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Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino1, M.E. Carlotti1*, E. Pelizzetti2, D. Vione2, M. Trotta1, L. Battaglia1 Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria n° 9, 10125 Turin, Italy 2 Dipartimento di Chimica Analitica, Università degli Studi di Torino, via Pietro Giuria n° 5, 10125 Turin, Italy *Correspondence: [email protected]
1
Solid lipid nanoparticles (SLNs) have been introduced as a novel carrier system for drugs and cosmetics. It has also been found that SLNs show physical UV blocking action on their own. Cetyl palmitate, glyceryl behenate and palmitic acid SLNs, all loaded with retinyl (vitamin A) palmitate, were prepared and introduced in a hydroxyethylcellulose gel. SLNs in hydroxyethylcellulose gel protect retinyl palmitate from the photodegradation induced by UVA and UVB radiation. This effect is probably due to the light-scattering properties of SLN. Nanoparticles also protect vitamin A palmitate from thermal degradation. DSC thermal analytical examination, Z potential determination and particle size measurement confirmed the solid character of the nanoparticles, their easy dispersion and their mean diameters in the nanometer range. Key words: Solid lipid nanoparticles – Retinyl palmitate – Photodegradation – Thermal degradation – Light-scattering.
Solid lipid nanoparticles (SLNs) are particles with a mean diameter between approximately 50 and 1000 nm. The two basic production methods for SLN are hot and cold homogenization techniques, but SLN can also be produced by microemulsion technique [1]. Many different drugs have been incorporated in SLN and recently they have been introduced as a novel carrier system for cosmetics, as incorporation of active products into the solid matrix of SLN can protect them against degradation [2]. It has been found that incorporation of tocopherol acetate into SLN prevents chemical degradation, reduces side effects and increases the UV blocking capacity [3]; also coenzyme Q10 was stabilized by introduction in cetyl palmitate SLN [4]. SLNs formulated with either wax or glyceride bulk material and incorporating retinol have been compared by Jenning and Ghola [5], with respect to drug encapsulation efficiency. Retinol is chemically unstable in water and rather stable in lipid phases; thus rapid degradation of retinol indicates rapid drug expulsion from the carrier, while good stability indicates an effective encapsulation in the lipid phase of the nanoparticles. This method can be proposed as an alternative to DSC or microscopy, frequently employed to evaluate the entrapment efficiency of an active in nanoparticles. Glyceryl behenate gave superior entrapment compared to palmitate, cetyl palmitate and solid paraffin. The entrapment increased with decreasing polarity of the molecule (tretinoin < retinol < retinyl palmitate). The encapsulation efficiency was successfully enhanced by formulating SLN from mixtures of liquid and solid lipids [6]. Glyceryl behenate SLN loaded with vitamin A and incorporated in a hydrogel and in an o/w cream have been tested with respect to their influence on drug penetration into porcine skin. Best results were obtained with retinol loaded SLN incorporated in the o/w cream, which showed retarding drug expulsion [7]. The stabilization effect of SLN on retinol has been investigated using different lipids and different surfactants. Unfa-
vorable lipids (e.g. acid lipids) can lead to a less pronounced stabilization. Preparing particles different in size, the smallest particles with the largest interface area had the highest stabilization effect. Highly crystalline solid nanoparticles can act as particulate UV blocking agents by scattering the light efficiently [8, 9]. Solid lipid nanoparticles (SLNs) have been also introduced as a new generation of UV blockers. Cetyl palmitate SLNs have the ability of reflecting and scattering UV radiations on their own thus leading to photoprotection without the need of molecular sunscreens. Incorporation of sunscreens leads to a synergistic photoprotection [10-12]. In a previous paper [13], we studied the photostability of vitamin A and of its palmitic ester in ethanol and in octyl octanoate solutions. The vitamins were irradiated alone and upon addition of sunscreens and butylated hydroxy toluene. The photostability of vitamins was also studied in an o/w fluid emulsion with and without butylated hydroxy toluene. Butylated hydroxy toluene inhibited the photodegradation of both vitamins, suggesting that oxygen may be involved in their degradation. The aim of the present paper was to prepare retinyl palmitate loaded SLN, to introduce them in a hydroxy ethyl cellulose gel and to evaluate their protective effect against photochemical and thermal degradation of the active.
I. EXPERIMENTAL 1. Materials
All-trans-retinyl palmitate (FW 524.80) was purchased from Sigma and methyl alcohol (HPLC grade) was from Carlo Erba. Tego Care 450 (polyglyceryl-3-methyl glucose distearate) and Natrosol MR (hydoxyethylcellulose) were purchased from ACEF SpA. Cetyl palmitate (hexadecyl hexadecanoate, 98% p/p) was from Aldrich while glyceryl dibehenate (EP-Glyceryl Behenate NF) was from Gattefossé. Palmitic acid (hexadecanoic 159
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
2.2.2. Palmitic acid SLNs In a first tube, palmitic acid and Epikuron 200 were melted at 65°C, then butyl alcohol was added under magnetic stirring and the tube was plugged to avoid alcohol evaporation. In a second tube, sodium taurocholate was dispersed in 6 ml of warm (65°C) filtered water. When the mixture in the first tube was completely homogeneous, the contents of the second tube were added under magnetic stirring and maintaining the temperature at 65°C till the contents of the tube was clear. This indicates that the microemulsion has formed. The microemulsion was then added to 7.5 ml filtered cold water in an ice bath to obtain the precipitation of the nanoparticles by thermal shock. The composition of the final SLNs dispersion was a result of successive modifications.
acid) was purchased from Merck. Dermol M-5 (caprylic/capric triglyceride) was from Prodotti Gianni SpA and Amphisol K (potassium cetyl phosphate) was from Roche. Sodium taurocholate was from Prodotti Chimici Alimentari SpA and Epikuron 200 (phosphatidylcholine) was from Lucas Meyer. The homogenizers used were Silverson SL2 and Ultra Turrax T25 basic (IKA Labortechnik, Germany). Irradiation tests were carried out in Pyrex glass cells (5-ml solutions) under a solarbox equipped with a TLK05-40W UVA lamp (Philips, The Netherlands) and another equipped with a TL12RST40T12-40W UVB lamp (Philips, The Netherlands). The photodegradation of retinyl palmitate was followed by spectrophotometric analysis using a lambda 2 UV-Vis spectrophotometer (Perkin-Elmer, United States). Centrifuge 54117 (Eppendorf), centrifuge 4225 (AIC) and refrigerated centrifuge Allegra 64R (Beckman/Coulter, United States) were used to prepare samples for analysis. Differential scanning calorimetry (DSC) was performed on a power-compensation DSC-7 (Perkin-Elmer). Particle size analyses and Z potential measurements of SLN were performed by photon correlation spectroscopy (PCS) using a 90 Plus Particle Size analyzer (Brookhaven Instruments, New York, United States). Tests of shear deformation were performed using a rotational viscometer (Brookfield Model DV-II) with a SC-029 spindle. The measurements of pH were carried out with a HI 9321 microprocessor pH-meter (Hanna Instruments). A Sonica Ultrasonic cleaner, 2200 model, ETH (Soltec, Italy), was used for the dispersion of samples.
2.2.3. Retinoid loaded SLNs For all the three types of nanoparticles described above, the incorporation of retinyl palmitate was performed by adding the drug to the melted lipid phase during the first step of the SLN preparation. Particular care was taken that the temperature of the thermostatic bath, and obviously of the mixture, was exactly 65°C and not higher till the end of the preparation, to avoid retinyl palmitate degradation. The compositions of empty SLNs and of retinyl palmitate loaded SLNs are reported in Table I. Table I - Composition of empty SLN dispersions with different lipid phases and of retinyl palmitate loaded SLN.
2. Methods
2.1. Determination of molar absorptivity (ε) A concentrated stock solution (1.90 x 10-3 M) of retinyl palmitate in methyl alcohol (HPLC grade) was prepared to obtain the calibration curve and to determine values of ε on diluted samples. The spectra of diluted solutions (1.90 x 10-6 M, 9.52 x 10-7 M and 4.72 x 10-7 M) were recorded over the range 260-400 nm. All measurements were repeated thrice and the means were calculated to plot the graph. The ε value for retinyl palmitate at 323 nm in methyl alcohol was 5.20 x 104 M-1 cm-1. 2.2. Preparation of SLNs 2.2.1. Cetyl palmitate SLNs and glyceryl behenate SLNs The lipidic ingredients were melted at 65°C and transferred in a water bath at 65°C to avoid lipid crystallization. Warm water (70°C) containing Amphysol K as surfactant was added under homogenization to the melted lipid phase, maintaining the homogenization of the mixture in the thermostatic water bath for 15 min. The dispersion was then quickly cooled transferring it into an ice bath to promote the liposphere formation by thermal shock. The best formulation was obtained by successive modifications of the initial formulation. Amphysol K gave to the nanoparticles a surface negative charge that allowed their easy dispersion in water, due to electrostatic repulsion and depressed their mean diameters.
160
Sample
Components
Percentage (%)
Placebo cetyl palmitate SLN dispersion
cetyl palmitate Tego Care 450 Amphysol K water
5.0 3.6 0.5 90.9
Retinoid loaded cetyl palmitate SLN dispersion
cetyl palmitate Tego Care 450 Amphysol K retinyl palmitate water
5.0 3.6 0.5 0.91 89.99
Placebo glyceryl behenate SLN dispersion
Glyceryl behenate Dermol M 5 Tego Care 450 Amphysol K water
3.3 1.7 3.6 0.5 90.9
Retinoid loaded glyceryl behenate SLN dispersion
Glyceryl behenate Dermol M5 Tego Care 450 Amphysol K retinyl palmitate water
3.3 1.7 3.6 0.5 0.91 89.99
Placebo palmitic acid SLN dispersion
palmitic acid Epicuron 200 sodium taurocholate butanol water
5.4 4.1 6.9 2.2 81.4
Retinoid loaded palmitic acid SLN dispersion
palmitic acid Epicuron 200 sodium taurocholate retinyl palmitate butanol water
5.4 4.1 6.9 1.1 2.2 80.3
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
2.3. Preparation of the hydroxyethylcellulose gel as vehicle of retinyl palmitate and of SLNs 98.5 grams of filtered water were weight and heated to 60°C. Natrosol MR (1.5 g) was gradually added to hot water under stirring, in order to avoid agglomeration. The gel was brought to 25°C under stirring, then it was left to stand for some hours in order to eliminate the air bubbles. Later retinyl palmitate or aliquots of SLNs dispersions were added to the gel under stirring till the preparation was homogeneous. The final percentage of retinyl palmitate in the gel was 0.1% for all the samples.
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
After centrifugation (13000 rpm for 2 min), each supernatant was spectrophotometrically analyzed to measure the concentration of retinyl palmitate. 2.6. Rheology Rheological measurements were performed in order to study the influence of UV radiation on hydroxy ethyl cellulose. The samples subjected to rheological measurements before and after 60 and 120 min of UVA and UVB irradiation were the same used for the test of photostability. Rheological measurements were carried out using a Brookfield Model DV-II Digital viscometer at the temperature of 25 ± 0.1°C. Experiments were performed adopting the SC-029 spindle. The steady shear rate dependence of the shear stress was determined by subjecting 13 g of each sample to a series of increasing and then decreasing shear rates with data taken after 1 min shearing at each shear rate. The results were plotted on diagrams of shear stress (mPa) vs. shear rate (s-1).
2.4. Irradiation tests The hydroxyethylcellulose gels, prepared as described in Section 2.5, were then irradiated under UVA and UVB lamps. The radiation intensity under the lamps was measured under the same conditions adopted for samples irradiation (10 cm distance from the lamp) using a CO.FO.ME.GRA. multimeter equipped with a probe sensitive to radiation in the wavelength interval 290-400 nm. The radiation intensity emitted by the UVA lamp is 8.9 x 10-4 W cm-2, that emitted by the UVB lamp is 2.6 x 10-4 W cm-2. Comparing the power of radiation per unit area of the lamps with that of standard solar radiation (1.1 x 10-3 W cm-2 for UVA and 1.3 x 10-4 W cm-2 for UVB), it is clear that our UVA lamp shows a slightly lower irradiation intensity than standard sunlight UVA, while the UVB lamp emission is about twice as high as sunlight UVB. To compare our experiments with real sunlight exposure times, the MED values (minimum dose that produces sunburn) of solar UVB and UVA can be considered. In the literature [14], the UVA solar MED has been evaluated at about 20-50 J cm-2 and the UVB solar MED at about 0.0200.050 J cm-2. These values correspond to 1-3 min irradiation under the UVB lamp and 350-950 min (6-16 h) under the UVA one. Considering that the irradiation experiments typically lasted up to 120 min, irradiation with the UVA lamp has more relevance for comparison with real conditions. UVB irradiation can however be useful for process understanding. Retinyl palmitate and retinyl palmitate-loaded SLN in hydroxyethylcellulose gels were irradiated with the UVB and the UVA lamps. Aliquots (16 g) of the gel containing 0.1% retinyl palmitate alone or SLN-incorporated retinyl palmitate (0.1%) were irradiated and stirred in closed Pyrex containers for 120 min at 10-cm distance from the lamps. Samples (0.100 g) were taken every 20 min, diluted 1:100 in methyl alcohol and centrifuged (13000 rpm for 1 min). The supernatant was then spectrophotometrically analyzed (λ = 323 nm) to detect changes of absorbance, peak structure and molar concentration.
2.7. Differential scanning calorimetry Differential scanning calorimetry (DSC) was performed on a power compensation DSC apparatus (Perkin-Elmer). Thermograms were detected in the 25-90°C range at a heating rate of 5°C/min. Melting point corresponds to the maximum of the heating curve (heat flow vs. temperature). In order to bypass the interference of the surfactants (Epikuron 200 and Tego Care 450) on thermograms, cetyl palmitate SLN and palmitic acid SLN dispersions were washed. They were first centrifuged (15000 rpm; 8 ± 0.1°C), the supernatant containing the surfactant was removed and the nanoparticle pellets were re-suspended in 1 ml filtered water and centrifuged again. This washing procedure was repeated twice. The resulting nanoparticles suspension was dried over a vacuum for 24 h. Aliquots (15-16 mg) of the dry samples were weighed in aluminum pans and DSC scans were recorded. 2.8. Particle size and zeta potential analyses The average diameter, polydispersity index (PI) and zeta potential of each of the three types of SLN were determined by photon correlation spectroscopy (PCS), using a 90 Plus Particle Size Analyzer at a fixed angle of 90° and at a temperature of 25°C. Analyses of empty SLN were carried out in order to evaluate changes in nanoparticle size and in the electrophoretic mobility after drug incorporation. Prior to measurement SLN dispersions were diluted 1:2000 with filtered water for size determination or 1:1000 with filtered water and then 1:4 with KCl (1 mM) for zeta potential determination.
II. RESULTS AND DISCUSSION 1. Characterization of nanoparticles
2.5. Thermostability and stability over time of hydroxy ethyl cellulose gel The thermal stability of retinyl palmitate over time and the influence of temperature on the protection of retinyl palmitate, with and without SLN, were thus investigated in the dark. One container for each sample was stored in the dark under different temperature conditions: 25 and 40°C for one month. After 1, 3, 7, 15 and 30 days, a 0.100-g sample of each gel was taken from each container and diluted 1:100 in methyl alcohol.
1.1. Differential scanning calorimetry The solid state of the nanoparticles was investigated by DSC. The melting peak of pure cetyl palmitate SLN proved the solid character of the lipid matrix at room temperature (melting temperature higher than room temperature). The thermogram reported as an example in Figure 1 shows that the introduction of retinyl palmitate in cetyl palmitate SLN depressed the 161
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
The negativity of the Z potential value could be justified by the adsorption of cetyl phosphate (Amphisol K) on the surface of the nanoparticles. The retinyl palmitate introduced in the nanoparticles caused a low change of Z potential (respectively to -40 and -50 ± 2 mV). Palmitic acid nanoparticles had a high negative Z potential too (-52 ± 2 mV), probably due to the presence of phospholipids on their surface; the addition of retinyl palmitate resulted in a less negative potential (-45 ± 2 mV), probably because it is adsorbed at the interface and it replaces in part the phospholipids.
2. Test of stability
2.1. Irradiation tests Photostability of retinyl palmitate dispersed in hydroxyethylcellulose gel free and encapsulated in SLNs introduced in the gel was investigated. The possible protective action of nanoparticles on the photodegradation of retinyl palmitate was studied. Such an action might be linked to their light scattering power. Figures 2 and 3 show as an example the photodegradation upon UVA and UVB irradiation of retinyl palmitate in hydroxyethylcellulose gel and introduced in cetyl palmitate nanoparticles dispersed in gel. The curves of photodegradation are a mean of three determinations.
Figure 1 - DSC thermogram of cetyl palmitate SLNs with and without retinyl palmitate.
nanoparticle transition temperature from 50.35 to 48.75°C. The depression of the melting temperature after the addition of the active is one of the indicators of its inclusion in the lattice (retinyl palmitate is liquid at room temperature). Also with nanoparticles of palmitic acid a depression of melting point was observed after the introduction of retinyl palmitate. The inclusion of retinyl palmitate in the SLNs could be deduced also from the stability at 40°C and stability over time (30 days) of retinyl palmitate loaded on SLNs higher than that of retinyl palmitate free in the aqueous medium (gel), in agreement with the literature [5].
% concentration of retinyl palmitate
0,14
1.2. Particle size determination The empty SLN dispersions showed, after PCS analysis, a particle size distribution that is completely in the nanometer range for all the three types of lipospheres: empty cetyl palmitate SLNs had a mean diameter of 620 nm (polidispersity 0.219); empty gliceryl behenate SLNs had a mean diameter of 774 nm (polydispersity 0.281). The mean diameter of palmitic acid nanoparticles, obtained from hot microemulsions, show a very low polidispersity (0.068) and was 450 nm, smaller than those of cetyl palmitate and of gliceryl behenate. SLNs of cetyl palmitate loaded with retinyl palmitate had a mean diameter of 690 (polidispersity 0.275); SLNs of glyceryl behenate loaded with retinyl palmitate had a mean diameter of 845 nm (polidispersity 0.291); SLNs of palmitic acid loaded with retinyl palmitate had a mean diameter of 0.474 (polidispersity 0.064). In all three cases the introduction of retinyl palmitate in SLNs did not change the particle size distribution radically.
Retinyl palmitate in SLNs in gel
Retinyl palmitate in gel
0,12 0,1 0,08 0,06 0,04 0,02 0 0
20
40
60
80
100
120
time (minutes)
Figure 2 - Photodegradation of retinyl palmitate under UVA irradiation in hydroxyegthylcellulose gel and in cetyl palmitate SLNs vehicled in the gel. 0,16
Retinyl palmitate in SLNs in gel
Retinyl palmitate in gel
% concentration of retinyl palmitate
0,14
1.3. Zeta potential measurements Zeta potential of the empty cetyl palmitate and glyceryl behenate SLNs had a mean value (respectively of - 46 and -51 ± 2 mV ) negative enough to inhibit the aggregation of the nanoparticles because of their own negative charge.
0,12 0,1 0,08 0,06 0,04 0,02 0 0
20
40
60 t (minutes)
80
100
120
Figure 3 - Photodegradation of retinyl palmitate under UVB irradiation in hydroxyegthylcellulose gel and in cetyl palmitate SLNs vehicled in the gel.
162
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
We found that cetyl palmitate nanoparticles protected the vitamin A palmitate from photodegradation under UVA for a 45% and under UVB for a 16%, which are values higher than or comparable to the protective effect of 17% found, from those authors, for PGLA nanoparticles versus 2-ethyl-hexyl-pmethoxy-cinnamate. Glyceryl behenate SLNs protected retinyl palmitate for a 24% under UVA and for a 29% under UVB, and palmitic acid SLNs protected retinyl palmitate under UVA for a 13% and under UVB for a 26%. Also these data are in agreement with those found in the literature [14]. Under UVB irradiation the lipospheres of glyceryl behenate offered a protection to vitamin ester against photodegradation slightly higher than that of cetyl palmitate because of a slight difference in their light scattering properties. In fact the light scattering is the main mechanism involved in the protection of the active from irradiation. The photodegradation of retinyl palmitate alone in the gel was slightly higher under UVA than under UVB, probably due to the absorbance maximum of vitamin A palmitate at 323 nm (in the UVA range).
Table II - Percentage of degradation of retinyl palmitate dispersed in the hydroxyethylcellulose gel and introduced in SLNs vehicled in the gel (120 min irradiation). Sample
Percentage of vitamin degradation under UVA
Percentage of vitamin degradation under UVB
Retinyl palmitate in gel
74
60
Retinyl palmitate in cetyl palmitate SLN in gel
29
44
Retinyl palmitate in glyceryl behenate SLN in gel
50
31
Retinyl palmitate in palmitic acid SLN in gel
61
38
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
The encapsulation of retinyl palmitate in nanoparticles highly improves the photostability of the vitamin ester under both UVA and UVB irradiation when compared with the hydroxy ethyl cellulose gel without SLN. Table II reports the percentage of photodegradation, after 120 min UVA and UVB irradiation, of retinyl palmitate dispersed in the gel alone and loaded on the nanoparticles vehicled in the gel. All-trans-retinyl palmitate under UV irradiation hydrolyzes to all-trans-retinol that undergoes a series of cis-trans isomerization with the resulting formation of 11-cis-retinal, all-trans-retinal, all-trans-retinoic acid (irritant for the skin) and 4-hydroxy-ketoretinoic acid [14]. The reaction of photodegradation of retinyl palmitate involves oxygen, in fact it was observed that a mixture of a radical scavenger (vitamin E) with an antioxidant (vitamin C) was able to break the chain reaction [15, 16]. The protection effect towards retinyl palmitate degradation offered by SLNs is probably due to the light scattering and reflecting properties of the nanoparticles as reported in literature [10, 12]. Actually, light scattering can relevantly decrease the radiation intensity that is responsible for retinyl palmitate photodegradation. However, there is evidence that light scattering is not the only effect involved. The lipospheres of cetyl palmitate offered a remarkably higher protection to retinyl palmitate from photodegradation, under UVA, than that of glyceryl behenate and palmitic acid SLNs; this difference in the protection effect between cetyl palmitate, glyceryl behenate and palmitic acid might be due to the different polarity between the hydrophobic core of the nanoparticles and the aqueous medium (gel). Some authors found that oxidation of vitamin A was higher when it was in an aqueous medium than when it was vehicled in liposomes [17]. Other authors found that vitamin A palmitate was slightly less stable under irradiation when it was in o/w emulsions than in octyl octanoate, possibly because it was located at o/w interface and the aqueous medium could influence the stability of vitamin A palmitate [18]. A photoprotective effect under UVB radiations of poly-D,Llactides-co-glycolide (PGLA) nanoparticles encapsulation on trans-2-ethyl-p-methoxy cinnamate (EHMC) was found [19]. The photodegradation of the sunscreen agent in the emulsion vehicle was reduced by encapsulation into PGLA nanoparticles (the extent of degradation was 35.3% for the sunscreen loaded onto nanoparticles compared to 52.3% for free trans-EHMC).
4.2. Thermostability in hydroxy ethyl cellulose gel of retinyl palmitate (free and introduced in SLNs) The stability of retinyl palmitate in hydroxyethylcellulose gel, free and loaded onto nanoparticles vehicled in the gel was investigated at 25 and 40°C in the dark. Figure 4, reported as an example, shows the degradation of retinyl palmitate free in the hydroxyethylcellulose gel and encapsulated in cetyl palmitate nanoparticles introduced in the gel. The plots are a mean of three determinations. All the examined lipospheres highly improved the stability over time of the vitamin both at 25 and 40°C. Palmitic acid nanoparticles showed the best efficiency in protecting the vitamin ester, under comparable experimental conditions, perhaps for high protection properties of phospholipids. Table III reports the percentage of degradation of retinyl palmitate after 30 days at 25 and at 40°C in hydroxyethylcellulose gel and in the nanoparticles introduced in the gel. The study of the thermal decomposition of the vitamin can be a method to investigate its encapsulation as the retinoids are less stable in an aqueous medium than in a lipid phase, their higher stability in SLN can indicate a higher partition of 0,12 retinyl palmitate loaded SLN, in HEC gel, at 40°C
% conc. of retinyl palmitate
0,1 0,08
retinyl palmitate free, in HEC gel, at 40°C
0,06
0,04
retinyl palmitate loaded SLN, in HEC gel, at 25°C
0,02
0 0
5
10
15
20
time (days)
25
30
35
retinyl palmitate free, in HEC, gel at 25°C
Figure 4 - Thermostability at 25 and 40°C of retinyl palmitate free or inglobated in cetyl palmitate SLNs, vehicled in hydroxyethylcellulose gel.
163
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Table II - Percentage of degradation after 30 days of storage of retinyl palmitate alone in the hydroxyethylcellulose gel and included in SLN vehicled in the gel at 25 and 40°C. Percentage of retinyl palmitate degradation at 25°C
Percentage of retinyl palmitate degradation at 40°C
Retinyl palmitate in gel
74
83
Retinyl palmitate in cetyl palmitate SLN in gel
28
37
Retinyl palmitate in glyceryl behenate SLN in gel
23
Retinyl palmitate in palmitic acid SLN in gel
20
20000 shear stress (mPa)
Sample
22500 17500 15000 12500 10000 7500 5000 2500 0 0
5
10
15 shear rate (s
26 24
20 -1
25
30
)
upper curve before irr.
back curve before irr.
upper curve after 60' irr.
back curve after 60' irr.
upper curve before 120' irr.
back curve after 120' irr.
Figure 5 - Flux rheograms of hydroxyethylcellulose gel wiith retinyl palmitate before irradiation and after 60 and 120 s or UVA irradiation.
the vitamin in the lipid phase of the nanoparticles. In fact, the protection against thermodegradation mainly consists in parting the active from the water phase and from the oxygen. Both at 25 and 40°C the lipospheres of gliceryl behenate and palmitic acid were more protective than the cetyl palmitate ones. From these data it can be confirmed that the nanoparticles of gliceryl behenate, with a small amount of liquid oil, can give a better encapsulation efficiency of the retinoids than those of cetyl palmitate [5]. Moreover, nanoparticles of palmitic acid are smaller than those obtained with the other lipids, which increases their protection effect as reported in the literature [8].
place upon direct irradiation. However the viscosity values at each shear rate, deduced from the ratio shear stress/shear rate, decreased only slightly with irradiation. In any case, the decrease of the hydroxyethylcellulose gel viscosity is low. Accordingly, it can be concluded that the studied hydroxyethylcellulose gel does not compete as substrate of photodegradation with retinyl palmitate that degrades in a greater extent. * * * Lipospheres of different composition have been obtained and loaded with retinyl palmitate. They have a negative surface, as shown by the high negative value of Z potential that inhibits their aggregation. The smallest lipospheres (about 480 nm mean diameter) were obtained with palmitic acid, using the technology of microemulsions. DSC analysis demonstrates that the nanoparticles are actually solid at room temperature and that an effective loading of retinyl palmitate in SLN takes place. The depression of the SLN melting temperature upon addition of vitamin A ( liquid at room temperature) indicates that the latter is able to interact with the lipid core. The lipospheres protected the vitamin ester very well from the photodegradation induced by UV radiations and from the thermal degradation induced by the storage at 40°C in the dark. The protection from photodegradation is mainly due to the light scattering by SLN. A secondary effect due to the hydrophobicity higher in the SLNs than in the aqueous medium also exists. Retinyl palmitate is chemically unstable in water and rather stable in the lipid phase. The good thermostability of retinyl palmitate in SLNs indicates its effective encapsulation in the nanoparticles lipid phase, in agreement with the literature data [5] according to which parting the active from water and oxygen increases its stability. The consistence of the gels containing the nanoparticles was maintained under UV irradiation, hydroxyethylcellulose does not therefore compete as a substrate of photodegradation with retinyl palmitate.
4.3. Rheology We have studied the time evolution of the rheological properties of the samples under irradiation. The systems were the same as those adopted for the photo and thermal degradation of retinyl palmitate. Flux rheograms of the gels with retinoid-loaded SLNs were investigated before and after UVA-UVB irradiation. Under illumination the components of the gel can be expected to undergo photodegradation that can result, for example, in a decrease of the viscosity of the gels. The photodegradation of the gel with retinoid-loaded lipospheres (of cetyl palmitate, of glyceryl behenate, of palmitic acid) was thus monitored plotting the flux rheograms before and after UVA-UVB irradiation time. Figure 5 shows the flux rheograms of the gel containing cetyl palmitate lipospheres loaded with retinyl palmitate. The rheological behavior of all the gel systems considered was pseudoplastic, which is clearly shown by the trend of shear stress over time at increasing shear rates. These systems have thixotropic hysteresis areas that increase with the extension of illumination time, denoting a slower re-structuration ability of the systems after UV irradiation. This limited de-structuration behavior is more evident under UVA then under UVB irradiation for all the three gel systems considered. However, after UVA-UVB irradiation only a slight loss of viscosity of the systems and a slight decrease of the consistence of them were observed. From the rheological behavior it can be seen that a slight photolysis of the components of the gel systems possibly takes
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Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
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GASCO M.R. - Solid lipid nanoparticles from warm microemulsions. - Pharm.Technol., 9 (11), 52- 58, 1997. MULLER R.H., MADER K., GHOLA S. - Solid lipid nanoparticles for controlled drug delivery. A review of the state of art. - Eur. J. Pharm.and Biopharm., 50, 161-177, 2000. WISSING S.A., MULLER R.H. - A novel sunscreen system based on tocopherol acetate incorporated in solid lipid nanoparticles. - Int. J.Cosm. Sci., 23, 233-243, 2001. MULLER R.H., DINGLER A. - Feste Lipid-Nanoparticles (Lipopearls) als neuartiger carrier fur kosmetiche un dermatologische. - Wirkstoffe, P.2 Wiss., 49, 11-15, 1998. JENNING V., GHOLA S. - Encapsulation of retinoids in solid lipid nanoparticles (SLN). - J. Microenc.,18 (2), 149-158, 2001. JENNING V., GHOLA S. - Comparison of wax and glyceride solid lipid nanoparticles (SLN). - Int. J. Pharm., 196, 219-222, 2000. JENNING V., SHAFER-KORTING M., GHOLA S. - Vitamin A loaded solid lipid nanoparticles for topical use: drug release properties. - J. Control. Rel., 66, 115-126, 2000. MULLER R.H., RADKE M., WISSING S.A. - Solid lipid nanoparticles (SLN) and nano-structured lipid carriers (NLC) in cosmetic and dermatological products. - Adv. Drug Del. Syst. Rev., 54, 135-155, 2002. LOUKAS Y.L., JAYOSEREKA P., GREGORIADIS G. - Novel liposome-base multilcomponent systems for the protection of photolabile agents. - Int. J. Pharm., 117 (1), 85-94, 1995. WISSING S.A., MULLER R.H. - Solid lipid nanoparticles (SLN)-a novel carrier for UV blockers. - Pharmazie, 56 (10), 783-786, 2001.
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WISSING S.A., MULLER R.H. -The development of an improved carrier system for sunscreen formulations based on crystalline lipid nanoparticles. - Int. J. Pharm., 242 (1-2), 373-375, 2002. WISSING S.A., MULLER R.H. - Cosmetic applications for solid lipid nanoparticles (SLN). - Int. J. Pharm., 254, 65-68, 2003. CARLOTTI M.E., ROSSATTO V., GALLARATE M. - Vitamin A and vitamin A palmitate stability over time and under UVA and UVB radiation. - Int. J. Pharm., 240, 85-94, 2002. VALHQUIST A. - What are natural retinoids? - Dermatology, 199, 302-307, 1999. DREHER F., GABARD B., SHWINDT D.A., MAIBACH H.I. Topical melatonin in combination with vitamin E and C protects skin from ultraviolet-induced erythema. A human study in vivo. - Br. J. Dermatol., 139, 332-339, 1998. SICS H., STOHE W., SINQUIST A.R. - Antioxidant functions of Vitamin E and C, beta-carotene and other carotenoids. - Ann. NY Acad. Sci., 669, 7-20, 1992. ARSIC I., VIDOVIC S., VULITA G. - Influence of liposomes on the stability of vitamin A incorporated in polyacrylate hydrogels - Int. J. Cosm. Sci., 21, 219-225, 1999. TSUNODA T., TAKABASHY K. - Stability of all trans retinol in cream. - J. Soc. Cosm. Chem., 46, 191-198, 1995. PERUGINI P., SIMEONI S., SCALIA S., GENTA I., MODENA T., CONTI B. - Effect of nanoparticles encapsulation on the photostability of the sunscreen agent, 2-ethyl-p-methoxy-cinnamate. - Int. J. Pharm., 246, 37-45, 2002.
MANUSCRIPT Received 9 March 2004, accepted for publication 14 October 2004.
165
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino1, M.E. Carlotti1*, E. Pelizzetti2, D. Vione2, M. Trotta1, L. Battaglia1 Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria n° 9, 10125 Turin, Italy 2 Dipartimento di Chimica Analitica, Università degli Studi di Torino, via Pietro Giuria n° 5, 10125 Turin, Italy *Correspondence: [email protected]
1
Solid lipid nanoparticles (SLNs) have been introduced as a novel carrier system for drugs and cosmetics. It has also been found that SLNs show physical UV blocking action on their own. Cetyl palmitate, glyceryl behenate and palmitic acid SLNs, all loaded with retinyl (vitamin A) palmitate, were prepared and introduced in a hydroxyethylcellulose gel. SLNs in hydroxyethylcellulose gel protect retinyl palmitate from the photodegradation induced by UVA and UVB radiation. This effect is probably due to the light-scattering properties of SLN. Nanoparticles also protect vitamin A palmitate from thermal degradation. DSC thermal analytical examination, Z potential determination and particle size measurement confirmed the solid character of the nanoparticles, their easy dispersion and their mean diameters in the nanometer range. Key words: Solid lipid nanoparticles – Retinyl palmitate – Photodegradation – Thermal degradation – Light-scattering.
Solid lipid nanoparticles (SLNs) are particles with a mean diameter between approximately 50 and 1000 nm. The two basic production methods for SLN are hot and cold homogenization techniques, but SLN can also be produced by microemulsion technique [1]. Many different drugs have been incorporated in SLN and recently they have been introduced as a novel carrier system for cosmetics, as incorporation of active products into the solid matrix of SLN can protect them against degradation [2]. It has been found that incorporation of tocopherol acetate into SLN prevents chemical degradation, reduces side effects and increases the UV blocking capacity [3]; also coenzyme Q10 was stabilized by introduction in cetyl palmitate SLN [4]. SLNs formulated with either wax or glyceride bulk material and incorporating retinol have been compared by Jenning and Ghola [5], with respect to drug encapsulation efficiency. Retinol is chemically unstable in water and rather stable in lipid phases; thus rapid degradation of retinol indicates rapid drug expulsion from the carrier, while good stability indicates an effective encapsulation in the lipid phase of the nanoparticles. This method can be proposed as an alternative to DSC or microscopy, frequently employed to evaluate the entrapment efficiency of an active in nanoparticles. Glyceryl behenate gave superior entrapment compared to palmitate, cetyl palmitate and solid paraffin. The entrapment increased with decreasing polarity of the molecule (tretinoin < retinol < retinyl palmitate). The encapsulation efficiency was successfully enhanced by formulating SLN from mixtures of liquid and solid lipids [6]. Glyceryl behenate SLN loaded with vitamin A and incorporated in a hydrogel and in an o/w cream have been tested with respect to their influence on drug penetration into porcine skin. Best results were obtained with retinol loaded SLN incorporated in the o/w cream, which showed retarding drug expulsion [7]. The stabilization effect of SLN on retinol has been investigated using different lipids and different surfactants. Unfa-
vorable lipids (e.g. acid lipids) can lead to a less pronounced stabilization. Preparing particles different in size, the smallest particles with the largest interface area had the highest stabilization effect. Highly crystalline solid nanoparticles can act as particulate UV blocking agents by scattering the light efficiently [8, 9]. Solid lipid nanoparticles (SLNs) have been also introduced as a new generation of UV blockers. Cetyl palmitate SLNs have the ability of reflecting and scattering UV radiations on their own thus leading to photoprotection without the need of molecular sunscreens. Incorporation of sunscreens leads to a synergistic photoprotection [10-12]. In a previous paper [13], we studied the photostability of vitamin A and of its palmitic ester in ethanol and in octyl octanoate solutions. The vitamins were irradiated alone and upon addition of sunscreens and butylated hydroxy toluene. The photostability of vitamins was also studied in an o/w fluid emulsion with and without butylated hydroxy toluene. Butylated hydroxy toluene inhibited the photodegradation of both vitamins, suggesting that oxygen may be involved in their degradation. The aim of the present paper was to prepare retinyl palmitate loaded SLN, to introduce them in a hydroxy ethyl cellulose gel and to evaluate their protective effect against photochemical and thermal degradation of the active.
I. EXPERIMENTAL 1. Materials
All-trans-retinyl palmitate (FW 524.80) was purchased from Sigma and methyl alcohol (HPLC grade) was from Carlo Erba. Tego Care 450 (polyglyceryl-3-methyl glucose distearate) and Natrosol MR (hydoxyethylcellulose) were purchased from ACEF SpA. Cetyl palmitate (hexadecyl hexadecanoate, 98% p/p) was from Aldrich while glyceryl dibehenate (EP-Glyceryl Behenate NF) was from Gattefossé. Palmitic acid (hexadecanoic 159
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
2.2.2. Palmitic acid SLNs In a first tube, palmitic acid and Epikuron 200 were melted at 65°C, then butyl alcohol was added under magnetic stirring and the tube was plugged to avoid alcohol evaporation. In a second tube, sodium taurocholate was dispersed in 6 ml of warm (65°C) filtered water. When the mixture in the first tube was completely homogeneous, the contents of the second tube were added under magnetic stirring and maintaining the temperature at 65°C till the contents of the tube was clear. This indicates that the microemulsion has formed. The microemulsion was then added to 7.5 ml filtered cold water in an ice bath to obtain the precipitation of the nanoparticles by thermal shock. The composition of the final SLNs dispersion was a result of successive modifications.
acid) was purchased from Merck. Dermol M-5 (caprylic/capric triglyceride) was from Prodotti Gianni SpA and Amphisol K (potassium cetyl phosphate) was from Roche. Sodium taurocholate was from Prodotti Chimici Alimentari SpA and Epikuron 200 (phosphatidylcholine) was from Lucas Meyer. The homogenizers used were Silverson SL2 and Ultra Turrax T25 basic (IKA Labortechnik, Germany). Irradiation tests were carried out in Pyrex glass cells (5-ml solutions) under a solarbox equipped with a TLK05-40W UVA lamp (Philips, The Netherlands) and another equipped with a TL12RST40T12-40W UVB lamp (Philips, The Netherlands). The photodegradation of retinyl palmitate was followed by spectrophotometric analysis using a lambda 2 UV-Vis spectrophotometer (Perkin-Elmer, United States). Centrifuge 54117 (Eppendorf), centrifuge 4225 (AIC) and refrigerated centrifuge Allegra 64R (Beckman/Coulter, United States) were used to prepare samples for analysis. Differential scanning calorimetry (DSC) was performed on a power-compensation DSC-7 (Perkin-Elmer). Particle size analyses and Z potential measurements of SLN were performed by photon correlation spectroscopy (PCS) using a 90 Plus Particle Size analyzer (Brookhaven Instruments, New York, United States). Tests of shear deformation were performed using a rotational viscometer (Brookfield Model DV-II) with a SC-029 spindle. The measurements of pH were carried out with a HI 9321 microprocessor pH-meter (Hanna Instruments). A Sonica Ultrasonic cleaner, 2200 model, ETH (Soltec, Italy), was used for the dispersion of samples.
2.2.3. Retinoid loaded SLNs For all the three types of nanoparticles described above, the incorporation of retinyl palmitate was performed by adding the drug to the melted lipid phase during the first step of the SLN preparation. Particular care was taken that the temperature of the thermostatic bath, and obviously of the mixture, was exactly 65°C and not higher till the end of the preparation, to avoid retinyl palmitate degradation. The compositions of empty SLNs and of retinyl palmitate loaded SLNs are reported in Table I. Table I - Composition of empty SLN dispersions with different lipid phases and of retinyl palmitate loaded SLN.
2. Methods
2.1. Determination of molar absorptivity (ε) A concentrated stock solution (1.90 x 10-3 M) of retinyl palmitate in methyl alcohol (HPLC grade) was prepared to obtain the calibration curve and to determine values of ε on diluted samples. The spectra of diluted solutions (1.90 x 10-6 M, 9.52 x 10-7 M and 4.72 x 10-7 M) were recorded over the range 260-400 nm. All measurements were repeated thrice and the means were calculated to plot the graph. The ε value for retinyl palmitate at 323 nm in methyl alcohol was 5.20 x 104 M-1 cm-1. 2.2. Preparation of SLNs 2.2.1. Cetyl palmitate SLNs and glyceryl behenate SLNs The lipidic ingredients were melted at 65°C and transferred in a water bath at 65°C to avoid lipid crystallization. Warm water (70°C) containing Amphysol K as surfactant was added under homogenization to the melted lipid phase, maintaining the homogenization of the mixture in the thermostatic water bath for 15 min. The dispersion was then quickly cooled transferring it into an ice bath to promote the liposphere formation by thermal shock. The best formulation was obtained by successive modifications of the initial formulation. Amphysol K gave to the nanoparticles a surface negative charge that allowed their easy dispersion in water, due to electrostatic repulsion and depressed their mean diameters.
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Sample
Components
Percentage (%)
Placebo cetyl palmitate SLN dispersion
cetyl palmitate Tego Care 450 Amphysol K water
5.0 3.6 0.5 90.9
Retinoid loaded cetyl palmitate SLN dispersion
cetyl palmitate Tego Care 450 Amphysol K retinyl palmitate water
5.0 3.6 0.5 0.91 89.99
Placebo glyceryl behenate SLN dispersion
Glyceryl behenate Dermol M 5 Tego Care 450 Amphysol K water
3.3 1.7 3.6 0.5 90.9
Retinoid loaded glyceryl behenate SLN dispersion
Glyceryl behenate Dermol M5 Tego Care 450 Amphysol K retinyl palmitate water
3.3 1.7 3.6 0.5 0.91 89.99
Placebo palmitic acid SLN dispersion
palmitic acid Epicuron 200 sodium taurocholate butanol water
5.4 4.1 6.9 2.2 81.4
Retinoid loaded palmitic acid SLN dispersion
palmitic acid Epicuron 200 sodium taurocholate retinyl palmitate butanol water
5.4 4.1 6.9 1.1 2.2 80.3
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
2.3. Preparation of the hydroxyethylcellulose gel as vehicle of retinyl palmitate and of SLNs 98.5 grams of filtered water were weight and heated to 60°C. Natrosol MR (1.5 g) was gradually added to hot water under stirring, in order to avoid agglomeration. The gel was brought to 25°C under stirring, then it was left to stand for some hours in order to eliminate the air bubbles. Later retinyl palmitate or aliquots of SLNs dispersions were added to the gel under stirring till the preparation was homogeneous. The final percentage of retinyl palmitate in the gel was 0.1% for all the samples.
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
After centrifugation (13000 rpm for 2 min), each supernatant was spectrophotometrically analyzed to measure the concentration of retinyl palmitate. 2.6. Rheology Rheological measurements were performed in order to study the influence of UV radiation on hydroxy ethyl cellulose. The samples subjected to rheological measurements before and after 60 and 120 min of UVA and UVB irradiation were the same used for the test of photostability. Rheological measurements were carried out using a Brookfield Model DV-II Digital viscometer at the temperature of 25 ± 0.1°C. Experiments were performed adopting the SC-029 spindle. The steady shear rate dependence of the shear stress was determined by subjecting 13 g of each sample to a series of increasing and then decreasing shear rates with data taken after 1 min shearing at each shear rate. The results were plotted on diagrams of shear stress (mPa) vs. shear rate (s-1).
2.4. Irradiation tests The hydroxyethylcellulose gels, prepared as described in Section 2.5, were then irradiated under UVA and UVB lamps. The radiation intensity under the lamps was measured under the same conditions adopted for samples irradiation (10 cm distance from the lamp) using a CO.FO.ME.GRA. multimeter equipped with a probe sensitive to radiation in the wavelength interval 290-400 nm. The radiation intensity emitted by the UVA lamp is 8.9 x 10-4 W cm-2, that emitted by the UVB lamp is 2.6 x 10-4 W cm-2. Comparing the power of radiation per unit area of the lamps with that of standard solar radiation (1.1 x 10-3 W cm-2 for UVA and 1.3 x 10-4 W cm-2 for UVB), it is clear that our UVA lamp shows a slightly lower irradiation intensity than standard sunlight UVA, while the UVB lamp emission is about twice as high as sunlight UVB. To compare our experiments with real sunlight exposure times, the MED values (minimum dose that produces sunburn) of solar UVB and UVA can be considered. In the literature [14], the UVA solar MED has been evaluated at about 20-50 J cm-2 and the UVB solar MED at about 0.0200.050 J cm-2. These values correspond to 1-3 min irradiation under the UVB lamp and 350-950 min (6-16 h) under the UVA one. Considering that the irradiation experiments typically lasted up to 120 min, irradiation with the UVA lamp has more relevance for comparison with real conditions. UVB irradiation can however be useful for process understanding. Retinyl palmitate and retinyl palmitate-loaded SLN in hydroxyethylcellulose gels were irradiated with the UVB and the UVA lamps. Aliquots (16 g) of the gel containing 0.1% retinyl palmitate alone or SLN-incorporated retinyl palmitate (0.1%) were irradiated and stirred in closed Pyrex containers for 120 min at 10-cm distance from the lamps. Samples (0.100 g) were taken every 20 min, diluted 1:100 in methyl alcohol and centrifuged (13000 rpm for 1 min). The supernatant was then spectrophotometrically analyzed (λ = 323 nm) to detect changes of absorbance, peak structure and molar concentration.
2.7. Differential scanning calorimetry Differential scanning calorimetry (DSC) was performed on a power compensation DSC apparatus (Perkin-Elmer). Thermograms were detected in the 25-90°C range at a heating rate of 5°C/min. Melting point corresponds to the maximum of the heating curve (heat flow vs. temperature). In order to bypass the interference of the surfactants (Epikuron 200 and Tego Care 450) on thermograms, cetyl palmitate SLN and palmitic acid SLN dispersions were washed. They were first centrifuged (15000 rpm; 8 ± 0.1°C), the supernatant containing the surfactant was removed and the nanoparticle pellets were re-suspended in 1 ml filtered water and centrifuged again. This washing procedure was repeated twice. The resulting nanoparticles suspension was dried over a vacuum for 24 h. Aliquots (15-16 mg) of the dry samples were weighed in aluminum pans and DSC scans were recorded. 2.8. Particle size and zeta potential analyses The average diameter, polydispersity index (PI) and zeta potential of each of the three types of SLN were determined by photon correlation spectroscopy (PCS), using a 90 Plus Particle Size Analyzer at a fixed angle of 90° and at a temperature of 25°C. Analyses of empty SLN were carried out in order to evaluate changes in nanoparticle size and in the electrophoretic mobility after drug incorporation. Prior to measurement SLN dispersions were diluted 1:2000 with filtered water for size determination or 1:1000 with filtered water and then 1:4 with KCl (1 mM) for zeta potential determination.
II. RESULTS AND DISCUSSION 1. Characterization of nanoparticles
2.5. Thermostability and stability over time of hydroxy ethyl cellulose gel The thermal stability of retinyl palmitate over time and the influence of temperature on the protection of retinyl palmitate, with and without SLN, were thus investigated in the dark. One container for each sample was stored in the dark under different temperature conditions: 25 and 40°C for one month. After 1, 3, 7, 15 and 30 days, a 0.100-g sample of each gel was taken from each container and diluted 1:100 in methyl alcohol.
1.1. Differential scanning calorimetry The solid state of the nanoparticles was investigated by DSC. The melting peak of pure cetyl palmitate SLN proved the solid character of the lipid matrix at room temperature (melting temperature higher than room temperature). The thermogram reported as an example in Figure 1 shows that the introduction of retinyl palmitate in cetyl palmitate SLN depressed the 161
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
The negativity of the Z potential value could be justified by the adsorption of cetyl phosphate (Amphisol K) on the surface of the nanoparticles. The retinyl palmitate introduced in the nanoparticles caused a low change of Z potential (respectively to -40 and -50 ± 2 mV). Palmitic acid nanoparticles had a high negative Z potential too (-52 ± 2 mV), probably due to the presence of phospholipids on their surface; the addition of retinyl palmitate resulted in a less negative potential (-45 ± 2 mV), probably because it is adsorbed at the interface and it replaces in part the phospholipids.
2. Test of stability
2.1. Irradiation tests Photostability of retinyl palmitate dispersed in hydroxyethylcellulose gel free and encapsulated in SLNs introduced in the gel was investigated. The possible protective action of nanoparticles on the photodegradation of retinyl palmitate was studied. Such an action might be linked to their light scattering power. Figures 2 and 3 show as an example the photodegradation upon UVA and UVB irradiation of retinyl palmitate in hydroxyethylcellulose gel and introduced in cetyl palmitate nanoparticles dispersed in gel. The curves of photodegradation are a mean of three determinations.
Figure 1 - DSC thermogram of cetyl palmitate SLNs with and without retinyl palmitate.
nanoparticle transition temperature from 50.35 to 48.75°C. The depression of the melting temperature after the addition of the active is one of the indicators of its inclusion in the lattice (retinyl palmitate is liquid at room temperature). Also with nanoparticles of palmitic acid a depression of melting point was observed after the introduction of retinyl palmitate. The inclusion of retinyl palmitate in the SLNs could be deduced also from the stability at 40°C and stability over time (30 days) of retinyl palmitate loaded on SLNs higher than that of retinyl palmitate free in the aqueous medium (gel), in agreement with the literature [5].
% concentration of retinyl palmitate
0,14
1.2. Particle size determination The empty SLN dispersions showed, after PCS analysis, a particle size distribution that is completely in the nanometer range for all the three types of lipospheres: empty cetyl palmitate SLNs had a mean diameter of 620 nm (polidispersity 0.219); empty gliceryl behenate SLNs had a mean diameter of 774 nm (polydispersity 0.281). The mean diameter of palmitic acid nanoparticles, obtained from hot microemulsions, show a very low polidispersity (0.068) and was 450 nm, smaller than those of cetyl palmitate and of gliceryl behenate. SLNs of cetyl palmitate loaded with retinyl palmitate had a mean diameter of 690 (polidispersity 0.275); SLNs of glyceryl behenate loaded with retinyl palmitate had a mean diameter of 845 nm (polidispersity 0.291); SLNs of palmitic acid loaded with retinyl palmitate had a mean diameter of 0.474 (polidispersity 0.064). In all three cases the introduction of retinyl palmitate in SLNs did not change the particle size distribution radically.
Retinyl palmitate in SLNs in gel
Retinyl palmitate in gel
0,12 0,1 0,08 0,06 0,04 0,02 0 0
20
40
60
80
100
120
time (minutes)
Figure 2 - Photodegradation of retinyl palmitate under UVA irradiation in hydroxyegthylcellulose gel and in cetyl palmitate SLNs vehicled in the gel. 0,16
Retinyl palmitate in SLNs in gel
Retinyl palmitate in gel
% concentration of retinyl palmitate
0,14
1.3. Zeta potential measurements Zeta potential of the empty cetyl palmitate and glyceryl behenate SLNs had a mean value (respectively of - 46 and -51 ± 2 mV ) negative enough to inhibit the aggregation of the nanoparticles because of their own negative charge.
0,12 0,1 0,08 0,06 0,04 0,02 0 0
20
40
60 t (minutes)
80
100
120
Figure 3 - Photodegradation of retinyl palmitate under UVB irradiation in hydroxyegthylcellulose gel and in cetyl palmitate SLNs vehicled in the gel.
162
Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
We found that cetyl palmitate nanoparticles protected the vitamin A palmitate from photodegradation under UVA for a 45% and under UVB for a 16%, which are values higher than or comparable to the protective effect of 17% found, from those authors, for PGLA nanoparticles versus 2-ethyl-hexyl-pmethoxy-cinnamate. Glyceryl behenate SLNs protected retinyl palmitate for a 24% under UVA and for a 29% under UVB, and palmitic acid SLNs protected retinyl palmitate under UVA for a 13% and under UVB for a 26%. Also these data are in agreement with those found in the literature [14]. Under UVB irradiation the lipospheres of glyceryl behenate offered a protection to vitamin ester against photodegradation slightly higher than that of cetyl palmitate because of a slight difference in their light scattering properties. In fact the light scattering is the main mechanism involved in the protection of the active from irradiation. The photodegradation of retinyl palmitate alone in the gel was slightly higher under UVA than under UVB, probably due to the absorbance maximum of vitamin A palmitate at 323 nm (in the UVA range).
Table II - Percentage of degradation of retinyl palmitate dispersed in the hydroxyethylcellulose gel and introduced in SLNs vehicled in the gel (120 min irradiation). Sample
Percentage of vitamin degradation under UVA
Percentage of vitamin degradation under UVB
Retinyl palmitate in gel
74
60
Retinyl palmitate in cetyl palmitate SLN in gel
29
44
Retinyl palmitate in glyceryl behenate SLN in gel
50
31
Retinyl palmitate in palmitic acid SLN in gel
61
38
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
The encapsulation of retinyl palmitate in nanoparticles highly improves the photostability of the vitamin ester under both UVA and UVB irradiation when compared with the hydroxy ethyl cellulose gel without SLN. Table II reports the percentage of photodegradation, after 120 min UVA and UVB irradiation, of retinyl palmitate dispersed in the gel alone and loaded on the nanoparticles vehicled in the gel. All-trans-retinyl palmitate under UV irradiation hydrolyzes to all-trans-retinol that undergoes a series of cis-trans isomerization with the resulting formation of 11-cis-retinal, all-trans-retinal, all-trans-retinoic acid (irritant for the skin) and 4-hydroxy-ketoretinoic acid [14]. The reaction of photodegradation of retinyl palmitate involves oxygen, in fact it was observed that a mixture of a radical scavenger (vitamin E) with an antioxidant (vitamin C) was able to break the chain reaction [15, 16]. The protection effect towards retinyl palmitate degradation offered by SLNs is probably due to the light scattering and reflecting properties of the nanoparticles as reported in literature [10, 12]. Actually, light scattering can relevantly decrease the radiation intensity that is responsible for retinyl palmitate photodegradation. However, there is evidence that light scattering is not the only effect involved. The lipospheres of cetyl palmitate offered a remarkably higher protection to retinyl palmitate from photodegradation, under UVA, than that of glyceryl behenate and palmitic acid SLNs; this difference in the protection effect between cetyl palmitate, glyceryl behenate and palmitic acid might be due to the different polarity between the hydrophobic core of the nanoparticles and the aqueous medium (gel). Some authors found that oxidation of vitamin A was higher when it was in an aqueous medium than when it was vehicled in liposomes [17]. Other authors found that vitamin A palmitate was slightly less stable under irradiation when it was in o/w emulsions than in octyl octanoate, possibly because it was located at o/w interface and the aqueous medium could influence the stability of vitamin A palmitate [18]. A photoprotective effect under UVB radiations of poly-D,Llactides-co-glycolide (PGLA) nanoparticles encapsulation on trans-2-ethyl-p-methoxy cinnamate (EHMC) was found [19]. The photodegradation of the sunscreen agent in the emulsion vehicle was reduced by encapsulation into PGLA nanoparticles (the extent of degradation was 35.3% for the sunscreen loaded onto nanoparticles compared to 52.3% for free trans-EHMC).
4.2. Thermostability in hydroxy ethyl cellulose gel of retinyl palmitate (free and introduced in SLNs) The stability of retinyl palmitate in hydroxyethylcellulose gel, free and loaded onto nanoparticles vehicled in the gel was investigated at 25 and 40°C in the dark. Figure 4, reported as an example, shows the degradation of retinyl palmitate free in the hydroxyethylcellulose gel and encapsulated in cetyl palmitate nanoparticles introduced in the gel. The plots are a mean of three determinations. All the examined lipospheres highly improved the stability over time of the vitamin both at 25 and 40°C. Palmitic acid nanoparticles showed the best efficiency in protecting the vitamin ester, under comparable experimental conditions, perhaps for high protection properties of phospholipids. Table III reports the percentage of degradation of retinyl palmitate after 30 days at 25 and at 40°C in hydroxyethylcellulose gel and in the nanoparticles introduced in the gel. The study of the thermal decomposition of the vitamin can be a method to investigate its encapsulation as the retinoids are less stable in an aqueous medium than in a lipid phase, their higher stability in SLN can indicate a higher partition of 0,12 retinyl palmitate loaded SLN, in HEC gel, at 40°C
% conc. of retinyl palmitate
0,1 0,08
retinyl palmitate free, in HEC gel, at 40°C
0,06
0,04
retinyl palmitate loaded SLN, in HEC gel, at 25°C
0,02
0 0
5
10
15
20
time (days)
25
30
35
retinyl palmitate free, in HEC, gel at 25°C
Figure 4 - Thermostability at 25 and 40°C of retinyl palmitate free or inglobated in cetyl palmitate SLNs, vehicled in hydroxyethylcellulose gel.
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Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
J. DRUG DEL. SCI. TECH., 15 (2) 159-165 2005
Table II - Percentage of degradation after 30 days of storage of retinyl palmitate alone in the hydroxyethylcellulose gel and included in SLN vehicled in the gel at 25 and 40°C. Percentage of retinyl palmitate degradation at 25°C
Percentage of retinyl palmitate degradation at 40°C
Retinyl palmitate in gel
74
83
Retinyl palmitate in cetyl palmitate SLN in gel
28
37
Retinyl palmitate in glyceryl behenate SLN in gel
23
Retinyl palmitate in palmitic acid SLN in gel
20
20000 shear stress (mPa)
Sample
22500 17500 15000 12500 10000 7500 5000 2500 0 0
5
10
15 shear rate (s
26 24
20 -1
25
30
)
upper curve before irr.
back curve before irr.
upper curve after 60' irr.
back curve after 60' irr.
upper curve before 120' irr.
back curve after 120' irr.
Figure 5 - Flux rheograms of hydroxyethylcellulose gel wiith retinyl palmitate before irradiation and after 60 and 120 s or UVA irradiation.
the vitamin in the lipid phase of the nanoparticles. In fact, the protection against thermodegradation mainly consists in parting the active from the water phase and from the oxygen. Both at 25 and 40°C the lipospheres of gliceryl behenate and palmitic acid were more protective than the cetyl palmitate ones. From these data it can be confirmed that the nanoparticles of gliceryl behenate, with a small amount of liquid oil, can give a better encapsulation efficiency of the retinoids than those of cetyl palmitate [5]. Moreover, nanoparticles of palmitic acid are smaller than those obtained with the other lipids, which increases their protection effect as reported in the literature [8].
place upon direct irradiation. However the viscosity values at each shear rate, deduced from the ratio shear stress/shear rate, decreased only slightly with irradiation. In any case, the decrease of the hydroxyethylcellulose gel viscosity is low. Accordingly, it can be concluded that the studied hydroxyethylcellulose gel does not compete as substrate of photodegradation with retinyl palmitate that degrades in a greater extent. * * * Lipospheres of different composition have been obtained and loaded with retinyl palmitate. They have a negative surface, as shown by the high negative value of Z potential that inhibits their aggregation. The smallest lipospheres (about 480 nm mean diameter) were obtained with palmitic acid, using the technology of microemulsions. DSC analysis demonstrates that the nanoparticles are actually solid at room temperature and that an effective loading of retinyl palmitate in SLN takes place. The depression of the SLN melting temperature upon addition of vitamin A ( liquid at room temperature) indicates that the latter is able to interact with the lipid core. The lipospheres protected the vitamin ester very well from the photodegradation induced by UV radiations and from the thermal degradation induced by the storage at 40°C in the dark. The protection from photodegradation is mainly due to the light scattering by SLN. A secondary effect due to the hydrophobicity higher in the SLNs than in the aqueous medium also exists. Retinyl palmitate is chemically unstable in water and rather stable in the lipid phase. The good thermostability of retinyl palmitate in SLNs indicates its effective encapsulation in the nanoparticles lipid phase, in agreement with the literature data [5] according to which parting the active from water and oxygen increases its stability. The consistence of the gels containing the nanoparticles was maintained under UV irradiation, hydroxyethylcellulose does not therefore compete as a substrate of photodegradation with retinyl palmitate.
4.3. Rheology We have studied the time evolution of the rheological properties of the samples under irradiation. The systems were the same as those adopted for the photo and thermal degradation of retinyl palmitate. Flux rheograms of the gels with retinoid-loaded SLNs were investigated before and after UVA-UVB irradiation. Under illumination the components of the gel can be expected to undergo photodegradation that can result, for example, in a decrease of the viscosity of the gels. The photodegradation of the gel with retinoid-loaded lipospheres (of cetyl palmitate, of glyceryl behenate, of palmitic acid) was thus monitored plotting the flux rheograms before and after UVA-UVB irradiation time. Figure 5 shows the flux rheograms of the gel containing cetyl palmitate lipospheres loaded with retinyl palmitate. The rheological behavior of all the gel systems considered was pseudoplastic, which is clearly shown by the trend of shear stress over time at increasing shear rates. These systems have thixotropic hysteresis areas that increase with the extension of illumination time, denoting a slower re-structuration ability of the systems after UV irradiation. This limited de-structuration behavior is more evident under UVA then under UVB irradiation for all the three gel systems considered. However, after UVA-UVB irradiation only a slight loss of viscosity of the systems and a slight decrease of the consistence of them were observed. From the rheological behavior it can be seen that a slight photolysis of the components of the gel systems possibly takes
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Protective effect of SLNs encapsulation on the photodegradation and thermal degradation of retinyl palmitate introduced in hydroxyethylcellulose gel S. Sapino, M.E. Carlotti, E. Pelizzetti, D. Vione, M. Trotta, L. Battaglia
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MANUSCRIPT Received 9 March 2004, accepted for publication 14 October 2004.
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