Lipospheres for the cosmetic delivery of glycolic acid were prepared by the melt method using tristearin as the lipid phase and hydrogenated soybean phosphatidylcholine as the emulsifier. The lipospheres, sized from 5 to 40 micron, contained a rather high glycolic acid loading level probably due to a partial polymorphic modification of the lipid and determined glycolic acid sustained release pattern

CAMERONI, Riccardo

COPPI, Gilberto

IANNUCCELLI, Valentina

SERGI, Santo

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

J. Appl. Cosmetol. 19, 113-119 (October/December 2001) PREPARATION ANO IN VITRO CHARACTERIZATION OF LIPOSPHERES AS A CARRIER FOR THE COSMETIC APPLICATION OF GLYCOLIC ACID V. lannuccelli, G. Coppi, S. Sergi, R. Cameroni Department of Pharmaceutical Sciences. University of Modena and Reggio Emilia - ltaly Received: July 200 7. Key words: glycolic acid, liposphere. po/ymorphism, release, tristearin. Summary Lipospheres for the cosmetic delivery of glycolic acid were prepared by the melt method using tri-stearin as the lipid phase and hydrogenated soybean phosphatidylcholine as the emulsifier. The most favourable conditions leading to the highest liposphere yield involved triglyceride/phospholipid ra-tios of 4: I or 5: l and a phospho lipid concentration of at least 2%. The lipospheres, sized from 5 to 40 µ m, contained a rather high glycolic acid loading leve! probably due to a partia l polymorph ic modification of the lipid and determined glycolic acid sustained release pattern. Riassunto Tramite la tecnica di fusione sono state preparate liposfere per il rilascio di acido glicolico ad uso cosmetico. A questo scopo è stata impiegata tristearina quale fase lipidica e fosfatidilcol ina di soia idrogenata quale stabilizzante. Le condizioni operative migliori, in grado di determinare la resa più alta in liposfere, hanno previsto rapporti 4: l o 5: I tra trigliceride e fosfolipide e una concentrazione in fosfolipide di a lmeno il 2%. Le liposfere così ottenute, di dimensioni comprese tra i 5 e i 40 µm, presentavano elevati livelli di caricamento in acido glicolico, probabi lmente attribuibili ad una par-ziale trasformazione polimo1fa del lipide e profil i di rilascio di ti po sostenuto. 113 Preparation and in vitro charocterization of lipospheres as a carrier tor the cosfr!etic application INTRODUCTION Lipospheres represent a recent fat-based micro-particulate system developed for parenteral ( I), ora! (2) and topical (3) delivery of bioactive compounds. The recent focusing of the cosmetic technology on such a lipid carrier is due to its ability to modulate the cutaneous permeation by retaining or sustaining the release of active sub-stances to the stratum corneum. This is an im-portant feature, since an increase of the effecti-veness of compounds involving or not skin ab-sorption as well as a decrease of side locai or systemic effects could be achieved. Moreover, lipospheres represent a microparticulate system utilizing naturally occurring lipids having che-mical affinity with cutaneous components (tri-g lycerides and phospholipids). Lipospheres con-s ist of water dispersible, solid microparticles composed of a solid fat core, stabilized by one Iayer of phospholipid molecules at the surface. They have some advantages over other micro-particulate system s, including liposomes and microspheres, for example, better physical sta-bility, low cost of ingredients, ease of prepara-tion and scal e- up, high di s pers ibility in an aqueous medium, high entrapment of hydropho-bic substances, controlled particle size and non-toxicity. Lipospheres having particular structure and names were developed for cosmetic compa-n ies, whereas lipid nanoparticles were registe-red under trade names as LipopearisTM or SLNTM (4). A range of cosmetic ingredi e nts l ike coenzyme QlO, vitamin E and its derivatives, retino! and sunscreen agents have been incorpo-rated into Iipid nanoparticles (5). In this regard, the present study describes the preparation and the characterization of liposphe-res for the cutaneous delivery of g lycolic acid in order to reduce the skin damage and improve the effectiveness of this compound. In particu-lar, lipospheres were obtained by the melt method using tristearin as the lipid phase and hydrogenated soybean phosphatidylcholine as 114 the emulsifier. The formed lipospheres were evaluated for yield, morphology, size, thermal properties, entrapment capacity and g lycolic acid release patte rn. MATERIALS ANO METHODS Materials The following chemicals were obtained from commerciai suppliers and used without further purification. Tristearin as the lipid phase and glycolic acid as the active substance were sup-plied by Fluka Chemie (Buchs, Switzerland), hydrogenated soybean phosphat idylcholine as the emulsifier was supplied by Lucas Meyer (Hamburg, Germany). All solvents and o ther products were analytical grade. Methods Unloaded liposphere preparation. Tristearin ( 1.6 g) was melted at 80°C and hot phosphate buffer solution at pH 7.4 containing hydrogenated soy-bean phosphatidylcholine was added in a ratio of Jipid to phospholipid (lip/pho) ranging from 2:1to5:1. The ratio of lipidic phase to aqueous phase ranged from I :25 to I: 1 O (Method I) or it was kept constant at I :25 (Method II) (Table I). The mixture was homogenized at 13,000 rpm by Ultra-Turrax (T25 Basic IKA-Werk, Labor-technik, Staufen, Germany). After 3 min the O/A emulsion was rapidly cooled under stirring to below 20°C. The formed lipospheres were washed with water, recovered by centrifugation at 4,000 rpm and freeze-dried. Loaded liposphere preparation . A 4: l lip/pho ra-tio according to the Method I was selected for the loaded lipospheres. Practically, glycolic acid ( 1.2 g) was mixed with tristearin e 1.6 g), melted and homogenized in presence of 20 ml of 2 % hydrogenated phosphatidylcholine pH 7.4 pho-sphate buffer solution, as described above. V lannucce/11, G. Coppi, S. Sergi, R. Cameroni Table I Liposphere formulation parameters Lipididaqueous phase Lip/pho Phospholipid ratio ratio (%) 1:25 2:1 2 1:17 3:1 2 1:12 4:1 2 1:10 5:1 2 Method I Liposphere yield, The yield in lipospheres was calculated on the weight of the supernatant frac-tion recovered by centrifugation compared with the whole sample. Morphological and particle size analysis. Lipo-sphere morphological structure was examined by both optical microscope and Scanning Elec-tron Microscope (SEM, XL-40, Philips, Eindho-ven, The Netherland). The particle size was de-term in ed by computerized image analys is (IMG-VIEW, CIGS, University of Modena and Reggio Emilia) of at least 200 lipospheres on SEM micrographs. Determination of tristearin/water apparent parti-tion coefficient of g lycol ic acid. A known volu-me of g lycolic acid water solution was added to a known amount of melted tristearin . The rnix-ture was maintained at 80°C under stirring unti! equilibrium was reached, Glycolic acid concen-tration was determined in the aqueous phase by spectrophotometrical analysis at a wavelength of 220 nm (model Lambda 3B, Perkin-Elmer, Norwalk, CT, USA), Entrapment capacity, Glycolic acid content was determined by placing a weighted amount of Joaded lipospheres in water, After 48 h glycolic acid concentrations in the filtered solutions we-re assayed by spectrophotometry, Thermal analysis. Thermograms of commerciai tristearin, commerciai g lycolic acid, (1 O: l) tri-stearin/glycolic acid physical mixture and loa-Lipididaqueous phase Lip/pho Phospholipid ratio ratio (%) 1:25 2:1 2 1:25 3:1 1.3 1:25 4:1 1 1:25 5:1 0.8 Method Il ded lipospheres were recorded on a differential scanni ng calorimeter (DSC-4, Perkin-Elmer, Norwalk, CT, USA) coupled with a computeri -zed data station (Perkin-Elmer). Samples (about 10 mg) were heated in crimped alurninum pans at a scanning rate of I 0°C/m.in using dry nitro-gen fl ow (30 ml/m.i n), Glycolic acid dissolution and release. Drug dis-solution and release from the loaded lipospheres were examined by using a column-type appara-tus (Apparatus 4, USP XXIV) (Dissotest CE- I , Sotax, Base!, Switzerland) in 100 ml of pH 7.4 phosphate buffer at a fl ow rate of 25 ml/min and a temperature of 37±0.2°C. Ali experiments we-re carried out under sink conditions by determi-ning the amount of glycolic acid released spec-trophotometrically at fixed time intervals. AII the data were averaged on three deterrninations. RESULTS ANO DISCUSSION Lipospheres can be produced by melt or solvent technique. A melt process was preferred to avoid the risk of organic solvent residuals. Ya-rious lipid/phospholipid (lip/pho) ratios ranging from 2: 1 to 6: l were used by other authors and the formation of different phospholipid structu-res such as liposomes is described for higher phospholipid contents. Moreover, the deterrni-nation of the surface phospholipid showed that 79-90% of the phospholipid polar heads were on the surface of the lipospheres prepared from lip/pho at a 2: I to 4: I. w/w ratio (3). 115 Preparation and in vitro characterization of lipospheres as a carrier far the cosmetic application To examine the effect of such a parameter on li-posphere y ield, morphology and size, lip/pho ratios ranging from 2: l to 5: l were obtained by keeping constant both the phospholipid concen-tration (2%, w/v) ( Method I) a nd the internal/ex ternai phase ratio ( l :25) (Method II). According to the Method I, lipospheres having ---- lOOµm Fig. 1 SEM 111icrograph of lipospheres recovered in the .rnpema1a111 by Jhe Me1/10d I. l O\; rm, ' t q "" • 2'l% ' I 10" '" ' '<1% A (l·l ) (~I~ (11·21) (~·41) (4Hll) (' 1·191} Pvtid• ,;,, l)w>) e (I·~ ~-10) (11-l~ ~14~ ~·~~ ~1-lffi) Pcticl• sbo l)w>) spherical shape, smooth surface and without ag-gregates were recovered in the supernatant by centrifugation (Fig. n. I). On the contrary, the lower fraction as well as the whole samples ob-tained by the Method II resulted in unshaped aggregates (Fig. n. 2). 100 µm Fig. 2 SEM 111icrograph of lipospheres obtained by 1he Method Il. B l)'/, f W/, • q :rr;. (1·5) (6-10) (ll·ll) (ll-40) (41-6)) (61·100) Pcrticll s;,. l)w>) D lQll ( '"" • q lc.1' t l(.1' ''"" r ~ '" .c::7 (1-l) ~-I~ (11-31) ~1.<J) (~ I.fil) ~1·100) Pcti<ll sbo l)w>) Fig. 3 Particle si:::.e dis1ribwio11 of lipospheres ob1ai11ed by the Melhod I according 10 the /ip/pho ratio: A) 2:1 ; B) 3:1 ; C) 4:1, D) 5:1. 116 The lip/pho ratio did not appear to affect signifi -cantly both average and dis tribution size of the liposphe res (Table II, Fig, n. 3), Lipospheres si-zed between I and 100 ~tm , the most population (80-90%) being in the size range of 5-40 ~tm , considered useful for topica! applications (3). T he yie ld in lipospheres, calculated by conside-ring the percentage of supernatant on the whole samples o bta ined by the Method I, increased w ith increasing the lip/pho ratio un ti I 4 : 1 ratio (Tabi e I I). The refore , the most favo u rab le lip/pho rat ios were found to be 4: I or 5: I , Table II Yield a11d particle size lipospheres obtained according to the Method I Lip/pho Particle size Yield ratio (µm) (%) 2:1 20.0±14.3 44.1±2.2 3:1 16.1±9.3 64.1±3.1 4:1 15.9±13.3 82.0±4.1 5:1 15.2±8.5 86.7±4.3 Therefore, 4: I lip/pho ratio was selected to ob-ta in lipospheres loaded with glycolic ac id. The loaded lipospheres were examined for morpho-logy, size distribution, water dispersibility, en-trapment capacity, thermal behaviour and glyco-lic acid re lease pattern. No sig nifi cant d ifferences in morphology and Fig, 4 Oprica l mic rograph of an aqueous s11spe11sio11 of loaded lipospheres (X 400). V lannuccelli, G. Coppi, S. Sergi, R. Cameroni size were found in comparison with the unloa-ded mi cropartic les. Th e loaded liposphe res showed a high dispersibili ty in water (Fig, n. 4). Si nce topica! p rodu c ts d o n ' t s up po rt hi g h amount of solid components owing to applicabi-lity and feel of use fa i Iure, a relatively high en-trapment capacity should be required to achieve the necessary dose. Severa! fac tors can affect the entrapment capacity such as active substan-ce partition coeffi c ient and lip id po ly morphic form and crystalli nity degree (5-6), Though g ly-colic acid was characterized by a high solubil ity in melt tristearin (apparent partition coeffic ient between melt tristearin and water at 80°C resul-ted 0 .08), the entrapment capacity of liposphe-res was found ra th e r hig h (34.06 ± 6 .44 % w/w), This can be re lated to both the emulsifica-tion short pe riod and the re-partitioning of the substance into the lipid phase wi th decreasing temperature of the water phase during the emul-sion cooling. When recrystallization temperatu-re of the li pid was reached, a solid lipid will en-trap the substance which is present at this tem-perature (5). In addi t ion, the c rystallization o f tris tearin in liposphe res led to partia l lipid poly-mo rphic modifications. In fact, as thermograms show (Fig. n. 5), the s table 13-form (m.p. 57.55 ± 0.50°C, enthalpy of mel ti ng 9 1.92 ± 4.48 cal/g) "" 60 .. 100 ., .. 100 ., Fig. 5 DSC thennograms: A) rristeari11; B) glycolic acid; C) hydrogenared pl10spharidylcl1oli11e; D) (10: i) trisreari11/glycolic acid phys ical mixrure; E) loaded lipospheres. 117 Preporot1on ond in vitro choroctenzot1on of ltpospheres os o camer far the cosmetic opplicat1on transform partially in the unstable a (m.p. 48.92 ± 0.20°C, enthalpy of melting approximately between 6 and 30 cal/g) and B' (m.p. 51.37 ± l .59°C) forms. The unstable a-form is charac-terized by a lattice with disordered chains and imperfections that mean increased possibility to include and retain molecules (5). Moreover, the coexisting B-form presents a decreased cristall i-nity degree (enthalpy of melting 22.41 ± 3.42 cal/g) with imperfections offering further space to accommodate the active substance. Thermograms of lipospheres did not show the characteri stic melting endotherm of glycolic acid, which is well evident in a 10: I physical mixture, suggesting a complete molecular d i-spersion of the substance inside the lipospheres. The lipid matrix so obtained provided prolon-ged release profiles (tso = 30-90 min) compared with g lycolic acid dissolution rate (tso = 2 min) (Fig. n. 6). No "burst effect" was observed that could means the absence of glycolic acid at the liposphere surface. Ml 1S H ~ CO tS ,. MS 1a U S 1S• 1U U• Fig. 6 Glycolic acid dissolution and release profile from Lipospheres. CONCLUSIONS The present in vitro study involving the prepara-tion and the characterization of lipospheres loa-ded with glycolic acid revealed lipid/phospholi-pid ratio and lipid crystalline structure as para-meters to be considered in achieving appropria-118 te liposphere yield and active substance incor-poration, the latest being of particular interest for hydrophilic compound entrapment. ACKNOWLEDGEMENTS This work was supported by a grant from the Ministero dell ' Istruzione, dell 'Università e della Ricerca (MIUR). V lannuccelli, G. Coppi, S. Serg1, R. Cameroni References 1) Amselem S., Alving C.R., Domb A.J. (1996) Lipospheres for vaccine delivery. In: Microparti-culate systems for the delivery of proteins and vaccines, Cohen S., Bernstein H. Ed., Marce! Dekker, New York, p. J 49-168. 2) Vyas S.P., Singh R., Dimitrijevic D. (1997) Development and characterization of nifedipine li-pospheres. Phannazie; 52: 403-404. 3) Domb A.J., Bergelson L., Amselem S. (1996). Lipospheres for controlled delivery of substan-ces. In: Microencapsulation - Methods and industriai appl ications, Benita S. Ed., Marce! Dekker, New York, p. 377-4 10. 4) Dingler A., Blum R.P., Niehus H., Miiller R.H., Gohla S. (1999) Solid lipid nanoparticles (SLWM/LipopearJ sTM) - a pharmaceutical and cosmetic carrier for the application of vitamin E in derma! products. J. Microencapsulation; 16:751-767. 5) Miiller R.H., Mader K., Gohla S. (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. Ew: J. Pharm. Biopharm; 50: 161- 177. 6) Cavalli R., Caputo O., Gasco M.R. (1993) Solid lipospheres of doxorubicin and idarubicin . lnt. J. Pharm.; 89: R9-R 12. Author Address: Valentina lannuccelli Department of Pharmaceutical Sciences Via G. Campi, 183 41100 Modena - ltaly Phone: +39 59 2055151 Fax: +39 59 2055131 e-mail: iannuccelli.valentina@unimo.it 119 

Preparation and in vitro characterization of lipospheres as a carrier for the cosmetic application of glycolic acid

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