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* Department of Chemistry, Università di Firenze, Sesto Fiorentino, Italy; Groupe de Dynamique des Phases Condensées, Université de Montpellier II, Montpellier, France; Institut Laue Langevin, Grenoble, France; Department of Chemical and Biosystem Sciences, Università di Siena, Siena, Italy; and || DRECAM/SCM, Lions, Commissariat à l'Énergie Atomique, Saclay, Gif sur Yvette, France
Correspondence: Address reprint requests to Dr. Sandra Ristori, Dept. of Chemistry, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy. Tel.: 39-055-457-3048; Fax: 39-055-457-3385; E-mail: ristori{at}unifi.it.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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; Hawthorne et al., 2001; Soloway et al., 1998; Barth, 2003), especially suited for radio-resistant and highly invasive tumors. It is based on boron accumulation into neoplastic cells and subsequent irradiation of patients with a thermal or epithermal neutron beam. Natural isotopic composition of boron is 20% 10B and 80% 11B. Of these two species, 10B has by far the larger absorption cross section for neutrons, so that the relevant fission process taking place in BNCT can be written as (Sjöland et al., 1997)
 | (1) |
109 nuclei of 10B have to be located inside each tumor cell for the therapy to be effective (Fairchild and Bond, 1985). If boron delivery takes place in the cell nucleus, the number of required 10B nuclei is at least one order-of-magnitude lower (Ryynänen et al., 2000; Braun et al., 2003). Still, huge amounts of boron are needed and much effort is currently being focused on finding appropriate carriers.
Liposomes are generally recognized as efficient, nontoxic drug vectors, able to concentrate the active molecules at delivery sites (Lasic and Papahadjopoulus, 1998). In the specific case of targeting tumor tissues, it has been demonstrated that liposomes of appropriate size (i.e., 100 nm mean diameter) can extravasate through their leaky endothelium and penetrate the defective outer membrane of neoplastic cells (Matsumura and Maeda, 1986; Shelly et al., 1992; Nagayasu et al., 1999). In particular, cationic liposomes may act as suitable carriers in BNCT, because they are known to form stable complexes with DNA, and are widely studied as nonviral vectors for gene therapy purposes (Lasic et al., 1997; Miller, 1998; Safinya, 2001).
Carboranyl-glucosydes have been synthesized to be used in BNCT (Tietze and Bothe, 1998; Giovenzana et al., 1999), and preliminary experiments in vitro have shown enhanced uptake by tumor cells (Tietze et al., 2001), with respect to healthy cells. This is probably due to the fact that sugars are among the building units most required by cells proliferating with abnormal rate. These boronated compounds also possess the favorable property, common to all carboranyl-derivatives, to carry 10 boron atoms per molecule, the chemical formula of the carborane unit being –C2B10H11.
In this work, our first aim was to establish if 1,2-dicarbadodecaborane, made by the icosahedral carborane cage, and two of its sugar derivatives (i.e., the glucosyl and lactosyl functionalized compounds), could be efficiently incorporated into cationic liposomes. To the best of our knowledge, cationic liposomes have not yet been used as carriers for borocompounds in BNCT, notwithstanding their appealing properties. A detailed investigation of the loaded liposome structural features was performed by small angle scattering of x rays (SAXS) and small angle scattering of neutrons (SANS). We found that the insertion of host molecules may have a drastic effect on the organization of the lipids within the bilayer and, consequently, that it can influence the size and shape of liposomes themselves. As mentioned above, both the size of the liposomes, and the possibility of their anchoring to the targeting species in a way that preserves their functionality, are critical requirements for drug delivery into tumor cells. Thus, to obtain the expected biomedical performance, and to avoid many unnecessary trial-and-error tests, the successful tailoring of drug carriers requires extensive physicochemical characterization. In turn, this entails determining the composition, global shape, and internal structure of the boron-loaded vectors.
Small angle scattering techniques are well-established methods for the study of self-assembling structures in a wide range of scale lengths, which is approximately from one nanometer to a few hundred nanometers (Lindner and Zemb, 2002). In particular, the combined use of x rays and neutrons, which rely on different contrast mechanisms, is suited for sugar-containing amphiphiles (Cantù et al., 1998; Zhang et al., 1999; Stradner et al., 2000; Irai et al., 2003). In fact, the polar head's high electron density allows the investigation of the hydrophilic region of the bilayer by x-ray scattering, whereas neutrons are more sensitive to the hydrophobic core (Wiener and White, 1991). From our neutron scattering curves the overall shape and size of liposomes could be calculated as well. Since molecular volumes, layer thicknesses, and scattering length densities are not independent variables (Hayter and Penfold, 1983), the parallel calculation of SAXS and SANS spectra, if performed at the absolute scale, also provides a useful means for obtaining an accurate description of the systems under study (Arleth et al., 1997; Dupuy et al., 1998).
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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1,2-Dicarbadodecaborane, henceforth referred to as orthocarborane (OCB, purity 98%) was purchased from Boron Biological (Mt. Airy, NC) and used as received. Its structure, in the ball-and-stick representation, is reported in Scheme 1.
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4)-ß-D-glucopyranoside (Lactosyl-carborane, LCOB, 4; see below) were a gift of professor Luigi Panza, University of Piemonte Orientale, Italy, and were synthesized as described elsewhere (Giovenzana et al., 1999).
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The three boronated compounds used in this work had the same carborane unit and differed only for the presence of one (or two) sugar ring(s).
Deuterated water (99.95% purity) was purchased from Isotech (Isotech North America, Williston, VT).
Liposome preparation
Liposomes made up by DOTAP and DOPE, in the weight ratio 1:1, were prepared as follows. Multilamellar dispersions were obtained by evaporating the solvent (CHCl3) from a 10–2 mol/dm3 stock solution and adding the appropriate amount of D2O. Monolamellar, monodisperse liposomes were obtained by the well-established procedure (MacDonald et al., 1991) of freeze-and-thaw (eight cycles of plunging in liquid nitrogen, then in a water bath at 50°C, and finally vortexing) followed by extrusion through polycarbonate membranes of 100-nm pore diameter (Liposofast apparatus; Avestin, Ottawa, Ontario, CN) with 27 passages. To prepare the composite systems, the appropriate amount of carborane, dissolved either in CHCl3 (OCB) or methanol (GCOB and LCOB), was added to the lipid chloroform solution before solvent evaporation. This allowed the best mixing among different molecules to obtain co-vesicled samples, i.e., samples in which lipids and carboranes were extruded together. In the case of LCOB, whose water solubility is considerably higher than the two other carboranes used in this work, addition after liposome formation was also possible. Non-co-vesicled samples were then obtained by depositing the LCOB/methanol solution in the bottom of a vial, followed by complete solvent evaporation and, finally, by the addition of the preformed liposome solution.
All samples contained a total lipid concentration of 4.2 10–2 mol/dm3 and were prepared in D2O. Although for x-ray experiment D2O is not necessary, the same systems were used for both SANS and SAXS. This was done to avoid any isotopic effect from the solvent, which would imply different hydrogen bond strength and, therefore, different sugar hydration (Zhang et al., 1999; Söderman and Joanson, 2000). Carborane content was invariably expressed as molar fraction with respect to the lipid+carborane ensemble.
Density measurements
Density values were used to obtain molecular volumes. GCOB and LCOB molecular volumes were not available from the literature and, although several theoretical studies (Hermansson et al., 1999; Türker, 2003; Martin et al., 1996) on crystallographic data (The Cambridge Structural Database; http://www.ccdc.cam.ac.uk/prods/csd/csd.html) could provide structural information on the carborane cage (i.e., OCB), it has to be noted that the volumes reported are extremely scattered, ranging from 70 to 80 Å3 per molecule (Valliant et al., 2002) to 503 Å3 per molecule (Türker, 2003). The reasons for this mismatch are not yet clear.
In the present work, the density of the carboranes was measured starting from diluted solutions in acetone (OCB, GCOB), n-hexane (OCB), or water (LCOB) with an Anton Paar (DMA 5000; Anton Paar, Graz, Austria) densimeter. This apparatus determines the resonant frequency in the oscillation of borosilicate U-tubes filled with the working solution, and thereby calculates the density of the liquid. Typically, four solutions at different concentration were used for each product, and the comparison with the pure solvent allowed us to extract the following solute density: OCB, d = 0.94 ± 0.05 Kg/dm3; GCOB, d = 1.92 ± 0.05 Kg/dm3; and LCOB, d = 1.99 ± 0.05 Kg/dm3.
Molecular volume of carboranes from theoretical calculations
Standard ab initio and density functional calculations were performed on OCB, GCOB, and LCOB isolated molecules by using the Gaussian 98 package (Frisch et al., 1998), implemented on a SGI ORIGIN 3000 workstation. Geometry optimization was obtained with the RHF and B3-LYP methods, and with the 6-31G** basis set. The single bond distances, obtained for the carbon and boron atoms of the cage, were in good agreement with the results of similar calculations carried out by Hermansson et al. (1999).
Excluded volumes were calculated on the optimized structures by the Connolly method (Connolly, 1994), using a molecular probe of 1.4 Å diameter (Chem3D 8.0 software, CambridgeSoft, Chicago, IL). The following volumes were thus obtained: OCB, 148 Å3/molecule; GCOB, 295 Å3/molecule; and LCOB, 423 Å3/molecule, which are to be compared with molecular volumes calculated from the density values of OCB, 255 Å3/molecule; GCOB, 291 Å3/molecule; and LCOB, 415 Å3/molecule.
The agreement is very good for GCOB and LCOB, whereas in the case of the simple carborane cage, the two methods gave different results.
SAXS measurements
Small angle x-ray scattering experiments were performed on a homemade setup (Zemb et al., 2003), using a rotating anode with a copper target as the radiation source. The design of the camera is such that it allows to measure signals as weak as a 10th of that of pure water. Samples were put into 1-mm-thick cells with Nalophan windows (Nalophan BF, Kalle, Wiesbaden, Germany), that gives very weak parasitic scattering. The geometry of this instrument is fixed, and the available q-range is 0.02–0.45 Å–1. Thanks to the highly monochromatic x-ray beam obtained by the optics before the sample, data reduction to the absolute scale can be performed in a simple way by using a semitransparent beam-stopper of calibrated attenuation. Thus, all SAXS spectra were acquired at the absolute scale. The detector used was a two-dimensional gas detector (Gabriel, 1977). Due to a quasipunctual collimation and to the above-mentioned high monochromaticity of the incident beam, the scattering vector resolution 
q was 0.004 Å–1, which did not require any desmearing procedure.
Each spectrum was accumulated during 4 h, to reach a satisfactory signal/noise ratio.
SANS measurements
Small angle neutron scattering experiments were performed on the research nuclear reactor ORPHEE, using the PAXY instrument of the Laboratoire Léon Brillouin (Saclay, France). To cover a wide q-range, three different configurations were used. To reach low q-values, the selected neutron wavelength 
was 12 Å and the sample-detector distance D was set to 5 m. The intermediate q-value configuration was = 4 Å and D = 5 m, whereas the large q-range was obtained by using = 4 Å and D = 1 m. The overall q-range obtained for the three configurations was 0.0047–0.55 Å–1. Neutron collimation before the sample was defined by three different diaphragms and tuned when changing the sample-detector distance. The detector used was a two-dimensional BF3-gas detector. Finally, note that the size of the detector pixel was constant and that all spectral points were taken into account to build the apparatus function R(q,q), which was used in the fitting procedure, as described below. Sample transmission was measured for all three configurations used in this work, and this allowed an original, in situ determination of the boron content, due to the high neutron absorption cross section of 10B, as will be explained later in the text.
The flux of the direct beam was measured using a calibrated plexisheet as the attenuator for data reduction to the absolute scale. The absolute signal of H2O was found at the correct level in the high q-range. The lower q-range was calibrated by overlapping the signal with the spectrum obtained in the intermediate q-range.
As described above, all liposome solutions were prepared in deuterated water, but the starting products contained 1H atoms (2% of the total hydrogen), and their exact amount had to be determined in order to subtract the correct value of incoherent scattering from the raw data. This calculation was performed by comparing the experimental spectra with the signal scattered by mixtures of H2O-D2O in the weight ratio 0:100 to 3:97. As shown in Lindner and Zemb (2002), the flat background arising from incoherent scattering is able to appreciably affect the signal only in the intermediate and large q-range, where the coherent signal tends to be 0.
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DATA TREATMENT AND ANALYSIS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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h,e) is higher than the scattering length density of water (s,e) and, in turn, the latter is above the value of the hydrophobic core (c,e). Due to this subtle balance, the intensity scattered at low q is quite weak and a low background setup, such as ours, is required. The q-range accessible on this SAXS apparatus (0.02–0.45 A–1) allowed us to probe the inner structure of the bilayers, but the outer size and shape of liposomes were not accessible. Thus, the scattered intensity in the q-range of interest can be written as (Cantù et al., 1998)
 | (2) |
is the surface extension of the bilayers per unit volume of solution (note that with the chosen definitions, each side of the bilayer contributes separately to ).
The amount of bilayer surface was calculated knowing the weight fraction of lipids and carboranes in solution. The density of mixed bilayers was calculated from the values obtained by density measurements, and taking 0.92 Kg/dm3 for the lipid mixture. Data were fitted at the absolute scale by using Eq. 2, averaged over polydispersity in the thicknesses tc. A Gaussian distribution (having half-height width 
tc) was used. Thus, fitting to the experimental data was done by varying the five parameters of tc; th; tc; c,e; and h,e. The step in tc,h was 0.4 Å, whereas the step in tc was 0.2 Å.
Agreement between the model and the experimental points was estimated by a 
2 test:
 | (3) |
2 on the logarithm of the intensity considerably improved the data fitting at high q where the intensity was low. The best-fit values are reported in Table 1.
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h,e, c,e and h,n, c,n for x rays and neutrons, respectively) have to be calculated with the same molecular volume for each bilayer part and with the same hydration rate for the hydrophilic portion. Finally, note that the fitting procedure in the case of neutron scattering curves mostly involved the thickness of the hydrophobic layer tc, since the scattering length density of the hydrated polar head region was very close to the solvent value. However, SANS fittings still allowed us to determine both the form and shape of liposomes, as discussed in the next section.
Method for SANS fitting at the absolute scale
For spherically symmetric vesicles, the scattering cross section per unit volume V can be written as
 | (4) |
/Vbilayer, where is the volume fraction of lipids in the sample and Vbilayer the total bilayer volume of a vesicle. Scattering from our dilute vesicle solutions did not exhibit any correlation peak; therefore, the structure factor S(q) could be assumed to be 1 in the whole q-range investigated.
The form factor of three concentric shells with radii Ri and length scattering densities i, i = 1,3 (i = 1 being the external layer) is given by Pedersen (1997) as
 | (5) |
 | (6) |
 | (7) |
The general fitting method was as follows: first, SAXS data were simulated using Eq. 2, and this gave the best-fittings values reported in Table 1. Second, the amount of water hydrating the polar heads was deduced, following the procedure described in Results and Discussion, below. Third, from the partial molar volume and deduced composition of each part of the bilayer (including the number of water molecules bound to each polar head), the neutron scattering length densities of both the heads (h,n) and the hydrophobic core (c,n) were calculated.
Note that for the liposomes studied in this work, a one-shell model gave fits of equivalent quality to those obtained with the proposed three-shell model. In fact, the polar head thickness was small compared to the thickness of the hydrophobic core and differences between the two models showed up only at large q (>0.2 Å–1), that is, in a domain where it was difficult to extract the scattered signal from the incoherent background. Nevertheless, we chose to use the three-shell model to be consistent with the x-ray data analysis.
The SANS experimental intensity suffers from a smearing of Eq. 4 by the size distribution of scattering objects and by the instrumental resolution. The form and relative weight of these two contributions are detailed elsewhere (Grillo, 2000) and the final equation we chose for our modeling procedure can be written as
 | (8) |
q,q') is represented as a Gaussian function and the polydispersity G(Ri,
*,r') is characterized by a log-normal distribution with a full width at half-maximum of 2R0*. The same 2 test was used to estimate the quality of the fitting, as explained in Method for SAXS Fitting at the Absolute Scale, above.
The differences in instrumental resolution between the configurations used to cover the broad q-range were constrained to separately fit the small angles (4.0 10–3 <q (Å–1) <0.09) and the large angles (q (Å–1) >0.09) data. The range of small q allowed us to extract the radius of liposomes and their polydispersity in size. The thicknesses of the different layers that constitute the liposome membrane were obtained from the fitting of SAXS data, as mentioned before. The only unknown parameters were Ri (the inner radius), and . The program varied Ri by 0.1 Å steps and * by 0.05 steps between given minimal and maximal approximate values. The value 2 was evaluated for each iteration and the parameters giving the smallest 2 were retained. We estimated the relative error on the radius Ri as 5%.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Carborane insertion into lipid bilayers is expected to take place easily, because of the well-known lipophilic character of the carborane cage (Escher et al., 1980; Pli
ka et al., 1981). Indeed, several authors have suggested using the –C2B10H11 unit to increase the penetration of drugs inside the cell membrane, without any reference to boron-related therapies (Yamamoto and Endo, 2001; Endo et al., 2001). Moreover, the choice of using dioleoyl hydrocarbon chains allowed us to work with fluid bilayers, since for this kind of lipid the L
Lß transition is known to occur below 0°C (Cevc, 1993). In principle, this should favor local deformations to accommodate host molecules, even though the latter have a completely different shape, as in the case of the icosahedral carborane cage.
In this work, boron concentration in carborane-containing liposomes was calculated from neutron transmission values, taking into account that, in the thermal range of energy (E 
0.3 eV), the absorption of neutrons by 10B can be totally attributed to the fission reaction (Eq. 1). This assumption does not bring any relevant limitation to analytical methods, and it has been adopted for boron determination in chromatography (Kvítek et al., 1975), where high accuracy is required. Solid-state neutron detectors based on carboranes have already been proposed (Robertson et al., 2002), and have been shown to work with high sensitivity, even when the starting carboranes are not isotopically enriched with 10B.
Nevertheless, up to now, the transmission method for quantitative determination of boron (Szegedi et al., 1990) has rarely been used.
Transmission can be written as
 | (9) |
a is the neutron absorption cross section. Transmission was measured for all the samples in each experimental configuration and normalized with respect to pure liposomes, dividing by their transmission value in the same experimental conditions. This could be done because lipid content was kept constant in all the preparations. The obtained values thus contained only the absorption due to glycosyl carboranes, which are made of C, H, O, and B atoms. Neutron absorption cross sections for all the isotopes of these elements are at least 3–4 orders-of-magnitude lower than the 10B value (see tables reported in Neutron News, 1992, 3:29–37); therefore, they were neglected. This approximation, though fully justified a priori by tabulated cross-sections and isotopic compositions of the involved atomic species, was also validated a posteriori by the results obtained in our calculation, as shown in the following.
In the thermal range, a is inversely proportional to the neutron speed—that is, to the square-root of the energy (E = 1/2 mv2), and the proportionality constant for 10B is 611 (Hughes and Schwartz, 1958). The neutron speed v was calculated from the associated wavelength (v = 
/m, where m is the neutron mass and the Plank's constant), thus obtaining v = 985.1 m/s (a = 8.586 10–12 m2) for = 4.03 Å, and v = 330.8 m/s (a = 2.557 10–12 m2) for = 12.0 Å.
The cell thickness d was 2 mm. Assuming that all concentrations in the final samples were the same as those in the starting solution— that is, before liposome extrusion—all quantities in Eq. 9 were known and the absorption coefficient µ = na a could be calculated.
Fig. 1 a shows the experimental absorption coefficients of GCOB-loaded liposomes and the corresponding theoretical lines as a function of carborane content. The higher error reported for = 12.0 Å was a consequence of the lower flux obtainable at low neutron energy in the PAXY beam line.
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However, in the case of the simple icosahedral cage (OCB), only a partial incorporation could be obtained, as shown in Fig. 1 b. This meant that the presence of the polar head played an important role in lipid-carborane mixing, and that dipole-dipole interactions added a favorable contribution. Indeed, homogeneous mixing of a sugar-based amphiphile with a tetraalkylammonium double-chain surfactant has been reported in the literature up to a high glycolipid content (molar fraction 0.75) (Ricoul et al., 1998).
Structure of the liposomes
The five types of liposomes that were examined are presented in a sequence, going from pure liposomes to the more complex systems.
Liposomes without carborane addition
The SAXS and SANS scattering diagrams of plain DOTAP/DOPE liposomes are shown in Figs. 2 and 3, respectively, together with the corresponding best fits, which were obtained using the physical characteristics listed in Table 1.
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). Moreover, the oscillations in the SAXS diagrams were marked, showing that the bilayer thickness was well defined. This could be quantified by the small width of the distribution thicknesses extracted from the fitting procedure, which is <7% for the total bilayer. Secondly, from the SANS fitting we found that DOTAP/DOPE liposomes were actually spherical objects, and that their size could be determined from the oscillation at low q. The obtained value for the outer radius was 445 Å and the width of its log-normal distribution was fairly low ( = 0.14).
More details on the structure of liposomes could be gained by performing a deeper analysis of SAXS data. In fact, from the mass density dm (kg/m3) of the bilayer, a total molecular volume vm (Å3) of the DOPE/DOTAP mixed surfactant could be calculated as
 | (10) |
 | (11) |
 | (12) |
 | (13) |
Using Eqs. 10 and 11, we slightly adjusted dm to get the correct scattering length density of the hydrophobic core. Then, from Eq. 12, the number of water molecules n per hydrophilic head was obtained, by matching the calculated value and measured value of the hydrophilic scattering length density h,e. Finally, the resulting hydrophilic thickness was deduced from Eq. 13. The best agreement was obtained using dm = 0.965 kg/m3. The results of this calculation are reported in Table 2.
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On the other hand, the SAXS diagrams corresponding to the four different OCB/liposomes systems at different loading ratios were very close to one another, as shown in Fig. 5, where they are plotted together with the pure liposome spectrum.
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c,e of the bilayer hydrophobic region to an appreciable extent. In fact, when the carborane cage bearing the ZOCB = 74 electron is inserted in the oleoylic part, an additional volume vin,c is to be considered to obtain the new scattering length density of
 | (14) |
c,e = 7.80 1010 cm–2 leads to an insertion volume of 185 A3 per molecule. This is an intermediate value between the volume obtained from ab initio calculations and the volume deduced from density measurements. As the OCB molecule is inserted in the lipophilic core, the volume it occupies inside the bilayer can be minimized by a rearrangement of the hydrocarbon chains around it, which are in a fluid state. There is no constraint on the location of the OCB molecule within the hydrophobic core. This no longer holds for the two other cases (GCOB and LCOB). Accordingly, the insertion volume obtained from the simulation of SAXS spectra (volume of the cage insertion) may represent a minimum value.
Addition of glucosyl carborane to DOTAP/DOPE liposomes
The SAXS scattering diagrams of liposomes containing glucosyl carborane are reported in Fig. 6, together with the best-fits obtained using the parameters listed in Table 1.
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The fitting procedure was guided by the following a priori: neutron absorption measurements have shown that all the GCOB molecules contained in the initial mixture were also present in the liposome dispersion after extrusion. Considering that GCOB solubility in water is very low (<2.0 10–4 mol/l) and that these molecules do not form micelles in water solution, it was assumed that GCOB molecules were all inserted in the bilayers. Taking the density of pure GCOB as measured from the acetone solutions (d = 1.92 ± 0.05 g/cm3), the density of each GCOB/liposome sample was calculated, and the obtained values are reported in the first column of Table 1.
From the above experimental data and scattering curve simulation, several conclusions could be drawn. First, upon GCOB addition, a small but appreciable increase in the thickness of the hydrophilic bilayer region (th) was observed. The SAXS scattering length was higher than in pure liposomes, but it remained substantially constant when GCOB content was increased.
Considering that neutron absorption data ensure that full uptake has occurred, we conclude that the hydrated sugar heads of GCOB were inserted in the hydrophilic region of the liposome bilayer, without extra protrusion. This was due to the small size of the glucosyl group, which is able to fit into the 5.5 Å of the hydrophilic layer up to very high loading without a remarkable thickness variation.
Such speculation could be made more quantitative using the procedure outlined above. Again, we assumed that the carborane cage was accommodated by the oleoylic chains in the bilayer. Nevertheless, because it was anchored to an hydrophilic head, its location was forced close to the interface between the hydrophilic and hydrophobic parts. Therefore, the effective volume vin,c required within the oleoyl chains could be different (and certainly greater) than in the pure OCB insertion. Again, Eq. 14 was used to derive the insertion volume of the carborane cage attached to a sugar head. The results are reported in Table 2. Obviously, they were slightly larger with respect to the case of OCB, when the cage is alone, but the volume depended only slightly on the rate X of insertion.
The same type of analysis could be done for the hydrophilic part where the sugar head was located. It gave ZG = 95 electrons in an insertion volume of vin,h. The three types of heads are hydrated by n water molecules each, on average. This lead to a scattering length density h,e equal to
 | (15) |
vin,h or n. The insertion volume of the sugar head was considered constant and equal to its value, and deduced as follows. The density measurement showed that the GCOB molecular volume was equal to 291 Å3. The OCB molecular volume determined from density measurement was 255 Å3. One solution would be to take 36 Å3 as the insertion molecular volume for the sugar head, but this value was obviously too small. Nevertheless, both theoretical simulation and SAXS fitting yielded a lower value for the insertion volume of the orthocarborane (150 Å3 and 185 Å3, respectively). The latter value was chosen because it came from an experimental measurement. Therefore, the insertion volume occupied by one sugar head in the hydrophilic part was taken equal to 106 Å3.
Finally, depending on the insertion rate, the hydrophilic thicknesses are then deduced by
 | (16) |
At a larger scale, the effect on the size of the vesicle was more subtle. Indeed, for low GCOB loading, a small decrease of the outer radius was observed, whereas for a higher loading ratio the size of the vesicles slightly increased (from 44 nm to 50 nm, in the X = 0.5 system). Although a discussion of the observed vesicle sizes is not within the aims of this article, a possible explanation for the nonmonotonic evolution of the vesicle radius with carborane content is offered as follows. The final size is an equilibrium (possibly metastable) between forces favoring small vesicles (e.g., those exerted during extrusion, entropy of mixing, etc.) and the bending rigidity of the bilayer (see Helfrich's expression, in Helfrich, 1978). It has been shown that the introduction of another species with different spontaneous curvature in the bilayer (Safran, 2002; Porte and Appell, 1981) gives an additional degree of freedom for the exchange between the two monolayers. Hydration forces can also play a key role in determining the bilayer rigidity and final curvature (McIntosh, 1996). This allows the bilayer to readjust more easily to outer constraints. At high carborane content, such an effect is apparently compensated by the cage rigidifying the bilayer and by crowding of the sugar heads on the surfaces.
Finally, the most important result is that DOTAP/DOPE vesicles were not deformed or disrupted, but retained their spherical shape even at high GCOB-loading ratios (one carborane molecule to each lipid molecule). This observation allows us to state that DOTAP/DOPE liposomes containing glucosyl carborane are good candidates as boronating agents for BNCT.
Addition of lactosyl carborane to DOTAP/DOPE liposomes (co-vesicled system)
The SAXS scattering diagrams of liposomes containing co-vesicled lactosyl carborane are reported in Fig. 7. These curves were simulated with the best-fit parameters listed in Table 1.
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h,e remained almost constant and even slightly decreased from 1.32 1011 cm–2 for XLCOB = 0 to 1.28 1011 cm–2 in for XLCOB = 0.5. As in the GCOB case, due to the cage insertion, the electronic density of the hydrophobic core slightly increased, from 7.6 1010 cm–2 to 7.8 1010 cm–2. Furthermore, a measurable decrease of the hydrophobic thickness was observed at high loading (tc = 24 Å for X = 0.5). Imposing a numerical agreement between the calculated and measured scattering length density of both the hydrophobic and hydrophilic parts allowed us to determine the insertion volume of the carborane cage vin,h anchored to a double sugar head, the lactosyl head insertion volume vin,h, the number of water molecules n per head, and the thicknesses of the hydrophilic part th. The results are all reported in Table 2 and again, the internal consistence among different data was extremely good. It can be observed that the insertion volume of the sugar-anchored carborane cage was higher than for the pure carborane cage. Nevertheless, the volume was not higher than in the GCOB case and the only further adaptation that was imposed by the double sugar head was a lower thickness of the hydrophobic core. This could mean that the arrangement of the oleylic chain around the carborane cages was not exactly the same in the two cases (GCOB and LCOB). The double sugar could not fit the pure liposome hydrophilic layer and therefore the polar head was protruding into water beyond the moieties of the phospholipid heads. The lactosyl units also brought a large amount of water with them. This is indeed a positive feature for medical application of liposomes loaded with LCOB, because the presence of sugar on the outer surface is thought to enhance the uptake by tumor cells. SANS spectra simulations, performed with the best-fit parameters reported in Table 1, showed that the outer radius of liposomes containing LCOB increased monotonically with increasing carborane content, although being smaller than the radius of pure liposomes, except for the highest molar fraction (XLCOB = 0.50). This behavior was in agreement with the case of GCOB-containing liposomes, and was therefore explained by the same arguments mentioned in the previous section.
Moreover, polydispersity, which was invariably low and close to the value of pure liposomes, strongly pushed toward substantial retention of the spherical shape after lactosyl carborane insertion. No major stress and deformations took place in the host structure, which showed itself, once again, to be stable and flexible enough to accommodate large quantities of guest sugar-carborane molecules. For the highest insertion rate, the total number of boron atoms present in the liposome could be deduced by
 | (17) |
The above calculation indicates that, in the case where this type of carrier is able to localize close to the cell nucleus, just 100–200 of them would be enough to destroy a malignant cell through neutron capture.
Addition of lactosyl carborane after liposome formation (not co-vesicled systems)
The presence of all LCOB molecules in the samples was assessed by neutron transmission experiments. The SAXS and SANS curves, of liposomes containing LCOB added after extrusion, were not exactly the same as the corresponding systems where LCOB had been added before liposome formation. An example of this situation is given in Fig. 8, where the SAXS diagrams of the two XLCOB = 0.5 systems are reported, together with the corresponding simulations.
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It was clear that sugar heads were protruding beyond the heads of the phospholipids and that the orthocarborane cage was also inserted in the hydrophobic region, in the case of carborane added to preformed liposomes. Therefore the structure of the bilayer was close to that obtained with co-extrusion. Finally, the outer radius of liposomes, determined from SANS curve simulations, increased with increasing LCOB content (as happened in co-vesicled samples), but its value was invariably lower than in the case of covesicled sample.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Of the three carboranes investigated, only OCB—which does not possess a polar part—showed incomplete loading, although the maximum value reached (approximately one carborane molecule to each three lipids) should not be considered a poor insertion rate.
Glucosyl carborane and lactosyl carborane were fully uptaken by DOTAP/DOPE liposomes to a high molecular fraction (x = 0.5; that is, one carborane molecule for each lipid). Extensive data treatment, based on the simultaneous fitting of the SAXS and SANS curves at the absolute scale, showed that the sugar moieties were included in the polar head layer of liposomes. In the case of LCOB, definite protrusion of the lactose double-ring was observed and the corresponding protrusion length was calculated.
Some differences were found in the composite structures according to different loading procedures, but in no case were the original liposomes disrupted. On the contrary, a substantial retention of their global size and shape was demonstrated.
The results obtained in this work indicate that DOTAP/DOPE liposomes, loaded with sugar-derived carboranes, can be considered potent boron carriers for BNCT purposes. In fact, it was calculated that only 100–200 of the loaded liposomes need to enter a tumor cell to reach the minimum number of boron atoms necessary for the therapy.
Thus, considering the targeting effect toward the nucleus, brought about by cationic lipids and the availability of the sugar moieties on the liposome surface, the potential favorable properties of this loaded vectors were demonstrated.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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A special acknowledgment is given to Dr. Thomas Zemb (Commissariat à l'Énergie Atomique, Saclay, France) for his valuable input and for providing the opportunity to develop this research project.
Submitted on July 15, 2004; accepted for publication October 7, 2004.
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSDATA TREATMENT AND ANALYSISRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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