidation. In the case of w / o microemulsions this part of the AP molecule is located in the internal aqueous phase while in o / w microemulsions it is in the external phase. Further, oxygen is considered to be an order of magnitude more soluble in oil than in water, and therefore it distributes preferentially into the oil phase. Indeed, the w / o interface could act as a physical barrier to oxygen diffusion into the internal aqueous phase (Gallarate et al., 1999), which additionally Table 3 Pearson coefficients for the case of zero, first and second order degradation kinetics of AP in different carrier systems Carrier system

Zero order a

First order b

Second order c

o / w ME w / o ME HSL NSL SLN

0.8931 0.9442 0.9789 0.9197 0.9837

0.9432 0.9809 0.9905 0.9868 0.9831

0.9796 0.9916 0.9736 0.9901 0.9541

a

Extent of a linear relationship between A t and t. Extent of a linear relationship between ln A t and t. c Extent of a linear relationship between 1 /A t and t where A t is a fraction of non-degraded AP at determined time. b

185

supports the better stability of the active component in the w / o type of microemulsions. To eliminate the effect of oxygen present, the stability of AP was determined in the same ME, but oxygen was replaced by argon, which is inert and heavier than air. The kinetics of degradation of AP, with and without de-airing, are shown in Fig. 3. When flooded with argon, AP was found to be more stable in both ME. However, the effect of de-airing is more prominent in the case of o / w ME, in accordance with the assumption that the stability of AP is highly dependent on the oxygen dissolved in the aqueous phase. The results of AP stability in the different colloidal carrier systems correlate with the content of structured lipid, which is higher in SLN than in liposomes. Although microemulsions contain the highest content of lipids, they were not found to be the best carriers. Due to the liquid state of lipids, the AP molecules can presumably penetrate easily to the aqueous phase.

3.2. Characterization of the environment of C16 -Tempo in colloidal carriers The size and charge of the particles were determined to ensure that the physical characteristics of the colloidal carrier systems prepared with AP and with the spin probe were similar over the experimental period. The size of the particles ranged from 150620 nm (polydispersity index (PI): 0.660.1) for nanoparticles to 260610 nm for NSL liposomes (PI: 0.4060.05) and 310640 nm for HSL liposomes (PI: 0.4560.05); zeta potential was 260610 mV for HSL liposomes, 25565 mV for NSL liposomes and 24565 mV for SLN. All colloidal dispersions remained stable during the period of investigation. The dispersions prepared with AP and those with C 16 -Tempo did not differ significantly in their particle size, particle size distribution or zeta potential. The ascorbyl palmitate molecule comprises two moieties, the lipophilic chain and the hydrophilic head. C 16 Tempo is a very similar molecule in that it consists of the same lipophilic chain and a cyclic paramagnetic nitroxide part and is a membrane-localized amphiphilic spin probe. This makes C 16 -Tempo a useful research tool, since it provides a marker for EPR investigation and enables the location and dynamics of AP in carrier systems to be determined independently. AP and C 16 -Tempo molecules are compared in Fig. 1. Similarities of both molecules are confirmed by the computer programme Chemscape Chime V 2.6 SP3 which specifies lipophilic (Fig. 1b) and electrostatic potential. From their structures and features it was expected that both AP and C 16 -Tempo molecules would be localized in a similar manner. Additional confirmation of the hydrophilic–lipophilic characteristics of these molecules is provided by comparison of the calculated partition coefficients (Rekker and Mannhold, 1992): log P for C 16 Tempo is 8.86, while for AP it is 7.19.

186

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

Our previous EPR experiments with colloidal carriers showed that spin probes can be localised inside the lipid compartments, at the interfaces or in the water phase of the system, in a manner depending on their lipophilicity and structure (Ahlin et al., 2003). From the EPR spectra we determined the nitrogen isotropic hyperfine splitting constant, a N , and the rotational correlation time, tC . The EPR spectra of C 16 -Tempo in different dispersions (Fig. 4) and the derived parameters (Fig. 5) are compared. The threeline spectra correspond to the C 16 -Tempo nitroxide group and their width depends on the environmental characteristics and rate of rotational reorientation. C 16 -Tempo incorporated in colloidal carriers shows mostly isotropic motion in non-structured media and microemulsions, but anisotropic motion in SLN and liposomes. The spectra demonstrate a differently restricted nitroxide motion, resulting from the different location of the nitroxide group within the dispersion structures.

3.2.1. Influence of environment polarity It is well known that the overall degree of hyperfine splitting of nitroxide free radicals is dependent on the polarity of the environment in which the nitroxide resides. This feature can be useful, since it can reveal whether the SP is situated in water, the hydrophobic interior region or at the interface in lipid bilayers or other nanostructures. The overall hyperfine splitting, a N , is a parameter rather sensitive to the environmental polarity of the SP’s nitroxide group. It ranges from |1.4 mT for non-polar to |1.7 mT for a highly polar environment (Marsh, 1981). C 16 -Tempo is practically insoluble in water but freely soluble in concentrated ethanol and lipids. Fig. 5a shows a N values in solvents and in colloidal dispersions. It is noteworthy that, based on our experiments, a N for C 16 -

Tempo is lowest in Miglyol (1.5460.01 mT) followed by water (1.58260.005 mT) and ethanol (1.61260.001 mT). The result for water was unexpected, and indicates spin probe aggregation. The formation of supramolecular structure in water caused by the amphiphilic structure of SP results in a lower a N value. The environment of the nitroxide groups is most polar in HSL liposomes, followed by NSL liposomes and SLN. In liposomes, the SP nitroxide heads are located in a more polar environment in the presence of hydrogenated lecithin than in non-hydrogenated lecithin. Comparing NSL liposomes and SLN, which have the same lipid at the interface, a N is slightly smaller for SLN. This indicates immersion of the SP deeper into the phospholipid layers in SLN. In both types of microemulsion, SP molecules show similar a N values which are most similar to that in Miglyol. Regardless of the type of ME, the nitroxide groups are located in environments with very similar polarity. Comparison of the results in Fig. 5a and Fig. 2 leads to the conclusion that the more polar the environment of sensitive groups (at the particle interface), the more unstable they are. Indeed the determination of a N provides an additional indication of the potential stabilization offered by a system. The results of this study suggest that the tempyl palmitate molecules are sufficiently flexible to be capable of bending in a lipophilic environment, thus causing the hydrophilic part (nitroxide and, by analogy, ascorbate) to remain more or less distant from the interface, and accounting for their different motion.

3.2.2. Spin probe motion in different colloidal carriers Information on the rotational mobility of the nitroxide group of SP can be quantified in terms of the rotational

Fig. 4. Representative EPR spectra of C 16 -Tempo incorporated in different carrier systems (HSL, hydrogenated soybean lecithin liposomes; ME, microemulsions; NSL, non-hydrogenated soybean lecithin liposomes; SLN, solid lipid nanoparticles) and in solvents (Miglyol and ethanol).

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

Fig. 5. EPR parameters obtained from spectra of C 16 -Tempo in different colloidal dispersions and solvents ((a) a N , hyperfine splitting constant; (b) tC , rotational correlation time).

correlation time. Due to different locations of C 16 -Tempo molecules in the dispersions, tc also differs. tc provides information about the influence of the SP environment on the dynamics of the nitroxide group. The results of tc represent the fast motion regime (5.10 211 ,tc ,1.10 29 s). The smaller the value of tc , the less restricted is the motion

187

of the SP. The fastest movement was observed in ethanol, followed by Miglyol and microemulsions. The value tc in water is in agreement with that of a N , which shows that the motion of C 16 -Tempo in water is restricted more than in ethanol, Miglyol and even in microemulsions. tC values are, like a N , smaller with microemulsions than with SLN and liposomes (Fig. 5b). The motion of C 16 -Tempo is therefore more restricted in the liposome bilayer (the highest tC ) than in SLN or in microemulsions. In ME, C 16 -Tempo molecules locate mostly in the interface and their nitroxide groups are in a less ordered environment resulting in almost the same rotational dynamics as in Miglyol, a viscous fluid. On the other hand, the lipid interactions contribute to lower a N values with microemulsions. No significant difference was detected in EPR spectra in w / o ME or o / w ME. In a control EPR experiment with binary and ternary mixtures of ME ingredients, SP showed higher motion, manifested by a relatively strong decrease in tC , than in structured systems. As shown in Fig. 5b, the SP in SLN have less restricted motion than the SP entrapped in liposomes. SP in liposomes are located in a more structured environment (comparing phospholipid layers only) than in SLN. Due to aggregation only a fraction of the non-integrated SP is detectable by EPR at room temperature. The remainder probably forms aggregates (Ahlin et al., 2003). In liposomes SP is located in the two layers of phospholipid molecules, whereas in SLN there are more possibilities. The influence of the bilayer structure on rotational motion was also investigated. SP entrapped in NSL liposomes with liquid-state bilayers and in HSL liposomes with gel-state bilayers shows significantly different tc values caused by its different depths in the bilayer (Fig. 5b). Small differences in rotational motion of a similar SP were also observed in studies in which the same types of formulations were investigated (Ahlin et al., 2000; Coderch et al., 2000). The latter authors showed that liposomes with hydrogenated phospholipids exhibit less motion of the saturated lipids in the two regions of the bilayer than those containing non-hydrogenated phospholipids, which confirms our findings. Additionally, the presence of cholesterol explains the differences in a N and tC values in NSL liposomes and SLN (Vrhovnik et al., 1998). Ahlin et al. (2000) studied the distribution of similar SPs (C 14 - and C 16 -Tempo) in the same type of SLN as in our study and obtained similar EPR results that confirmed the presence of liposome-like phospholipid layers on SLN surface. AP incorporated in SLN or in NSL liposomes was more stable than in HSL liposomes (Fig. 2). This is in accordance with the EPR results (Fig. 5a,b), which show that the sensitive groups are more stable in a less polar environment and show smaller mobility. This suggests that colloidal particles could increase the stability when they protect the sensitive groups of drug molecules. Whether or not carriers can achieve complete protection is addressed in the next section.

188

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

3.2.3. Accessibility of the nitroxide groups to reduction Like the nitroxide head of the SP, the hydrophilic head of AP is sensitive to reaction. AP is degraded by being oxidized or hydrolyzed, whereas nitroxides are subject to reduction. The kinetics of reduction provides additional information about the location and, consequently, exposure of the sensitive group to reactive species. Ascorbate (ASC) reduces nitroxide groups to diamagnetic hydroxylamine, which is not detectable by EPR. Due to its polar nature and charge ASC cannot penetrate the lipophilic part of the carrier system and can therefore reduce only those nitroxide groups located in water or exposed at water / lipid interfaces (outer surface of liposomes and SLN). Fig. 6 shows the kinetics of reduction of nitroxide by ASC in dispersions 10 days after their preparation. The kinetics were different in different carriers. The reduction in o / w ME followed first order kinetics, whereas the reaction in SLN and liposomes was almost second order. These findings are in agreement with the results of polarity and motion studies. SP was reduced faster if incorporated in HSL liposomes than in NSL liposomes. A more polar environment for the nitroxide head thus leads to faster reduction kinetics. These findings additionally support the AP stability results in the two types of liposome (Fig. 2). The method of liposome preparation used yields multilamellar vesicles where only the SP located on the outer surface was reduced in 60 min. After some time, SP would be expected to distribute from the inner to the outer monolayer. Liu et al. (1996) reported similar EPR kinetics when they determined the antioxidant activity of ascorbyl6-palmitate in synthetic surfactant vesicles. Reduction was fastest with o / w ME. The greater

accessibility to reduction is due to the faster rotational motion of the nitroxide groups and the mobility of the whole SP molecule resulting from its position in the weakly structured surfactant film. The reduction of SP in SLN is faster than that of SP in both types of liposomes, for the same reason as with ME. To determine the amount of SP in the solid core of SLN, intensities of EPR peaks were compared at room temperature and at 85 8C. At the higher temperature SP could also be detected in the solid core (Ahlin et al., 2000), in which 27.7% of the SP is incorporated. This fraction of drug contributes to its greater long-term stability in SLN.

4. Conclusion The information obtained by the EPR method with regard to stability is very exact because it enables us to reveal the effects on specific domains of nanostructured systems. The long-term stability of the model drug AP is shown to depend on the composition of, and drug distribution in, the colloidal carrier systems studied. In particular, the drug protecting ability depends on the location of the unstable part of the active compound in the carrier system. In AP, where the hydrophilic moiety is reactive to oxidation, the molecule is most stable in systems in which this part is least exposed to the hydrophilic environment. AP was more stable in SLN and NSL liposomes than in HSL liposomes and in ME. In the liquid-state carrier the active compound immerses deeper into the bilayer than in the gel-state particles, and so is better protected against degradation. Additionally, with SLN, the observed incorporation of a proportion of the drug into the solid lipid core is also predicted to lead to longer AP stability. The higher content of lipids in microemulsions does not guarantee higher AP stability, because motion inside the liquid carrier system is not prevented, as it is when solid lipids are used. For long-term AP stability, therefore, the content of solid lipids is also important. Although the obtained long-term AP stability in tested colloidal carriers is not adequate, knowledge and the determination of the crucial factors influencing stability enable several possible approaches for its enhancement. Dependence of stabilization of reactive compounds on the nature and composition of the carrier, as well as on the structure of the molecule itself, underlines the factors that have to be taken into account when formulating a carrier system.

Acknowledgements Fig. 6. Rate of reduction of C 16 -Tempo with 1 mmol / l sodium ascorbate. Colloid dispersions were 10 days old. Each point is an average of three measurements; standard deviation is less than 10%.

The authors express their sincere thanks for financial support from the Ministry of Education, Science and Sport of the Republic of Slovenia.

J. Kristl et al. / European Journal of Pharmaceutical Sciences 19 (2003) 181–189

References ˇ Ahlin, P., Kristl, J., Smid-Korbar, J., 1998. Optimisation of procedure parameters and physical stability of solid lipid nanoparticles in dispersions. Acta Pharm. 48, 257–267. ˇ ˇ Ahlin, P., Sentjurc, M., Strancar, J., Kristl, J., 2000. Location of lipophilic substances and ageing of solid lipid nanoparticles studied by EPR. Stp Pharm. Sci. 10 (2), 125–132. ˇ ˇ ˇ S., Strancar, Ahlin, P., Kristl, J., Pecar, J., Sentjurc, M., 2003. The effect of lipophilicity of spin labeled compounds on their distribution in solid lipid nanoparticle dispersions studied by EPR. J. Pharm. Sci. 92, 58–66. Austria, R., Semenzato, A., Bettero, A., 1997. Stability of vitamin C derivates in solution and topical formulations. J. Pharm. Biomed. Anal. 15, 795–801. Bagwe, R.P., Kanicky, J.R., Palla, J., 2001. Improved drug delivery using microemulsions: rationale, recent progress and new horizons. Crit. Rev. Ther. Drug Carrier Syst. 18 (1), 77–140. Coderch, L., Fonollosa, J., De Pera, M., Estelrich, J., De La Maza, A., Parra, J.L., 2000. Influence of cholesterol on liposome fluidity by EPR relationship with percutaneous absorption. J. Controlled Release 68, 85–95. Colven, R.M., Pinnell, S.R., 1996. Topical Vitamin C in aging. Clin. Dermatol. 14, 227–234. Gallarate, M., Carlotti, M.E., Trotta, M., Bovo, S., 1999. On the stability of ascorbic acid in emulsified systems for topical and cosmetic use. Int. J. Pharm. 188, 233–241. Kameyama, K., Sakai, C., Kondoh, S., 1996. Inhibitory effect of magnesium L-ascorbyl-2-phosphate (VC-PMG) on melanogenesis in vitro and in vivo. J. Am. Acad. Dermatol. 34, 29–33. Keller, K.L., Fenske, N.A., 1998. Uses of vitamin A, C and E and related compounds in dermatology: a review. J. Am. Acad. Dermatol. 39, 611–625.

189

Kibbe, A.H. (Ed.), 2000. Handbook of Pharmaceutical Excipients, 3rd Edition. American Pharmaceutical Association and Pharmaceutical Press, Washington and London. Lasic, D.D., 1998. Novel applications of liposomes. Trends Biotechnol. 16 (7), 307–321. Lawrence, M.J., Rees, G.D., 2000. Microemulsion-based media as novel drug delivery systems. Adv. Drug Deliv. Rev. 45, 89–121. Liu, X.Y., Guo, F.L., Wu, L.M., Liu, Y.Z., Liu, Z.L., 1996. Remarkable enhancement of antioxidant activity of vitamin C in an artificial bilayer by making it lipo-soluble. Chem. Phys. Lipids 83, 39–43. Marsh, D., 1981. Electron spin resonance: spin labels. In: Grell, E. (Ed.), Membrane Spectroscopy. Springer, Heidelberg, pp. 52–137. ¨ ¨ Muller, R.H., Mader, K., Gohla, S., 2000. Solid lipid nanoparticles (SLN) for controlled delivery—a review of the state of the art. Eur. J. Pharm. Biopharm. 50, 161–177. Philips, C.L., Combs, S.B., Pinell, S., 1994. Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts. J. Invest. Dermatol. 103, 228–232. Rekker, R.F., Mannhold, R., 1992. Calculation of Drug Lipophilicity: The Hydrophobic Fragmental Constant Approach. VCH, Weinheim. Reynolds, J.E.F. (Ed.), 1996. Martindale, The Extra Pharmacopoeia, 31st Edition. Royal Pharmaceutical Society, London. Silva, G.M., Campos, P.M., 2000. Ascorbic acid and its derivatives in cosmetic formulations. Cosmet. Toilet. 115, 59–62. ˇ Sˇ piclin, P., Gasperlin, M., Kmetec, V., 2001. Stability of ascorbyl palmitate in topical microemulsions. Int. J. Pharm. 222, 271–279. ˇ ˇ Vrhovnik, K., Kristl, J., Sentjurc, M., Smid-Korbar, J., 1998. Influence of liposome bilayer fluidity on the transport of encapsulated substances into the skin, studied by EPR. Pharm. Res. 15, 523–529. Zhai, H.Z., Maibach, H.I., 2001. Skin whitening agents. Cosmet. Toilet. 116 (1), 20–25.