- Email: [email protected]
Preparation of 100 nm diameter unilamellar vesicles containing zinc phthalocyanine and cholesterol for use in photodynamic therapy
Chemistry and Physics of Lipids 133 (2005) 69–78
Preparation of 100 nm diameter unilamellar vesicles containing zinc phthalocyanine and cholesterol for use in photodynamic therapy Carlos A.de Oliveiraa , Antonio E.H. Machadoa,∗ , Francisco B.T. Pessineb a
Instituto de Qu´ımica, Universidade Federal de Uberlˆandia, P.O. Box 593, 38400-902 Uberlˆandia, MG, Brazil b Instituto de Qu´ımica, Universidade Estadual de Campinas, Campinas, SP, Brazil Received 27 March 2004; received in revised form 28 August 2004; accepted 1 September 2004 Available online 12 October 2004
Abstract An efficient (89–95% yield) and low-cost procedure to prepare unilamellar vesicles was used to incorporate zinc phthalocyanine (ZnPc), a model compound used as a phototherapeutic agent in studies aiming the use of unilamellar vesicles as delivery system for photodynamic therapy (PDT). ZnPc was incorporated in the presence or absence of cholesterol (CHOL), which improved the stability of the delivery system. The net vesicles present a mean diameter around 1000 nm, whereas in the presence of CHOL, CHOL and ZnPc, or only ZnPc, a drastic reduction in its diameter, varying between 100 and 150 nm, was observed. The incorporation of only ZnPc also results in a considerable reduction in the diameter of the liposomes suggests that ZnPc, due to its high hidrophobicity, must share the same microenvironment occupied by CHOL molecules. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Liposomes; Zinc phthalocyanine; Cholesterol; Drug delivery; PDT
1. Introduction Liposomes are vesicle-like structures basically constituted of phospholipids organised as concentrical bilayers containing an aqueous compartment in their interior (Cevce, 1993). Due to their amphipatic characteristics, they can incorporate substances in the aqueous ∗ Corresponding author. Tel.: +55 34 3239 4143; fax: +55 34 3239 4208. E-mail addresses: [email protected] (C.A.de Oliveira), [email protected] (A.E.H. Machado).
compartment, the lipidic bilayer, or even distributed in both compartments. Considering this particularity, they have been recognised for their great potential as drug delivery systems (Anwer et al., 2000; Ostro and Cullis, 1989; Reddi et al., 1990). The use of vesicles in drug formulations for photodynamic therapy (PDT) is based on the observation that the affinity of photosensitisers (photodynamic agents) for neoplasic tissues increases as their degree of hydrophobicity increases, while their selective distribution is increased when incorporated in amphiphilic systems (Ricchelli et al., 1993). Due to the
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2004.09.010
70
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
high concentration of specialised receptors (Reddi et al., 1990), tumor tissues present a highly competitive mechanism to uptake low-density lipoproteins (LDL) leading to high rates of cholesterol (CHOL) incorporation. This characteristic can be used to formulate an efficient and specific drug delivery system. A further advantage of the presence of CHOL in the liposome particles is that it improves their mechanical stability, avoiding structural disintegration in the blood (Bali et al., 1983). It is known that the aggregation of photosensitiser molecules can make their photochemical activity less viable, mainly if the aim is their use in photodynamic therapy. This behaviour is attributed to the fact that the aggregates in the electronically excited state tend to be deactivated preferentially by non-radiative processes (Darwent et al., 1982). An efficient drug delivery system must prevent photosensitiser aggregation and selectively distribute into the target. Satisfactory results have been obtained by incorporation of the photosensitiser in oil emulsions, cyclodextrins and liposome suspensions (Garbo and Morgan, 1988). Such delivery systems have permitted photosensitisers to be used in in vitro essays, using tumor cell cultures, and in vivo, by intravenous injection, with a significant and selective accumulation of the drug into the tumor (Jori and Reddi, 1990). The pharmacokinetic characteristics of zinc phthalocyanine (ZnPc) make this molecule a promising second generation phototherapeutic agent (Valduga et al., 1992; Moser, 1998). In view of the above discussion and considering the advantages of Epikuron SH 200 over other commercial phospholipid preparations, this work investigated liposome formulations made from hydrogenated soy lecithin, containing or not containing CHOL, aiming the incorporation of ZnPc or other photodynamic agents for use as PDT materials.
hydrogenated soy phosphatidylcholine, was purchased from Lucas Meyer (98% purity), and maintained under vacuum and refrigeration prior its utilization. All other reagents were of analytical grade and used as received. CHOL (Riedel-De Haen AG) was purified before use following the next procedure: About 5 g of CHOL was dissolved in a small amount of hot methanol (323–333 K), added to 2 mL of hot chloroform and then filtered. The filtered solution was cooled to room temperature, crystallised at 277 K over night, and the crystals filtered off. This procedure was repeated three more times. The purified product was transferred to a dessicator and maintained under vacuum for 1 h to remove any traces of solvent. 2.1. Preparation of the liposomes The methodology used in this work is a modification of the ethanolic injection method, proposed by Batzri and Korn (1975). It consists in the controlled injection (1–2 L s−1 ) of an ethanolic solution of lipids (750 L, containing Epikuron SH 200 and CHOL) using a 1-mL insulin syringe into an excess of 0.9% aqueous NaCl solution. During the addition, the mixture was stirred at a temperature above the Epikuron phase transition temperature, (Tc = 328 K). As ZnPc is hydrophobic, it was injected combined with the phospholipid with or without CHOL. The formed liposomes were purified by dialysis using Sigma D-0405 dialysis tubes adopting two changes of the saline solution, at 1-h intervals between each change, under constant stirring, at 298 K. It is known that the stability of liposome-based formulations can be improved by changes in their lipidic composition and by the addition of certain substances, such as glucuronic acid (Oku, 1999) and polyethyleneglycol (PEG) (Mayhew et al., 1992). Therefore, the influence of the addition of a high molecular mass PEG (20.000 Da, Sigma) on the stability of the formulations was studied.
2. Materials and methods Zinc phthalocyanine was obtained from Sigma– Aldrich (97% purity) and used without further purification. 1 × 10−3 mol dm−3 ZnPc stock solutions in N,Ndimethylformamide (DMF, analytical grade), a compatible solvent for studies involving biologic media, were prepared prior to the experiments and maintained in the dark at 277 K. Epikuron 200 SH, a highly purified
2.2. Spectroscopic and fluorimetric characterization of free and ZnPc incorporated liposome A calibration curve to quantify the maximum ZnPc concentration incorporated into the liposomes was constructed. Dilutions in ethanol from the ZnPc stock solution were prepared, covering the concentration
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
interval between 10−7 and 10−5 mol dm−3 . The calibration curve was based on the absorbance maximum of the Q band of ZnPc in ethanol. UV–vis absorption spectra were recorded using a HP 8452A diode array spectrophotometer. Fluorescence spectra were obtained for liposome formulations using a SLM AMINCO SPF 500C spectrofluorimeter. The excitation and emission wavelengths were, respectively, 600 and 678 nm. 2.3. Size distribution of the liposomes The size distribution of the liposomes was measured by quasi-elastic dynamic light scattering, using a zeta plus photon correlation spectrometer from Brookhaven Instruments. 2.4. Ultrastructural characterization of the liposomes The structure as well as the lamellarities of the liposomes was evaluated by transmission electronic microscopy (TEM), using a Philips CM 100 electron microscope, operating at 80 kV. Copper grids of 200 mesh, coated with polyvinyl formvar or pioloform (dissolved at 0.8% (m/v) in dichloroethane) were used
71
to visualise the liposomes. A 3% (m/v) aqueous solution of uranyl acetate was used as contrast agent. 3. Results and discussion The ethanolic injection method, described by Batzri and Korn (1975) is a fast and simple procedure for liposome preparation, producing unilamellar vesicles without the aid of sonication, extrusion, or use of detergents. The presence of residual ethanol and the obtention of diluted liposome preparations are some of the disadvantages of this method. However, these inconveniences can be reduced by dialysis (Kremer et al., 1977). The size and homogeneity of the vesicles prepared by this method are sensitive to some variables such as temperature, stirring, rate of injection, and amount of ethanol and phospholipid (Kremer et al., 1977). 3.1. Spectroscopic characterization of free and encapsulated zinc phthalocyanine The liposome preparations, in 0.9% NaCl aqueous solution, show an intense characteristic band at 296 nm (Fig. 1).
Fig. 1. Representative absorption spectra of liposomes with or without CHOL (Epikuron/CHOL 3/1, m/m) and ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL; (b) with CHOL; (c) without CHOL and diluted (1/10, v/v) in DMF; (d) with CHOL and diluted (1/10, v/v) in DMF; (e) with or without CHOL and diluted (1/10, v/v) in ethanol. Inset: LUV absorption spectra (Epikuron/CHOL 3/1, m/m) without ZnPc, with (I) and without CHOL (II).
72
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
The incorporation of CHOL (0.75 g dm−3 ) introduces significant changes in the absorption spectrum, with the displacement of the absorption maximum to 234 nm, and a substantial reduction in the absorption intensity for wavelengths higher than 290 nm. In the absorption spectra of the large unilamellar vesicles (LUV) containing ZnPc, the absorption profile of the Q band of this compound is evident (Fig. 1). Dilutions of these formulations using ethanol or DMF show that ethanol affects more intensely the integrity of the vesicles. Ethanol dilutions (1/10, v/v), with or without CHOL) present the same behaviour observed for pure ethanolic solutions (Ribeiro et al., 2004), indicating that the presence of ethanol affects the structural integrity of the liposomes. These dilutions introduce a considerable blue shift in the emission spectral bands, when compared with the undiluted formulations. Preparations diluted with DMF under the same conditions tend to show a similar, but less intense behaviour, than that observed with ethanol. Aqueous liposome suspensions containing 2.25 g dm−3 of phospholipids and approximately 5 × 10−6 mol dm−3 of ZnPc, show absorption and
fluorescence spectra (Figs. 1 and 2) typical of the photosensitiser in the monomeric form (Ribeiro et al., 2004), in agreement with the work of Valduga et al. (1992) for ZnPc at concentrations lower than 5 × 10−6 mol dm−3 and 84 nm diameter LUV. After CHOL incorporation, the liposomes became more opaque and light scattering can be observed in the absorption spectrum, which is proportional to the frequency of the incident radiation. This does not interfere with the absorption spectrum of the ZnPc incorporated into the liposome, however, introduces changes in the emission spectra (Fig. 2). The CHOL incorporation reduces the ZnPc fluorescence intensity, inducing a discrete blue shift (3 nm) in the emission band. The decrease in the emission intensity is related to the opacity (internal filter effect) and not due to aggregation problems (Fig. 3). This blue shift is related to a decrease in polarity of the microenvironment that contains ZnPc, suggesting that the ZnPc molecules must be preferentially allocated in the lipidic bilayer, an expected behaviour due to their high hydrophobicity (Moser, 1998). Ribeiro et al. (2004) observed that the ZnPc fluorescence maxima tends to suffer a “blue shift” as solvent polarity decreases.
Fig. 2. Emission spectra of lioposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL; (b) with CHOL (0.75 g dm−3 ); (c) without CHOL and diluted (1/10, v/v) in ethanol; (d) with CHOL and diluted (1/10, v/v) in ethanol; (e) without CHOL and diluted (1/10, v/v) in DMF; (f) with CHOL and diluted (1/10, v/v) in DMF; (g) undiluted, without CHOL and ZnPc; (h) undiluted, with CHOL, without ZnPc.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
73
Fig. 3. Absorption spectra of liposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL and PEG; (b) with CHOL and without PEG; (c) without CHOL and with PEG; (d) with CHOL and PEG; (e) all previous conditions, diluted (1/10, v/v) in ethanol.
3.2. Size and stability of the liposome formulations Fig. 3 shows the absorption spectra related to these experiments. Despite the changes observed in the baseline (Fig. 3), the presence of CHOL or PEG 20,000 (0.1%) do not decrease the absorbance of the ZnPc Q band. In both situations, the site in which ZnPc is allocated becomes more hydrophobic, minimizing the possibility of ZnPc aggregation. After CHOL incorporation, formulations diluted with ethanol (1/10, v/v) showed the same behaviour, indicating an increase in liposome integrity. However, PEG incorporation did not further improve liposome integrity. On the other hand, the effect of PEG on the emission spectra is less pronounced than observed for CHOL (Fig. 4). As shown in Table 1, undialysed liposomes present a larger diameter when compared to those submitted to dialysis. This is probably related to the presence of ethanol residues in the undialysed formulations, which can promote liposome rupture favouring fusion. In the presence of PEG 20,000 (0.1%), size changes in the liposomes, dialysed or not, are not significant, suggesting that PEG inclusion increased their mechanical stability.
Even though the formulations with PEG increased liposome stability, and its association with CHOL results in particles with sizes around 100 nm (Table 1), the size of the liposomes containing only PEG (Fig. 5) was usually larger than that observed in the presence of only CHOL (Table 2). As shown in this figure, the absence of PEG in the LUV containing 5 × 10−6 mol dm−3 ZnPc resulted in a mean diameter of 117 nm. Even after long periods of storage, the size distribution of the dialysed formulations (Table 1) did not show significant changes. However, the most stable formulations are the ones containing CHOL. For undialysed preparation, without CHOL or PEG, storage showed an increase in the effective mean particle diameter, indicating residual ethanol can open some of the liposomes, favouring the formation of particles with higher diameters due to fusion of the smaller particles. Due to the stabilizing effect of the presence of PEG on the size distribution of the liposomes, the role of its concentration on the stability of formulations (with 2.25 g dm−3 of Epikuron SH 200) containing different amounts of ZnPc, was evaluated. As shown in Fig. 5, the results were not promising. These results show that the inclusion of ZnPc at a concentration near 5 × 10−6 mol dm−3 was capable to
74
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
Fig. 4. Effect of CHOL and PEG (0.1%) on the emission spectra of liposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL or PEG; (b) without CHOL, with PEG; (c) with CHOL, without PEG; (d) with CHOL and PEG.
reduce the diameter of the liposomes without CHOL to values considerably lower than the observed for LUV containing PEG and close to the value observed in the presence of CHOL (Table 1). However, the same cannot be said for the association of ZnPc and PEG (Fig. 5).
The influence of the Epikuron/CHOL ratio on the integrity of the liposomes was also evaluated (Table 2). In this case, all ratios tested gave liposomes with a diameter near 100 nm, and low polydispersity. Since the ethanolic injection method produces unilamellar
Table 1 Effect of the dialysis and storage (at 4 ◦ C) on the size distribution and polydispersity of the liposome preparations Condition
Mean diameter (nm)
Effective diameter (nm)
Polydispersity
Without CHOL, ZnPc and PEG, undialysed
994.1 ± 107.8a 1384.8 ± 158.3b 1641.6 ± 212.3c 280.8 ± 8.8a 111.1 ± 1.0a 117.2 ± 0.9a 597.7 ± 40.1a 534.7 ± 51.3b 630.9 ± 68.1c 301.7 ± 14.8a 336.8 ± 10.2b 373.4 ± 36.4c 106.3 ± 0.6a 107.3 ± 0.9b 107.2 ± 0.9c 113.2 ± 0,8a 112.2 ± 1.0b 112.2 ± 1.2c
940.9 1332.7 1573.7 287.4 111.2 117.2 589.8 523.4 667.8 291.3 326.7 431.1 106.4 107.2 107.2 113.2 112.5 112.3
0.338 0.331 0.302 0.322 0.181 0.146 0.326 0.330 0.253 0.347 0.358 0.202 0.176 0.183 0.193 0.130 0.166 0.157
Without CHOL, ZnPc and with PEG, undialysed With CHOL, without ZnPc and PEG, undialysed With CHOL, PEG and without ZnPc, undialysed Without CHOL, ZnPc and PEG, dialysed
Without CHOL, ZnPc and and with PEG, dialysed
With CHOL, without ZnPc and PEG, dialysed
With CHOL and PEG, without ZnPc, dialysed
a b c
After preparation. After 8 days of storage. After 14 days of storage.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
75
Fig. 5. Effect of PEG concentration on the size distribution of the liposome preparations without CHOL.
vesicles, they can be considered LUV. Formulations containing CHOL above the 3:1 (m/m) ratio became increasingly turbid. Also, for Epikuron concentrations above 2.25 g dm−3 , even in the presence of CHOL, a considerable increase in the diameter of the vesicles was observed. Considering that the purity of phosphatidylcholine in Epikuron SH 200 is 98% (Lucas, 2004), the Epikuron/CHOL molar ratio corresponding to a 3:1 (m/m) ratio, is 1.58/1.00, since the molecular mass of phosphatidylcholine is 733 g mol−1 . It is known that CHOL is unable to form bilayers. However, its insertion into phospholipid bilayers at levels below 33 mol% prevents crystallization of the acyl side chains, eliminating the physical characteristics of phase transition, providing fluidity to the membrane over a large temperature range (Demel and De Kruyff, Table 2 Influence of CHOL concentration on size distribution of the liposomes, in formulations made without ZnPc or PEG Epikuron/CHOL ratio, (m/m)
Mean diameter (nm)
Effective diameter (nm)
Polydispersity
Epikuron 9:1 5:1 3:1 2:1 1:1
717 ± 51 120 ± 1 104 ± 1 113 ± 1 136 ± 1 196 ± 2
709 119 104 113 136 195
0.402 0.234 0.158 0.140 0.155 0.191
1976). The mechanisms by which CHOL induces this plasticity in the liposomes is not well known. Probably it is associated with the interaction between the 3-␣hydroxy group from the sterol and the phospholipids from the lipidic bilayer (Demel and De Kruyff, 1976). The use of CHOL is justified by evidences that support the decrease of interaction between humoral components and cellular immune systems, such as serum proteins, opsonins and phagocytes, thus prolonging the circulation time of the liposomes (Love et al., 1990). Johnson (1975) demonstrated that macrophages are not able to incorporate liposomes containing high CHOL concentrations. However, Ginevra et al. (1990) showed that the presence of 20 mol% of CHOL in small unilamellar vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) optimizes the release of ZnPc to LDL, which is important to increase drug delivery selectivity to the tumoral cells. An interesting and highly reproducible result, obtained with different liposome formulations, was the drastic reduction in the size of the vesicles formed in the presence of CHOL when the Epikuron concentration was maintained around 2.25 g dm−3 . Liposomes without CHOL with a mean diameter of 1000 nm had their size reduced about 10 times after the incorporation of a small concentration of CHOL (Table 2). The ability of ethanol to favour the fusion of SUV prepared with DPPC, after storage at temperature lower than Tc , explains in part the fact that our undialysed
76
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
formulations possess a diameter higher than that observed for the dialysed ones. This effect became much more expressive after 14 days of storage at 277 K (Table 1). The liposome diameter reduction, observed after the addition of CHOL is probably due to its inser-
tion into the bilayer structure of the vesicle, substituting a monomer, similar to that which occurs with micelle solutions (De Paula et al., 2004). The diameter reduction of the liposomes to values near the ones obtained with CHOL inclusion, also obtained by the incorporation of ZnPc, suggests that the ZnPc may be
Fig. 6. TEM micrographs of a new liposome formulation (Epikuron/CHOL (3/1, m/m) and 5 × 10−6 mol dm−3 of ZnPc): (a) the size uniformity of liposomes (40,000×) can be seen. Inset: the lipidic bilayer of a less contrasted liposome can be seen at higher amplification (63,000×); (b) the morphology of some less contrasted liposome (40,000×) is detailed.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
77
Fig. 7. TEM micrographs of an old (2 months aging) liposome formulation (Epikuron/CHOL (3/1, m/m) and 5 × 10−6 mol dm−3 of ZnPc). Panoramic view, in which the size uniformity of the liposomes can be observed (32,000×). Inset: a 63,000× magnification of liposomes, where the lipidic bilayer and a slight drain of the vesicles can be observed.
preferentially located in the same microenvironment occupied by CHOL, most probably the lipidic bilayer. This localization of ZnPc is reasonable considering its high hydrophobicity (Ribeiro et al., 2004). Also, the spectroscopic evaluation of liposome formulations containing ZnPc and CHOL show that only in LUV noruptured by ethanol, the absorbance and fluorescence intensity values due to ZnPc were reduced. The average efficiency of ZnPc incorporation was near 90%.
the several liposome formulations investigated in this study. In the case of SUV structures, the CHOL inclusion into the lipidic bilayer at a 20 mol% concentration did not influence the size of the particles (Franks, 1976; Johnson, 1973). However, it has been observed that molar concentrations above 30 mol% induce a progressive increase in the vesicle diameter (Franks, 1976; Johnson, 1973), as SUV containing 50 mol% of CHOL exhibit an increase of about 30% in their diameter (Johnson, 1973).
3.3. Evaluation of the liposome preparations by transmission electronic microscopy Figs. 6 and 7 present a structural visualisation of the liposomes, showing the existence of lipidic bilayers, an indication of unilamellarity of the vesicles. Control tests using multilamellar large vesicles (MLV) prepared by the hydration of a dry lipidic film confirm this. In agreement with diameter measurements, the liposome formulations made without CHOL does not present size uniformity, contrary to the preparations containing CHOL (Epikuron/CHOL 3:1 (m/m)). Storage of LUV containing CHOL does not affect the uniformity of the particles. However, some LUV changes occur probably due to osmotic changes in the medium or the escape of part of the liquids. The loss of encapsulated drug with storage time was negligible for
4. Conclusions A fast (around 10 min), reproducible and low cost method for LUV preparation using Epikuron SH 200 at a 2.25 g dm−3 concentration, is proposed. The diameter of the LUV varies between 100 and 1000 nm. The lowest size particles were obtained by CHOL incorporation (0.75 g dm−3 ). A drastic reduction in the particle diameter could be observed by the incorporation of ZnPc (5 × 10−6 mol dm−3 ). The effect of ZnPc incorporation on the size of the LUV is comparable to the one obtained by the addition of CHOL. The addition of CHOL to ZnPc increased LUV stability. Besides, the
78
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
presence of CHOL induces stability to the liposome as a drug delivery system. The addition of PEG 20,000 (0.1%) did not imply in significant size reduction in the preparations. The estimated ZnPc incorporation efficiency is between 89 and 95%. The proposed method is very useful for incorporation of other hydrophobic drugs that can be used as photodynamic therapeutic agents.
Acknowledgements The authors thank the CNPq, FAPEMIG and FAPESP for financial support and fellowships. The Laborat´orio de Microscopia Eletrˆonica (UNESP, Rio Claro/SP and FMRP-USP, Ribeir˜ao Preto/SP), and the Centro de Microscopia Eletrˆonica, Departamento de Morfologia, UFU, are acknowledged fot the use of their experimental infrastructure. Dr. Divinomar Severino is acknowledged for valuable suggestions, and Thiago Padovani Xavier for some physical measurements.
References Anwer, K., Meaney, C., Kao, G., Hussain, N., Shelvin, R., Earls, R.M., Leonard, P., Quezada, A., Rolland, A.P., Sullivan, S.M., 2000. Cationic lipid-based delivery system for systemic cancer gene therapy. Cancer Gene Ther. 7, 1156–1164. Batzri, S., Korn, E.D., 1975. Interaction of phospholipid vesicles with cells – endocytosis and fusion as alternate mechanisms for uptake of lipid-soluble and water-soluble molecules. J. Cell Biol. 66, 621–634. Bali, A., Dhawan, S., Gupta, C.M., 1983. Stability of liposomes in circulation is markedly enhanced by structural modification of their phospholipid component. FEBS Lett. 154, 373–377. Cevce, G., 1993. Lipid properties as a basis for membrane modelling and rational liposome design. In: Gregoriadis, G. (Ed.), Liposome Technology, vol. 1. CRC Press, Boca Raton, pp. 1–36. Darwent, J.R., Douglas, P., Harriman, A., Porter, G., Richoux, M.C., 1982. Metal phthalocyanines and porphyrins as photosensitisers for reduction of water to hydrogen. Coord. Chem. Rev. 44, 83–126. Demel, R.A., De Kruyff, B., 1976. The function of sterols in membranes. Biochim. Biophys. Acta 457, 109–132. De Paula, R., Machado, A.E.H., Miranda, J.A., 2004. 3-Benzoxazol2-yl-7-(N,N-diethylamino)-chromen-2-one as fluorescent probe
for investigation of micellar microenvironments. J. Photochem. Photobiol., A: Chem. 165, 109–114. Franks, N.P., 1976. Structural analysis of hydrated egg lecithin and cholesterol bilayers.1. X-ray-diffraction. J. Mol. Biol. 100, 345–358. Garbo, G.M., Morgan, A.R., 1988. Delivery systems for hydrophobic drugs. J. Photochem. Photobiol. B: Biol. 1, 494–495. Ginevra, F., Biffanti, S., Pagnan, A., Biolo, R., Reddi, E., Jori, G., 1990. Delivery of the tumor photosensitizer zinc(II)phthalocyanine to serum-proteins by different liposomes — studies invitro and invivo. Cancer Lett. 49, 59–65. Johnson, S.M., 1973. Effect of charge and cholesterol on size and thickness of sonicated phospholipid vesicles. Biochim. Biophys. Acta 307, 27–41. Johnson, S.M., 1975. The inability of macrophages to digest liposomes containing a high proportion of cholesterol. Biochem. Soc. Trans. 3, 160–161. Jori, G., Reddi, E., 1990. In: Kessel, D. (Ed.), Photodynamic Therapy of Neoplasic Disease. CRC Press, Boca Raton, pp. 117– 130. Kremer, J.M.H., Esker, M.W.J., Pathmamanoharan, C., Wiersema, P.H., 1977. Vesicles of variable diameter prepared by a modified injection method. Biochemistry 16, 3932–3935. Love, W.G., Amos, N., Kellaway, I.W., Williams, B.D., 1990. Specific accumulation of cholesterol-rich liposomes in the inflammatory tissue of rats with adjuvant arthritis. Ann. Rheum. Dis. 49, 611–614. Lucas 2004. Meyer’s product information: Epikuron 200 SH. Mayhew, E., Lasic, D.D., Babbar, S., Martin, F.J., 1992. Pharmacokinetics and antitumor activity of epirubicin encapsulated in long-circulating liposomes incorporating polyethylene glycolderivatized phospholipids. Int. J. Cancer. 51, 302–315. Moser, J.G., 1998. Photodynamic Tumor Therapy, 2nd and 3rd Generation Photosensitizers, Harwood, The Netherlands. Oku, N., 1999. Anticancer therapy using glucuronate modified longcirculating liposomes. Adv. Drug. Deliv. Rev. 40, 63–73. Ostro, M.J., Cullis, P.R., 1989. Use of liposomes as injectable-drug delivery systems. Am. J. Hosp. Pharm. 46, 1576–1587. Reddi, E., Zhow, C., Biolo, R., Menegaldo, E., Jori, G., 1990. Liposome or LDL-administered Al(III)-phthalocyanine as a photodynamic agent for tumours. I. Pharmacokinetic properties and phototherapeutic efficiency. Br. J. Cancer. 61, 407–411. Ricchelli, F., Gobbo, S., Jori, G., Moreno, G., Vinzens, F., Salet, C., 1993. Photosensitisation of mitochondria by liposome-bound porphyrins. Photochem. Photobiol. 58, 53–62. Ribeiro, J., Xavier, T.P., Koshiyama, D.R., Ferreira, S.K., Severino, D., Miranda, J.A., Machado, A.E.H., 2004. Photophysics of zinc (II) phthalocyanine in homogeneous and microheterogeneous media, unpublished results. Valduga, G., Reddi, E., Jori, G., 1992. Steady-state and time-resolved spectroscopic studies on zinc(II) phthalocyanine in liposomes. J. Photochem. Photobiol. B: Biol. 16, 331–340.
Preparation of 100 nm diameter unilamellar vesicles containing zinc phthalocyanine and cholesterol for use in photodynamic therapy Carlos A.de Oliveiraa , Antonio E.H. Machadoa,∗ , Francisco B.T. Pessineb a
Instituto de Qu´ımica, Universidade Federal de Uberlˆandia, P.O. Box 593, 38400-902 Uberlˆandia, MG, Brazil b Instituto de Qu´ımica, Universidade Estadual de Campinas, Campinas, SP, Brazil Received 27 March 2004; received in revised form 28 August 2004; accepted 1 September 2004 Available online 12 October 2004
Abstract An efficient (89–95% yield) and low-cost procedure to prepare unilamellar vesicles was used to incorporate zinc phthalocyanine (ZnPc), a model compound used as a phototherapeutic agent in studies aiming the use of unilamellar vesicles as delivery system for photodynamic therapy (PDT). ZnPc was incorporated in the presence or absence of cholesterol (CHOL), which improved the stability of the delivery system. The net vesicles present a mean diameter around 1000 nm, whereas in the presence of CHOL, CHOL and ZnPc, or only ZnPc, a drastic reduction in its diameter, varying between 100 and 150 nm, was observed. The incorporation of only ZnPc also results in a considerable reduction in the diameter of the liposomes suggests that ZnPc, due to its high hidrophobicity, must share the same microenvironment occupied by CHOL molecules. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Liposomes; Zinc phthalocyanine; Cholesterol; Drug delivery; PDT
1. Introduction Liposomes are vesicle-like structures basically constituted of phospholipids organised as concentrical bilayers containing an aqueous compartment in their interior (Cevce, 1993). Due to their amphipatic characteristics, they can incorporate substances in the aqueous ∗ Corresponding author. Tel.: +55 34 3239 4143; fax: +55 34 3239 4208. E-mail addresses: [email protected] (C.A.de Oliveira), [email protected] (A.E.H. Machado).
compartment, the lipidic bilayer, or even distributed in both compartments. Considering this particularity, they have been recognised for their great potential as drug delivery systems (Anwer et al., 2000; Ostro and Cullis, 1989; Reddi et al., 1990). The use of vesicles in drug formulations for photodynamic therapy (PDT) is based on the observation that the affinity of photosensitisers (photodynamic agents) for neoplasic tissues increases as their degree of hydrophobicity increases, while their selective distribution is increased when incorporated in amphiphilic systems (Ricchelli et al., 1993). Due to the
0009-3084/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.chemphyslip.2004.09.010
70
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
high concentration of specialised receptors (Reddi et al., 1990), tumor tissues present a highly competitive mechanism to uptake low-density lipoproteins (LDL) leading to high rates of cholesterol (CHOL) incorporation. This characteristic can be used to formulate an efficient and specific drug delivery system. A further advantage of the presence of CHOL in the liposome particles is that it improves their mechanical stability, avoiding structural disintegration in the blood (Bali et al., 1983). It is known that the aggregation of photosensitiser molecules can make their photochemical activity less viable, mainly if the aim is their use in photodynamic therapy. This behaviour is attributed to the fact that the aggregates in the electronically excited state tend to be deactivated preferentially by non-radiative processes (Darwent et al., 1982). An efficient drug delivery system must prevent photosensitiser aggregation and selectively distribute into the target. Satisfactory results have been obtained by incorporation of the photosensitiser in oil emulsions, cyclodextrins and liposome suspensions (Garbo and Morgan, 1988). Such delivery systems have permitted photosensitisers to be used in in vitro essays, using tumor cell cultures, and in vivo, by intravenous injection, with a significant and selective accumulation of the drug into the tumor (Jori and Reddi, 1990). The pharmacokinetic characteristics of zinc phthalocyanine (ZnPc) make this molecule a promising second generation phototherapeutic agent (Valduga et al., 1992; Moser, 1998). In view of the above discussion and considering the advantages of Epikuron SH 200 over other commercial phospholipid preparations, this work investigated liposome formulations made from hydrogenated soy lecithin, containing or not containing CHOL, aiming the incorporation of ZnPc or other photodynamic agents for use as PDT materials.
hydrogenated soy phosphatidylcholine, was purchased from Lucas Meyer (98% purity), and maintained under vacuum and refrigeration prior its utilization. All other reagents were of analytical grade and used as received. CHOL (Riedel-De Haen AG) was purified before use following the next procedure: About 5 g of CHOL was dissolved in a small amount of hot methanol (323–333 K), added to 2 mL of hot chloroform and then filtered. The filtered solution was cooled to room temperature, crystallised at 277 K over night, and the crystals filtered off. This procedure was repeated three more times. The purified product was transferred to a dessicator and maintained under vacuum for 1 h to remove any traces of solvent. 2.1. Preparation of the liposomes The methodology used in this work is a modification of the ethanolic injection method, proposed by Batzri and Korn (1975). It consists in the controlled injection (1–2 L s−1 ) of an ethanolic solution of lipids (750 L, containing Epikuron SH 200 and CHOL) using a 1-mL insulin syringe into an excess of 0.9% aqueous NaCl solution. During the addition, the mixture was stirred at a temperature above the Epikuron phase transition temperature, (Tc = 328 K). As ZnPc is hydrophobic, it was injected combined with the phospholipid with or without CHOL. The formed liposomes were purified by dialysis using Sigma D-0405 dialysis tubes adopting two changes of the saline solution, at 1-h intervals between each change, under constant stirring, at 298 K. It is known that the stability of liposome-based formulations can be improved by changes in their lipidic composition and by the addition of certain substances, such as glucuronic acid (Oku, 1999) and polyethyleneglycol (PEG) (Mayhew et al., 1992). Therefore, the influence of the addition of a high molecular mass PEG (20.000 Da, Sigma) on the stability of the formulations was studied.
2. Materials and methods Zinc phthalocyanine was obtained from Sigma– Aldrich (97% purity) and used without further purification. 1 × 10−3 mol dm−3 ZnPc stock solutions in N,Ndimethylformamide (DMF, analytical grade), a compatible solvent for studies involving biologic media, were prepared prior to the experiments and maintained in the dark at 277 K. Epikuron 200 SH, a highly purified
2.2. Spectroscopic and fluorimetric characterization of free and ZnPc incorporated liposome A calibration curve to quantify the maximum ZnPc concentration incorporated into the liposomes was constructed. Dilutions in ethanol from the ZnPc stock solution were prepared, covering the concentration
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
interval between 10−7 and 10−5 mol dm−3 . The calibration curve was based on the absorbance maximum of the Q band of ZnPc in ethanol. UV–vis absorption spectra were recorded using a HP 8452A diode array spectrophotometer. Fluorescence spectra were obtained for liposome formulations using a SLM AMINCO SPF 500C spectrofluorimeter. The excitation and emission wavelengths were, respectively, 600 and 678 nm. 2.3. Size distribution of the liposomes The size distribution of the liposomes was measured by quasi-elastic dynamic light scattering, using a zeta plus photon correlation spectrometer from Brookhaven Instruments. 2.4. Ultrastructural characterization of the liposomes The structure as well as the lamellarities of the liposomes was evaluated by transmission electronic microscopy (TEM), using a Philips CM 100 electron microscope, operating at 80 kV. Copper grids of 200 mesh, coated with polyvinyl formvar or pioloform (dissolved at 0.8% (m/v) in dichloroethane) were used
71
to visualise the liposomes. A 3% (m/v) aqueous solution of uranyl acetate was used as contrast agent. 3. Results and discussion The ethanolic injection method, described by Batzri and Korn (1975) is a fast and simple procedure for liposome preparation, producing unilamellar vesicles without the aid of sonication, extrusion, or use of detergents. The presence of residual ethanol and the obtention of diluted liposome preparations are some of the disadvantages of this method. However, these inconveniences can be reduced by dialysis (Kremer et al., 1977). The size and homogeneity of the vesicles prepared by this method are sensitive to some variables such as temperature, stirring, rate of injection, and amount of ethanol and phospholipid (Kremer et al., 1977). 3.1. Spectroscopic characterization of free and encapsulated zinc phthalocyanine The liposome preparations, in 0.9% NaCl aqueous solution, show an intense characteristic band at 296 nm (Fig. 1).
Fig. 1. Representative absorption spectra of liposomes with or without CHOL (Epikuron/CHOL 3/1, m/m) and ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL; (b) with CHOL; (c) without CHOL and diluted (1/10, v/v) in DMF; (d) with CHOL and diluted (1/10, v/v) in DMF; (e) with or without CHOL and diluted (1/10, v/v) in ethanol. Inset: LUV absorption spectra (Epikuron/CHOL 3/1, m/m) without ZnPc, with (I) and without CHOL (II).
72
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
The incorporation of CHOL (0.75 g dm−3 ) introduces significant changes in the absorption spectrum, with the displacement of the absorption maximum to 234 nm, and a substantial reduction in the absorption intensity for wavelengths higher than 290 nm. In the absorption spectra of the large unilamellar vesicles (LUV) containing ZnPc, the absorption profile of the Q band of this compound is evident (Fig. 1). Dilutions of these formulations using ethanol or DMF show that ethanol affects more intensely the integrity of the vesicles. Ethanol dilutions (1/10, v/v), with or without CHOL) present the same behaviour observed for pure ethanolic solutions (Ribeiro et al., 2004), indicating that the presence of ethanol affects the structural integrity of the liposomes. These dilutions introduce a considerable blue shift in the emission spectral bands, when compared with the undiluted formulations. Preparations diluted with DMF under the same conditions tend to show a similar, but less intense behaviour, than that observed with ethanol. Aqueous liposome suspensions containing 2.25 g dm−3 of phospholipids and approximately 5 × 10−6 mol dm−3 of ZnPc, show absorption and
fluorescence spectra (Figs. 1 and 2) typical of the photosensitiser in the monomeric form (Ribeiro et al., 2004), in agreement with the work of Valduga et al. (1992) for ZnPc at concentrations lower than 5 × 10−6 mol dm−3 and 84 nm diameter LUV. After CHOL incorporation, the liposomes became more opaque and light scattering can be observed in the absorption spectrum, which is proportional to the frequency of the incident radiation. This does not interfere with the absorption spectrum of the ZnPc incorporated into the liposome, however, introduces changes in the emission spectra (Fig. 2). The CHOL incorporation reduces the ZnPc fluorescence intensity, inducing a discrete blue shift (3 nm) in the emission band. The decrease in the emission intensity is related to the opacity (internal filter effect) and not due to aggregation problems (Fig. 3). This blue shift is related to a decrease in polarity of the microenvironment that contains ZnPc, suggesting that the ZnPc molecules must be preferentially allocated in the lipidic bilayer, an expected behaviour due to their high hydrophobicity (Moser, 1998). Ribeiro et al. (2004) observed that the ZnPc fluorescence maxima tends to suffer a “blue shift” as solvent polarity decreases.
Fig. 2. Emission spectra of lioposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL; (b) with CHOL (0.75 g dm−3 ); (c) without CHOL and diluted (1/10, v/v) in ethanol; (d) with CHOL and diluted (1/10, v/v) in ethanol; (e) without CHOL and diluted (1/10, v/v) in DMF; (f) with CHOL and diluted (1/10, v/v) in DMF; (g) undiluted, without CHOL and ZnPc; (h) undiluted, with CHOL, without ZnPc.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
73
Fig. 3. Absorption spectra of liposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL and PEG; (b) with CHOL and without PEG; (c) without CHOL and with PEG; (d) with CHOL and PEG; (e) all previous conditions, diluted (1/10, v/v) in ethanol.
3.2. Size and stability of the liposome formulations Fig. 3 shows the absorption spectra related to these experiments. Despite the changes observed in the baseline (Fig. 3), the presence of CHOL or PEG 20,000 (0.1%) do not decrease the absorbance of the ZnPc Q band. In both situations, the site in which ZnPc is allocated becomes more hydrophobic, minimizing the possibility of ZnPc aggregation. After CHOL incorporation, formulations diluted with ethanol (1/10, v/v) showed the same behaviour, indicating an increase in liposome integrity. However, PEG incorporation did not further improve liposome integrity. On the other hand, the effect of PEG on the emission spectra is less pronounced than observed for CHOL (Fig. 4). As shown in Table 1, undialysed liposomes present a larger diameter when compared to those submitted to dialysis. This is probably related to the presence of ethanol residues in the undialysed formulations, which can promote liposome rupture favouring fusion. In the presence of PEG 20,000 (0.1%), size changes in the liposomes, dialysed or not, are not significant, suggesting that PEG inclusion increased their mechanical stability.
Even though the formulations with PEG increased liposome stability, and its association with CHOL results in particles with sizes around 100 nm (Table 1), the size of the liposomes containing only PEG (Fig. 5) was usually larger than that observed in the presence of only CHOL (Table 2). As shown in this figure, the absence of PEG in the LUV containing 5 × 10−6 mol dm−3 ZnPc resulted in a mean diameter of 117 nm. Even after long periods of storage, the size distribution of the dialysed formulations (Table 1) did not show significant changes. However, the most stable formulations are the ones containing CHOL. For undialysed preparation, without CHOL or PEG, storage showed an increase in the effective mean particle diameter, indicating residual ethanol can open some of the liposomes, favouring the formation of particles with higher diameters due to fusion of the smaller particles. Due to the stabilizing effect of the presence of PEG on the size distribution of the liposomes, the role of its concentration on the stability of formulations (with 2.25 g dm−3 of Epikuron SH 200) containing different amounts of ZnPc, was evaluated. As shown in Fig. 5, the results were not promising. These results show that the inclusion of ZnPc at a concentration near 5 × 10−6 mol dm−3 was capable to
74
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
Fig. 4. Effect of CHOL and PEG (0.1%) on the emission spectra of liposomes containing ZnPc (5 × 10−6 mol dm−3 ): (a) without CHOL or PEG; (b) without CHOL, with PEG; (c) with CHOL, without PEG; (d) with CHOL and PEG.
reduce the diameter of the liposomes without CHOL to values considerably lower than the observed for LUV containing PEG and close to the value observed in the presence of CHOL (Table 1). However, the same cannot be said for the association of ZnPc and PEG (Fig. 5).
The influence of the Epikuron/CHOL ratio on the integrity of the liposomes was also evaluated (Table 2). In this case, all ratios tested gave liposomes with a diameter near 100 nm, and low polydispersity. Since the ethanolic injection method produces unilamellar
Table 1 Effect of the dialysis and storage (at 4 ◦ C) on the size distribution and polydispersity of the liposome preparations Condition
Mean diameter (nm)
Effective diameter (nm)
Polydispersity
Without CHOL, ZnPc and PEG, undialysed
994.1 ± 107.8a 1384.8 ± 158.3b 1641.6 ± 212.3c 280.8 ± 8.8a 111.1 ± 1.0a 117.2 ± 0.9a 597.7 ± 40.1a 534.7 ± 51.3b 630.9 ± 68.1c 301.7 ± 14.8a 336.8 ± 10.2b 373.4 ± 36.4c 106.3 ± 0.6a 107.3 ± 0.9b 107.2 ± 0.9c 113.2 ± 0,8a 112.2 ± 1.0b 112.2 ± 1.2c
940.9 1332.7 1573.7 287.4 111.2 117.2 589.8 523.4 667.8 291.3 326.7 431.1 106.4 107.2 107.2 113.2 112.5 112.3
0.338 0.331 0.302 0.322 0.181 0.146 0.326 0.330 0.253 0.347 0.358 0.202 0.176 0.183 0.193 0.130 0.166 0.157
Without CHOL, ZnPc and with PEG, undialysed With CHOL, without ZnPc and PEG, undialysed With CHOL, PEG and without ZnPc, undialysed Without CHOL, ZnPc and PEG, dialysed
Without CHOL, ZnPc and and with PEG, dialysed
With CHOL, without ZnPc and PEG, dialysed
With CHOL and PEG, without ZnPc, dialysed
a b c
After preparation. After 8 days of storage. After 14 days of storage.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
75
Fig. 5. Effect of PEG concentration on the size distribution of the liposome preparations without CHOL.
vesicles, they can be considered LUV. Formulations containing CHOL above the 3:1 (m/m) ratio became increasingly turbid. Also, for Epikuron concentrations above 2.25 g dm−3 , even in the presence of CHOL, a considerable increase in the diameter of the vesicles was observed. Considering that the purity of phosphatidylcholine in Epikuron SH 200 is 98% (Lucas, 2004), the Epikuron/CHOL molar ratio corresponding to a 3:1 (m/m) ratio, is 1.58/1.00, since the molecular mass of phosphatidylcholine is 733 g mol−1 . It is known that CHOL is unable to form bilayers. However, its insertion into phospholipid bilayers at levels below 33 mol% prevents crystallization of the acyl side chains, eliminating the physical characteristics of phase transition, providing fluidity to the membrane over a large temperature range (Demel and De Kruyff, Table 2 Influence of CHOL concentration on size distribution of the liposomes, in formulations made without ZnPc or PEG Epikuron/CHOL ratio, (m/m)
Mean diameter (nm)
Effective diameter (nm)
Polydispersity
Epikuron 9:1 5:1 3:1 2:1 1:1
717 ± 51 120 ± 1 104 ± 1 113 ± 1 136 ± 1 196 ± 2
709 119 104 113 136 195
0.402 0.234 0.158 0.140 0.155 0.191
1976). The mechanisms by which CHOL induces this plasticity in the liposomes is not well known. Probably it is associated with the interaction between the 3-␣hydroxy group from the sterol and the phospholipids from the lipidic bilayer (Demel and De Kruyff, 1976). The use of CHOL is justified by evidences that support the decrease of interaction between humoral components and cellular immune systems, such as serum proteins, opsonins and phagocytes, thus prolonging the circulation time of the liposomes (Love et al., 1990). Johnson (1975) demonstrated that macrophages are not able to incorporate liposomes containing high CHOL concentrations. However, Ginevra et al. (1990) showed that the presence of 20 mol% of CHOL in small unilamellar vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) optimizes the release of ZnPc to LDL, which is important to increase drug delivery selectivity to the tumoral cells. An interesting and highly reproducible result, obtained with different liposome formulations, was the drastic reduction in the size of the vesicles formed in the presence of CHOL when the Epikuron concentration was maintained around 2.25 g dm−3 . Liposomes without CHOL with a mean diameter of 1000 nm had their size reduced about 10 times after the incorporation of a small concentration of CHOL (Table 2). The ability of ethanol to favour the fusion of SUV prepared with DPPC, after storage at temperature lower than Tc , explains in part the fact that our undialysed
76
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
formulations possess a diameter higher than that observed for the dialysed ones. This effect became much more expressive after 14 days of storage at 277 K (Table 1). The liposome diameter reduction, observed after the addition of CHOL is probably due to its inser-
tion into the bilayer structure of the vesicle, substituting a monomer, similar to that which occurs with micelle solutions (De Paula et al., 2004). The diameter reduction of the liposomes to values near the ones obtained with CHOL inclusion, also obtained by the incorporation of ZnPc, suggests that the ZnPc may be
Fig. 6. TEM micrographs of a new liposome formulation (Epikuron/CHOL (3/1, m/m) and 5 × 10−6 mol dm−3 of ZnPc): (a) the size uniformity of liposomes (40,000×) can be seen. Inset: the lipidic bilayer of a less contrasted liposome can be seen at higher amplification (63,000×); (b) the morphology of some less contrasted liposome (40,000×) is detailed.
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
77
Fig. 7. TEM micrographs of an old (2 months aging) liposome formulation (Epikuron/CHOL (3/1, m/m) and 5 × 10−6 mol dm−3 of ZnPc). Panoramic view, in which the size uniformity of the liposomes can be observed (32,000×). Inset: a 63,000× magnification of liposomes, where the lipidic bilayer and a slight drain of the vesicles can be observed.
preferentially located in the same microenvironment occupied by CHOL, most probably the lipidic bilayer. This localization of ZnPc is reasonable considering its high hydrophobicity (Ribeiro et al., 2004). Also, the spectroscopic evaluation of liposome formulations containing ZnPc and CHOL show that only in LUV noruptured by ethanol, the absorbance and fluorescence intensity values due to ZnPc were reduced. The average efficiency of ZnPc incorporation was near 90%.
the several liposome formulations investigated in this study. In the case of SUV structures, the CHOL inclusion into the lipidic bilayer at a 20 mol% concentration did not influence the size of the particles (Franks, 1976; Johnson, 1973). However, it has been observed that molar concentrations above 30 mol% induce a progressive increase in the vesicle diameter (Franks, 1976; Johnson, 1973), as SUV containing 50 mol% of CHOL exhibit an increase of about 30% in their diameter (Johnson, 1973).
3.3. Evaluation of the liposome preparations by transmission electronic microscopy Figs. 6 and 7 present a structural visualisation of the liposomes, showing the existence of lipidic bilayers, an indication of unilamellarity of the vesicles. Control tests using multilamellar large vesicles (MLV) prepared by the hydration of a dry lipidic film confirm this. In agreement with diameter measurements, the liposome formulations made without CHOL does not present size uniformity, contrary to the preparations containing CHOL (Epikuron/CHOL 3:1 (m/m)). Storage of LUV containing CHOL does not affect the uniformity of the particles. However, some LUV changes occur probably due to osmotic changes in the medium or the escape of part of the liquids. The loss of encapsulated drug with storage time was negligible for
4. Conclusions A fast (around 10 min), reproducible and low cost method for LUV preparation using Epikuron SH 200 at a 2.25 g dm−3 concentration, is proposed. The diameter of the LUV varies between 100 and 1000 nm. The lowest size particles were obtained by CHOL incorporation (0.75 g dm−3 ). A drastic reduction in the particle diameter could be observed by the incorporation of ZnPc (5 × 10−6 mol dm−3 ). The effect of ZnPc incorporation on the size of the LUV is comparable to the one obtained by the addition of CHOL. The addition of CHOL to ZnPc increased LUV stability. Besides, the
78
C.A.de Oliveira et al. / Chemistry and Physics of Lipids 133 (2005) 69–78
presence of CHOL induces stability to the liposome as a drug delivery system. The addition of PEG 20,000 (0.1%) did not imply in significant size reduction in the preparations. The estimated ZnPc incorporation efficiency is between 89 and 95%. The proposed method is very useful for incorporation of other hydrophobic drugs that can be used as photodynamic therapeutic agents.
Acknowledgements The authors thank the CNPq, FAPEMIG and FAPESP for financial support and fellowships. The Laborat´orio de Microscopia Eletrˆonica (UNESP, Rio Claro/SP and FMRP-USP, Ribeir˜ao Preto/SP), and the Centro de Microscopia Eletrˆonica, Departamento de Morfologia, UFU, are acknowledged fot the use of their experimental infrastructure. Dr. Divinomar Severino is acknowledged for valuable suggestions, and Thiago Padovani Xavier for some physical measurements.
References Anwer, K., Meaney, C., Kao, G., Hussain, N., Shelvin, R., Earls, R.M., Leonard, P., Quezada, A., Rolland, A.P., Sullivan, S.M., 2000. Cationic lipid-based delivery system for systemic cancer gene therapy. Cancer Gene Ther. 7, 1156–1164. Batzri, S., Korn, E.D., 1975. Interaction of phospholipid vesicles with cells – endocytosis and fusion as alternate mechanisms for uptake of lipid-soluble and water-soluble molecules. J. Cell Biol. 66, 621–634. Bali, A., Dhawan, S., Gupta, C.M., 1983. Stability of liposomes in circulation is markedly enhanced by structural modification of their phospholipid component. FEBS Lett. 154, 373–377. Cevce, G., 1993. Lipid properties as a basis for membrane modelling and rational liposome design. In: Gregoriadis, G. (Ed.), Liposome Technology, vol. 1. CRC Press, Boca Raton, pp. 1–36. Darwent, J.R., Douglas, P., Harriman, A., Porter, G., Richoux, M.C., 1982. Metal phthalocyanines and porphyrins as photosensitisers for reduction of water to hydrogen. Coord. Chem. Rev. 44, 83–126. Demel, R.A., De Kruyff, B., 1976. The function of sterols in membranes. Biochim. Biophys. Acta 457, 109–132. De Paula, R., Machado, A.E.H., Miranda, J.A., 2004. 3-Benzoxazol2-yl-7-(N,N-diethylamino)-chromen-2-one as fluorescent probe
for investigation of micellar microenvironments. J. Photochem. Photobiol., A: Chem. 165, 109–114. Franks, N.P., 1976. Structural analysis of hydrated egg lecithin and cholesterol bilayers.1. X-ray-diffraction. J. Mol. Biol. 100, 345–358. Garbo, G.M., Morgan, A.R., 1988. Delivery systems for hydrophobic drugs. J. Photochem. Photobiol. B: Biol. 1, 494–495. Ginevra, F., Biffanti, S., Pagnan, A., Biolo, R., Reddi, E., Jori, G., 1990. Delivery of the tumor photosensitizer zinc(II)phthalocyanine to serum-proteins by different liposomes — studies invitro and invivo. Cancer Lett. 49, 59–65. Johnson, S.M., 1973. Effect of charge and cholesterol on size and thickness of sonicated phospholipid vesicles. Biochim. Biophys. Acta 307, 27–41. Johnson, S.M., 1975. The inability of macrophages to digest liposomes containing a high proportion of cholesterol. Biochem. Soc. Trans. 3, 160–161. Jori, G., Reddi, E., 1990. In: Kessel, D. (Ed.), Photodynamic Therapy of Neoplasic Disease. CRC Press, Boca Raton, pp. 117– 130. Kremer, J.M.H., Esker, M.W.J., Pathmamanoharan, C., Wiersema, P.H., 1977. Vesicles of variable diameter prepared by a modified injection method. Biochemistry 16, 3932–3935. Love, W.G., Amos, N., Kellaway, I.W., Williams, B.D., 1990. Specific accumulation of cholesterol-rich liposomes in the inflammatory tissue of rats with adjuvant arthritis. Ann. Rheum. Dis. 49, 611–614. Lucas 2004. Meyer’s product information: Epikuron 200 SH. Mayhew, E., Lasic, D.D., Babbar, S., Martin, F.J., 1992. Pharmacokinetics and antitumor activity of epirubicin encapsulated in long-circulating liposomes incorporating polyethylene glycolderivatized phospholipids. Int. J. Cancer. 51, 302–315. Moser, J.G., 1998. Photodynamic Tumor Therapy, 2nd and 3rd Generation Photosensitizers, Harwood, The Netherlands. Oku, N., 1999. Anticancer therapy using glucuronate modified longcirculating liposomes. Adv. Drug. Deliv. Rev. 40, 63–73. Ostro, M.J., Cullis, P.R., 1989. Use of liposomes as injectable-drug delivery systems. Am. J. Hosp. Pharm. 46, 1576–1587. Reddi, E., Zhow, C., Biolo, R., Menegaldo, E., Jori, G., 1990. Liposome or LDL-administered Al(III)-phthalocyanine as a photodynamic agent for tumours. I. Pharmacokinetic properties and phototherapeutic efficiency. Br. J. Cancer. 61, 407–411. Ricchelli, F., Gobbo, S., Jori, G., Moreno, G., Vinzens, F., Salet, C., 1993. Photosensitisation of mitochondria by liposome-bound porphyrins. Photochem. Photobiol. 58, 53–62. Ribeiro, J., Xavier, T.P., Koshiyama, D.R., Ferreira, S.K., Severino, D., Miranda, J.A., Machado, A.E.H., 2004. Photophysics of zinc (II) phthalocyanine in homogeneous and microheterogeneous media, unpublished results. Valduga, G., Reddi, E., Jori, G., 1992. Steady-state and time-resolved spectroscopic studies on zinc(II) phthalocyanine in liposomes. J. Photochem. Photobiol. B: Biol. 16, 331–340.