Colloids and Surfaces B: Biointerfaces 49 (2006) 22–30
Pegylated tetraarylporphyrin entrapped in liposomal membranes A possible novel drug-carrier system for photodynamic therapy Mariusz K˛epczy´nski a , Kinga Nawalany a , Barbara Jachimska b , Marek Romek c , Maria Nowakowska a,∗ a Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krak´ ow, Poland Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-239 Krak´ow, Poland c Department of Cytology and Histology, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Krak´ ow, Poland b
Received 22 November 2005; received in revised form 3 February 2006; accepted 16 February 2006 Available online 6 March 2006
Abstract A system of poly(ethylene glycol) bound tetraarylporphyrin entrapped in liposomal membranes was investigated. The interactions between the 5-(4-hydroxymethylphenyl)-10,15,20-tritolylporphyrin (Po) covalently attached to the poly(ethylene glycol) chain (PEG-Po), and phosphatidylcholine liposomes in the aqueous solution were studied. The adsorption of the investigated polymer to lipid vesicles was confirmed by measurements of dynamic light scattering and zeta potential. Experimental results demonstrate that the diameter of liposomes increased and the absolute value of the zeta potential decreased after addition of PEG-Po. The binding constants (Kb ) of Po chromophores to liposome in pH range from 5.2 to 9.0 were determined using fluorescence spectroscopy. The degree of binding was found to be pH-independent and the average value was 24.6 ± 0.9 mg ml−1 . The acid–base properties of the porphyrin chromophores and their aggregation in an aqueous solution were also studied. pK values associated with imine-N protonation of the porphyrin core were found to be 2.59 and 0.68 at the ionic strength of 0.1 M. The equilibrium constant for dimerization, KD , was found to be 5 × 103 M−1 . © 2006 Elsevier B.V. All rights reserved. Keywords: Liposomes; Tetraarylporphyrin; Poly(ethylene glycol); Liposomal binding constant; Drug carrier system
1. Introduction Porphyrins and their analogues are the most commonly administered photosensitizers in the photodynamic therapy (PDT) of malignancies and some other diseases [1]. The ability of these dyes to be incorporated into the cells’ membranes is an important factor for efficient PDT. It is well known that tumors proliferate by receiving a supply of nutrients mainly from the neovasculature, vessels which have an enhanced permeability even to the macromolecules [2]. Therefore, when anticancer drugs conjugated to polymers are administered, they may easily permeate tumor tissues and accumulate in them [3]. Poly(ethylene glycol) (PEG) is a water-soluble polymer that exhibits protein resistance, low toxicity, and non-immunogenicity. For these reasons
∗
Corresponding author. Tel.: +48 12 6632250; fax: +48 12 6340515. E-mail address:
[email protected] (M. Nowakowska).
0927-7765/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2006.02.008
PEG is widely exploited as a polymer support in medical applications. Several PEG-porphyrin conjugations for PDT were developed and studied [4–8]. Lottner et al. [4,5] have proposed a potential photosensitizer for PDT based on hematoporphyrin or tetraarylporphyrin-platinum(II) complex with two or three PEG fragments bound to porphyrin core. Pegylated 5,10,15,20-tetrakis-(m-hydroxyphenyl)chlorine (mTHPC) with different number of PEG chains attached to chlorine ring has been proposed as an efficient photodynamic agent. Hornung et al. [6,7] have used tetrakis(m-methoxypolyethylene glycol) derivative of mTHPC for study of selective targeting in a rat ovarian cancer model. Grahn et al. [8] have proved the in vivo photodynamic activities of mTHPC with 1–3 PEG chains. It is well known that liposomes are widely studied for use in drug delivery systems. The liposomal encapsulation of anticancer drugs significantly alters the biodistribution and pharmacokinetics, resulting in some reduction in toxicities and in improvement of targeting to desired target tissues [9]. Liposomes are spherical vesicles formed by some amphiphilic compounds,
M. K˛epczy´nski et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 22–30
e.g., phosphatidylcholines and ceramides. The lipid bilayer surrounds the aqueous phase; therefore the liposomes may carry both lipophilic and water-soluble substances. Incorporation of lipids with covalently attached polyethylene glycol into liposomal bilayer membranes causes significant stabilization of liposome suspensions, prevents their aggregation and inhibits protein and cellular interactions with liposomes thus considerably prolonging their blood circulation time. These vesicles are known as Stealth liposomes or sterically stabilized liposomes (SSL) and are applied as an effective drug delivery support [10]. Taking into account all the above mentioned findings we have assumed that incorporation of porphyrin covalently attached to PEG polymer chain into liposomal bilayer should provide an effective drug delivery system for PDT. Previous studies by Iida et al. [11] on photophysical properties of PEG-linked free base or manganese complex of the tolyl or pentafluorophenylporphyrins with methylene groups spacer, incorporated into lipid bilayers, demonstrated that the investigated porphyrins were almost completely adsorbed on the lipid bilayer of egg phosphatidylcholine liposomes. In the present paper we report the detailed studies on interactions between 5-(4-hydroxymethylphenyl)-10,15,20tritolylporphyrin (Po) covalently attached to the chain end of water-soluble poly(ethylene glycol) (PEG-Po), and liposomes. We have used several different methods to observe these interactions. A dynamic light scattering method and zeta potential measurements were applied to follow changes in hydrodynamic diameter and the surface charge of liposomes after addition of PEG-Po, respectively. The visualization of vesicles was done with transmission electron microscopy. The fluorescence method was used to quantify the process of Po chromophores binding to the liposomal bilayer. Porphyrin used is a highly hydrophobic tetraarylporphyrin (TArP) dye, therefore it should easily enter into the liposomal membrane non-polar medium. Moreover, covalent attachment of Po to the end of PEG chains gives the sensitizer an amphiphilic character which is essential not only for its solubility in water but also for incorporation of Po into liposomes [12]. PEG-Po is a model of the macromolecular drug, which has been shown to be very effective in vivo studies [6–8]. As a result of PEG-Po incorporation into liposomal dispersion the system can gain a structure similar to that of the Stealth liposomes, thus it should be resistant against the plasma proteins.
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Aldrich Chemical Co. (Milwaukee, WI) at the highest possible purity. Benzaldehyde terminated poly(ethylene glycol)-5-(4hydroxymethylphenyl)-10,15,20-tritolylporphyrin (PEG-Po) with porphyrin chromophores at the end of the polymeric chain was obtained by the method described in literature [13]. The PEG-Po was characterized by UV spectroscopy and GPC chromatography. The number average molecular weight, M n , was determined by GPC to be 8000 g/mol. The content of Po was 0.68 wt.%. 2.2. Absorption and emission spectra UV–vis absorption spectra at 25 ◦ C of the samples were measured using a Hewlett-Packard 8452A diode-array spectrophotometer equipped with a HP 89090A Peltier temperature control accessory. Steady-state fluorescence spectra of the samples were recorded on an SLM-AMINCO 8100 Instruments spectrofluorimeter at room temperature. Emission spectra were corrected for the wavelength dependence of the detector response by using an internal correction function provided by the manufacturer. 2.3. Dynamic light scattering measurements A Malvern Nano ZS light-scattering apparatus (Malvern Instrument Ltd., Worcestershire, UK) was used for dynamic light scattering (DLS) measurements. The Nano ZS instrument incorporates non-invasive backscatter (NIBSTM ) optics. DLS technique measures the time-dependent fluctuations in the intensity of scattered light which occur because the particles are undergoing Brownian motion. Analysis of these intensity fluctuations enables the determination of the diffusion coefficients of the particles which are converted into a size distribution. The diffusion coefficient, D, is calculated from correlation function: g(τ) = 1 + exp(−2Dq2 τ)
(1)
2. Materials and methods
were τ is the sample time and q = (4πn/)sin(θ/2) the scattering vector of light; n the refractive index of the solution; λ the wavelength of the laser beam and θ is the scattering angle. The mean hydrodynamic radius of liposomes, RH , can be calculated from the diffusion coefficient according to the Einstein–Stokes equation:
2.1. Materials
D=
l-␣-Phosphatidylcholine (PC) type XIII-E from egg yolk (99%, solution of 100 mg/ml in ethanol) was obtained from Sigma Chemical Co. (St. Louis, MO). It was a mixture of lipids with the following fatty acid makeup: 33% C16:0 (palmitic), 13% C18:0 (stearic), 31% C18:1 (oleic), and 15% C18:2 (linoleic) (other fatty acids being minor contributors), which gives an average molecular weight of approximately 768 g/mol. Diethyl ether (>99.8%) was obtained from Fluka Chemie (Buchs, Switzerland). All other reagents were received from
were k is the Boltzmann constant, T the absolute temperature, and η is the viscosity of the medium. The time-dependence autocorrelation function of the photocurrent was acquired every 10 s, with 15 acquisitions for each run. The sample of solutions was illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173◦ was measured by an avalanche photodiode. The z-average diameter (dz) and the polydispersity index (PD) of the samples were automatically provided by the instrument using cumulant analysis.
kT 6πηRH
(2)
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M. K˛epczy´nski et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 22–30
2.4. Zeta potential measurements
2.6. Preparation of liposomes
The zeta potential of liposomes was measured with the Malvern Nano ZS using the technique of Laser Doppler Velocimetry (LDV). In this technique, a voltage is applied across a pair of electrodes at either end of the cell containing the particle dispersion. Charged particles are attracted to the oppositely charged electrode and their velocity was measured and expressed in unit field strength as an electrophoretic mobility. The zeta potential was calculated from the electrophoretic mobility, ue , using Henry equation [14]:
Phospholipid vesicles were prepared by extrusion or by sonication. The lipid solution in ethanol (50 l) was placed in a volumetric flask, and the ethanol was evaporated under flow of nitrogen. The lipid film was next dissolved in diethyl ether that was then reevaporated under nitrogen to complete dryness. About 10 mM phosphate buffer was added till a lipid concentration of 2.5 mg/ml was attained, and the sample was sonicated for 5 min at 20 ◦ C in a Bronsonic ultrasonic bath. The resulting suspension was then either extruded using the PPH Marker manual extruder by means of the 100 nm Nucleopore TrackEtch Membrane Whatman filters or sonicated for 10 min at 4 ◦ C with a probe sonicator (Sonics VC 130, Newtown, CT USA). For each pH value, a separate stock solution of liposomes was prepared. dz and PD of the liposomal vesicles vary for different preparation. For liposomes prepared by extrusion dz and PD were in the range of 104–109 nm and 0.025–0.09, respectively.
ue =
2ε ζ f (κa) , 3η
κ−1 =
ε kT 8πe2 I
1/2 (3)
were ζ is the zeta potential, ε the dielectric constant, η the viscosity and f (κa)-the Henry’s function of the dimensionless product of κ and a parameters. The units of κ, termed the Debye length, are reciprocal length and κ−1 is often taken as a measure of the “thickness” of the electrical double layer. The parameter ‘a’ refers to the particle radius. k is the Boltzmann constant, T the absolute temperature, I the ionic strength and e is the elementary charge of electron. For ionic strength of 5 × 10−3 M and for liposomes of diameter 105 nm (the mean diameter for the two types used) κa = 12.3 which corresponds to a Henry’s factor of 1.25 and for ionic strength of 0.1 M, κa = 55.1 giving f(κa) = 1.42.
2.5. Transmission electron microscopy A negative staining technique was used to visualize the liposomes. A 200 mesh copper grid coated Formvar/Carbon film (Pacific Grid-Tech, USA) was dipped in the sample dispersion and left for 20 min and excess of sample was blotted with filter paper. The samples were stained with 1% solution of uranyl acetate in water and then allowed to dry. The dried grids were analyzed in a JOEL 100 SX transmission electron microscope (Japan) at an accelerating voltage of 80 kV. Micrographs were taken at ×35 000 magnification and then they were scanned and processed with CorelDraw 9.0 software (Corel Corporation, Ottawa, Canada).
2.7. Determination of liposome-binding constants A spectroscopic titration technique was used to determine the binding constants (Kb ) of the porphyrin chromophores to lipid vesicles. Details of this technique were described previously [15]. To determine the incubation time required to reach equilibrium, the partitioning kinetics of the porphyrin moieties into liposomes were studied. After each addition of an aliquot of lipid, the system was equilibrated and emission spectra of porphyrin chromophore were recorded. Kb is given in units of mg ml−1 throughout this study. 3. Results and discussion 3.1. Properties of PEG-Po In these studies we used a homopolymer of ethylene glycol with 5-(4 -acryloyloxyphenyl)-10,15,20-tri(p-tolyl)porphyrin (PEG-Po). The method of synthesis used for PEG-Po allows obtaining the polymer required for our studies, which is the polymer with the porphyrin chromophores attached only to
Fig. 1. Chemical structure of PEG-Po polymer.
M. K˛epczy´nski et al. / Colloids and Surfaces B: Biointerfaces 49 (2006) 22–30
one end of the polymer chain. The chemical structure of PEG-Po is shown in Fig. 1. PEG-Po is well-soluble in water and in polar organic solvents such as chloroform, methanol and DMF. The polymer absorbs the light in visible spectral region. Its absorption spectrum is strongly pH dependent (see below). 3.1.1. Acid–base equilibrium of Po chromophores in the aqueous solutions of PEG-Po Protonated (mono- and dicationic) forms of porphyrins have been shown to possess no affinity for lipid membranes [16]. Therefore, the determination of exact values of the dissociation constants of the Po chromophores in our experimental conditions is of great importance for the study. Two sets of acid–base titrations were performed: beginning at the basic pH down to the acidic and backwards. The changes in the systems were followed by the measurements of the absorption spectra. Fig. 2a shows the selected spectra of PEG-Po in the Soret band spectral region of the aqueous solution containing 0.1 M KCl upon titrations. Data presented in Fig. 2b demonstrate that with the lowering of pH the intensity of the band centered at 422 nm decreases with the concomitant appearance of the band at 440 nm. The changes in the spectra reflect the acid–base
Fig. 2. (a) Changes in the absorption spectra of PEG-Po (cPo = 1.4 M in buffered solutions at 0.1 M KCl) as a function of pH. (b) Dependence of the absorbance at 440 nm () and at 422 nm () on pH.
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equilibria inside the porphyrin chromophore. The spectra do not show a consistent isosbestic point and thus more than one protonated porphyrin species must be present between pH 0.6 and 6.7. Two imine nitrogen atoms of a free-base porphyrin are able to attach protons forming mono- and dications. Using generally accepted convention that constants K3 and K4 are associated with monocation-free base and dication–monocation protonations of the imine groups, while the constants K1 and K2 correspond to the deprotonation of amine groups [17], the equilibria can be represented by the following expression: pK4
pK3
H2 Po2+ HPo+ Po
(4)
where H2 Po2+ and HPo+ denote the dicationic porphyrin species (two protons attached to the imino nitrogen atoms in the acidic medium) and the monocationic one, respectively. The dissociation constants are defined as K3 =
[Po][H+ ] , [HPo+ ]
K4 =
[HPo+ ][H+ ] [H2 Po2+ ]
(5)
The total concentration of all the species is expressed as c = [Po] + [HPo+ ] + [H2 Po2+ ]
(6)
By simple mathematical operations we obtained the following expressions for the concentration of the respective species as a function of pH: c [Po] = , β = 1 + 10(pK3 −pH) + 10(pK3 +pK4 −2pH) (7) β c [HPo+ ] = , χ = 1 + 10(pH−pK3 ) + 10(pK4 −pH) (8) χ c [H2 Po2+ ]= , γ = 1 + 10(pH−pK4 ) + 10(2pH−pK3 −pK4 ) (9) γ The constants K3 and K4 and absorption spectra of all three porphyrin forms, i.e., free-base, monocation and dication can be determined by an evolutionary factor analysis [18] with the above-mentioned mathematical model (Eqs. (6)–(9)). The experimentally measured absorption spectra were fitted to the model. The best fit was obtained for pK3 = 2.59 and pK4 = 0.68. The extracted absorption spectra of dicationic, monocationic and neutral forms are shown in Fig. 3. From the obtained values of dissociation constants it can be calculated that at pH higher than 5 all Po chromophores are present in the aqueous solution as the free-base form. The values of pK for several water-soluble TArPs have been determined previously and the influence of the peripheral charges surrounding the protonation sites was observed. Due to the electrostatic stabilization of dications, porphyrins with negatively charged peripheral groups are characterized by pK values higher by about four pH units than the non-ionic one. Therefore, one should compare our values of pK with porphyrins having non-charged or positively charged peripheral substituents. The dissociation constants are also sensitive to the ionic strength of medium and their values increase with an increase in ionic strength. For example, meso-tetrakis(4N-methylpyridyl)porphyrin (TMPyP) with positively charged groups has the values of pK3 and pK4 of 1.4 and
