Carborane Derivatives Loaded into Liposomes as Efficient Delivery

Oct 30, 2009 - liposomes as carriers for BNCT active compounds. Two carborane .... molar fraction, % ζ potential,a mV size,b nm. PIc. Cationic Liposo...
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J. Med. Chem. 2009, 52, 7829–7835 7829 DOI: 10.1021/jm900763b

Carborane Derivatives Loaded into Liposomes as Efficient Delivery Systems for Boron Neutron Capture Therapy† )

)

S. Altieri,‡,§ M. Balzi, S. Bortolussi,‡,§ P. Bruschi,‡ L. Ciani,^ A. M. Clerici,# P. Faraoni, C. Ferrari,# M. A. Gadan,‡, L. Panza,¥ D. Pietrangeli,[ G. Ricciardi,[ and S. Ristori*,^ Department of Nuclear and Theoretical Physics, University of Pavia and INFN, Pavia, Italy, §National Institute For Nuclear Physics (INFN), Section of Pavia, Italy, Department of Clinical Physiopathology, University of Florence, Florence, Italy, ^Department of Chemistry and CSGI, University of Florence, Florence, Italy, #Department of Surgery, University of Pavia, Pavia, Italy, National Commission for Atomic Energy (CNEA), Buenos Aires, Argentina, ¥DISCAFF, University of Oriental Piemonte, Novara, Italy, and [Department of Chemistry, University of Basilicata, Potenza, Italy )

‡

Received May 30, 2009

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of 10B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl β-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H2PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by R spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of 10B by at least 30 times more than that achieved by BPA.

† Contributed to mark the 100th anniversary of the Division of Medicinal Chemistry of the American Chemical Society. *To whom correspondence should be addressed. Phone: þ39-0554573048. Fax: þ39-055-4573036. E-mail: [email protected]. a Abbreviations: BNCT, boron neutron capture therapy; LCOB, oclosocarboranyl β-lactoside; H2PzCOB, 1-methyl-o-closocarboranyl-2hexylthioporphyrazine; BPA, borophenylalanine; LET, linear energy transfer; BSH, sodium borocaptate; DHD, DHD/K12/TRb; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOTAP, 1,2-dioleoyl-3-trimethylammoniumpropane (chloride salt); DOPA, 1,2-dioleoyl-sn-glycero-3phosphate (monosodium salt); PI, polydispersity index.

It represents a valuable option in the treatment of tumors that cannot be removed surgically or when the conventional therapies are ineffective. This is the case, for instance, with diffuse metastases: if the entire organ affected by metastases is irradiated after boron accumulation, all the tumor nodules and single cells are hit, without the need to know their precise number and distribution. The selectivity of BNCT is its main characteristic, and it depends critically on the possibility of obtaining a high ratio of the boron concentration in tumor cells to that in normal ones. Provided a sufficient amount of boron is accumulated in the tumor and a good concentration ratio is attained, the selectivity of the radiation dose delivered does not depend on the neutron irradiation field. To incorporate 10B atoms selectively inside cancer cells, different substances have been studied and employed.11 To date, however, only two boronated compounds have been approved for BNCT clinical practice: BPA (boronophenylalanine fructose)12 and BSH (sodium borocaptate).13 The former, in particular, has been shown to accumulate inside the cell nuclei, allowing higher efficiency in cell destruction. BSH is preferentially used in clinical trials for the treatment of cerebral tumors because it is able to pass the blood-brain barrier. Nowadays, BNCT potentialities are exploited in the treatment of an increasing number of pathologies. One major effort is focused on developing new boron carriers to obtain higher ratios of boron concentration between tumor and normal tissues, in comparison to what BPA and BSH can provide. For example, studies on animals and patients with

r 2009 American Chemical Society

Published on Web 10/30/2009

Introduction a

Boron neutron capture therapy (BNCT ) is an experimental form of binary radiotherapy1 based on the enrichment of tumor cells with 10B nuclei and on subsequent irradiation of the target with a thermal neutron beam. In fact, 10B has a large absorption cross section for thermal neutrons (3837 b). The products of the ensuing capture reaction, 10

B þ n f ½11 B f 7 Li þ R þ γ

(1Þ

are two high LET (linear energy transfer) particles, i.e., 7Li and R, both able to induce DNA damage in the cells where boron atoms are located. The range of R particles and lithium ions in tissues is comparable to a cell diameter. This ensures that irradiation can affect only those cells containing sufficient amounts of boron, while the surrounding cells are spared. BNCT has been applied all over the world to treat different neoplasias, such as glioblastoma, skin melanoma, head and neck cancer, mesothelioma, and diffuse liver metastases.2-10

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liver metastases treated with BPA showed a maximum ratio of about 6 between tumor and liver.2-14 If this ratio could be increased, a therapeutic radiation dose would be absorbed by the tumor, with minimal side effects for the normal tissues. In other words, by improving the selectivity of cell targeting, BNCT could be a safe and precise therapeutic strategy for tumors that are not curable with available techniques. This work presents the results obtained with two recently synthesized boron compounds that were loaded into liposomes and incubated with two different cell cultures, DHD/ K12/TRb (DHD) rat colon carcinoma and B16-F10 murine melanoma. The boron compounds employed were both carborane derivatives, namely, o-closocarboranyl β-lactoside (LCOB) and 1-methyl-o-carboranyl-2-hexylthioporphyrazine (H2PzCOB). The presence of a carborane (C2B10H12) unit in compounds to be used as BNCT agents is a desirable property because it is compatible with biomedical systems15,16 and it carries 10 boron atoms per molecule. Liposomes are among the most accredited drug carriers and, currently, the only one approved for clinical trials. They have proven to be suitable delivery agents for several boron compounds and thus have a strong potential for BNCT applications.17-19 It is generally accepted that optimizing biomedical applications requires extensive physicochemical characterization at the molecular and meso-scale level. Factors like overall size, surface charge, and drug insertion modality are of primary importance in understanding the interaction between loaded vectors and cellular or tissue targets. The liposomes used in this work were made by different lipid components, i.e., the positive DOTAP, the zwitterionic DOPC, and the negative DOPA, to obtain particles with varying surface charge. In addition to this, all liposomes contained the zwitterionic phospholipid DOPE as a fusogenic element.20 It has been shown in a previous study21 that DHD cells have a great capacity for incorporating BPA. Results suggest that the intracellular boron uptake is mediated by an active transport mechanism. It is thought to depend on the concentration used for the treatment, being only slightly dependent on the incubation period and showing a mild increase with increasing contact times. Under optimal conditions, the final boron concentration in cells is about 3 times the starting value used for the treatment. Compared to other kinds of tumor, melanomas are highly resistant to irradiation. Radiotherapy from conventional sources is rarely useful, especially for metastatic tumors. However, BNCT has shown considerable efficiency controlling this disease.22,23 In this context, B16-F10 melanoma cells form metastases in the lung of syngeneic rats, providing a good model of metastatic melanoma, and are thus a relevant system for studying boron delivery.24 In this work, boron uptake by tumor cells was determined by measuring R particle emission with the R spectrometry technique. Cells were deposited on Mylar disks and irradiated in the thermal column of the TRIGA Mark II reactor (University of Pavia). The 10B concentration was measured by analyzing the spectra of charged particles emitted in reaction 1, recorded on a thin silicon detector. The 10B concentration in DHD and B16-F10 cells incubated with liposomes containing LCOB and H2PzCOB was higher than the corresponding starting value and much higher than the 10B concentration obtained by BPA administration

Altieri et al. Table 1. Physicochemical Properties of Different Liposome Formulations ζ potential,a mV

B compd

molar fraction, %

LCOB H2PzCOB

0 25 0.70

LCOB H2PzCOB

Zwitterionic Liposomes 0 -20 25 -25 0.09 -14

LCOB H2PzCOB

0 25 0.13

a

Cationic Liposomes 45 50 56

Anionic Liposomes -60 -58 -52

size,b nm

PIc

140 150 240

0.12 0.15 0.32

150 160 300

0.12 0.15 0.58

140 150 450

0.13 0.16 0.49

Error ( 8 mV. b Error ( 5%. c (5%.

to the same cell lines. Enhanced boron accumulation in tumor cells and improved performance compared to a standard boronated molecule represent the first encouraging step for the establishment of new boron compounds and carriers that will be potentially much more effective than conventional agents in BNCT. Results and Discussion The main physicochemical properties of plain and carborane-loaded liposomes are given in Table 1 for typical molar fractions of LCOB and H2PzCOB. The surface charge (ζ potential) was dictated mainly by the lipid composition. Liposomes containing DOTAP or DOPA had a highly positive or negative charge, respectively, while the zwitterionic formulation (DOPC/DOPE) had an intermediate negative value. This was in agreement with observations reported in the literature25,26 It has been reported that aggregates of zwitterionic lipids are slightly negatively charged because they expose their phosphate groups to the outer surface or tend to absorb HO- ions from the aqueous medium. The inclusion of LCOB or H2PzCOB induced only small variations in surface charge values, consistent with the fact that both of these molecules are neutral. The plasma membranes of mammalian cells are usually negatively charged; thus, we believe that the carrier surface charge can play an important role in the process of drug delivery. The size of liposomes depended on the preparation method (extrusion or sonication) but also on the guest compound. In fact, while LCOB-loaded liposomes were generally stable for days or weeks, depending on the specific formulations, H2PzCOB-loaded liposomes had a tendency to grow and aggregate, as well as to release the boron compound within a few days. Therefore, aliquots of samples containing H2PzCOB were incubated with cells and characterized or analyzed for 10B concentration within three days of preparation. The values of the polydispersity index (PI) showed that the size distribution in plain liposomes was narrow and that this property was not altered much by LCOB insertion. On the other hand, liposomes loaded with H2PzCOB had a broader size distribution. The results of boron incorporation into the DHD and B16F10 cell lines are shown in Figure 1 for the three different liposome formulations loaded with LCOB (x = 25%) and for the corresponding solution of LCOB in the culture medium. The uptake ratio was calculated by normalizing the value of

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Table 2. Physicochemical Properties of Cationic Liposomes Loaded with Different Amounts of LOCB and H2PzCOBa molar fraction, %

0 15 25 50 0.70 1.00 1.65 1.80

ζ potential,b mV

size,c nm

PId

Liposomes B Compound: LCOB 45 48 50 56

140 140 150 150

0.12 0.14 0.15 0.15

B Compound: H2PzCOB 56 60 58 58

240 240 250 240

0.32 0.32 0.35 0.33

Figure 1. 10B uptake ratio of different liposome formulations loaded with LCOB at a mole fraction of 25%. The concentration of free or liposome-encapsulated LCOB was 9.25  10-5 M in the culture medium.

a Samples with 25% LCOB and 0.7% H2PzCOB were the same as those in Table 1. b Error ( 8 mV. c Error ( 5%. d (5%.

Figure 2. 10B uptake ratio of different liposomes loaded with H2PzCOB in DHD cells.

Figure 3. 10B uptake ratio of cationic liposomes loaded with LCOB at different molar fractions.

10

B ppm, obtained by neutron irradiation to the boron concentration in the incubation medium. It is evident that cationic liposomes are by far the most efficient delivery system for both types of cell lines, although remarkable boron accumulation was obtained by using LCOB alone. This could be due to the recognition mechanism exerted by the sugar unit of LCOB toward the membrane of DHD cells. By contrast, this uptake was not observed in melanoma cells. Figure 2 shows the uptake ratio obtained with H2PzCOBloaded liposomes in DHD cells. Enhanced efficacy was evidenced for the cationic formulation, as in the case of LCOB. The corresponding reference system (carborane in culture medium without liposomes) could not be prepared because H2PzCOB is not soluble in aqueous solution. Indeed, the uptake value obtained with this boron compound was significantly better than LCOB in the same cell line. This means that, notwithstanding the above-mentioned problems of liposome growth and decreased stability, liposomal H2PzCOB was able to increase the absolute value of boron uptake with respect to LCOB. By use of BPA as a boron carrier under comparable conditions (starting concentration of 4-7 ppm), uptake ratios of 0.07 and 0.2 were obtained for DHD and B16-F10 cells, respectively. To optimize the performance of cationic liposomes, we investigated in the uptake of DOTAP/DOPC/DOPE liposomes containing different amounts of boron compounds. Table 2 shows the physicochemical characteristics of cationic liposomes containing LCOB and H2PzCOB, prepared at different molar fractions of carborane derivatives.

As observed for the systems reported in Table 1, the incorporation of LCOB even at a very large mole fraction did not alter dramatically the liposome size or surface charge. A larger variation in size, but not in surface charge, was observed for H2PzCOB-loaded liposomes with respect to the empty vectors (the diameters were 240-250 nm instead of 140 nm). This difference was mainly attributed to the method of preparation. LCOB has a very favorable compatibility with liposomes, as reported in previous work,27 and we could use high molar ratios in our formulations, as mentioned above. The case was different for H2PzCOB, which could be incorporated into liposomes at much lower molar fractions, and shows a type of saturation at molar fractions lower than about 2%. The uptake ratios obtained with cationic liposomes containing three different LCOB molar fractions are shown in Figure 3. The observed trend of boron uptake did not increase monotonically with respect to the initial LCOB concentration for either type of cells. This suggested an interplay among different contributions. Similar nonmonotonic results were obtained for H2PzCOB on DHD cells, as shown in Figure 4. To understand this complex behavior, more physicochemical and biological investigations are required. Nevertheless, it was clear that using liposomes, particularly cationic liposomes, enhanced the uptake of 10B by cells and that the optimal loading molar fraction was not the same for the different cell lines. Conclusions Many severe tumors, such as gliomas and melanomas, are not yet treated satisfactorily by conventional therapeutic

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approaches, and in spite of recent remarkable improvements in radiological treatments, the progress of these pathologies are fatal in most cases. Thus, the search for innovative therapies to treat radioresistant tumors is an active field of research. BNCT is considered a viable option for these types of tumors, even if the general opinion is that to reach satisfactory clinical results, boron compounds and delivery systems more efficient than those currently approved should be designed. In this work we showed that carborane derivatives bearing lactosyl moieties were incorporated in model colon carcinoma and melanoma cell cultures at a high ratio, especially if liposomes were used as carriers. Carborane derivatives with a porphyrazine moiety were also delivered efficiently by liposomes. No significant toxic effects were observed for all the systems investigated. The best results were obtained with a cationic liposome formulation (DOTAP/DOPC/DOPE), and this was probably due to favorable electrostatic interactions with the negatively charged outer leaflet of mammalian plasma membranes. It should also be considered that cationic liposomes are known to drive guest drugs preferentially toward the cell nucleus,

Figure 4. 10B uptake ratio of cationic liposome loaded with different H2PzCOB molar fractions in DHD cells.

Altieri et al.

which could be an important additional advantage for the BNCT strategy. The measured uptake ratios were remarkably higher than those found for the BPA-fructose complex administered at the same concentration range. Experimental Section Materials. All chemicals and solvents (Aldrich Chemicals Ltd.) were of reagent grade and were used in the syntheses as supplied. 10B enriched 1-methyl-o-carborane was purchased from KATCHEM (Czech Republic). Solvents used were of spectroscopic or HPLC grade. THF was freshly distilled from sodium benzophenone ketyl under nitrogen. 1,2-Dioleoyl-3trimethylammoniumpropane (chloride salt) (DOTAP, purity >99%), 1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA, purity >99%), and 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE, purity >99%) were purchased from Avanti Polar Lipids, Inc., Alabaster, AL, and used without further purification. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC, purity >99%) was purchased from Northern Lipids, Inc., Vancouver, Canada. The molecular formulas of the lipids used in this work are shown in Scheme 1. Synthesis of LCOB. Lactosyl carborane was prepared according to a previously published procedure.27 Briefly, lactose octaacetate was glycosylated with propargyl alcohol in the presence of trimethylsilyl triflate. The obtained propargyl glycoside was boronated with 6,9-bis(acetonitrile)decaborane in toluene/acetonitrile at reflux. Careful deprotection with a diluted solution of sodium methoxide in methanol eventually afforded lactosyl carborane. These main passages are summarized in Scheme 2, while synthetic details are reported in the Supporting Information. It should be noted here that the high incorporation rate of LCOB into liposomes (see below) ensured that enough 10 B was present in the sample to carry out quantitative analysis without the use of enriched B10H12(CH3CN)2. Synthesis of H2PzCOB. 1-Methyl-o-carboranyl-2-hexylthioporphyrazine (H2PzCOB) was prepared by a slight modification of previously optimized synthetic strategies.28,29 10B enriched

Scheme 1. Molecular Formulas of the Lipids Used for the Liposome Formulations Studied in This Work

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Scheme 2. Main Steps in LCOB Synthesis

Scheme 3. Synthetic Path for H2PzCOB, 5a

a Reagents and conditions: (a) n-BuLi, THF, under Ar, -78 C to room temp, 1 h; (b) Br(CH2)6Br, THF, under Ar, -78 C to room temp, 2 h; (c) [dmmnt]Na2, MeOH/EtOH 8:2, 0 C to room temp, in the dark, 24 h; (d) n-PrOH, MgII(n-OPr)2, n-PrOH, 110 C, 24 h; (e) TFA, CHCl3; (f) NH3, pH 7, on ice.

1-methyl-o-carborane was used. The main steps of this synthesis are illustrated in Scheme 3, while synthetic details are reported in the Supporting Information. Liposome Preparation. Liposomes were made from DOTAP/ DOPC/DOPE, DOPA/DOPC/DOPE mixtures (1:1:1 molar ratio), and DOPC/DOPE mixtures (2:1 molar ratio). For simplicity, we will henceforth denote these formulations as cationic, anionic, and zwitterionic, respectively. The three different formulations were loaded with two o-closocarborane derivatives, LCOB and H2PzCOB, according to the coextrusion and sonication procedure, respectively. These methods have been shown to be the most appropriate for each of the two boron compounds investigated here.28-32 Briefly, a chloroform solution of lipids was mixed with the desired quantities of LCOB in acetone or H2PzCOB in chloroform to obtain the required molar ratios among all components. The solvent was then evaporated to dryness under vacuum overnight. The resulting film was swollen at room temperature with Milli-Q grade water. Upon vortexing, multilamellar vesicles were obtained and were submitted to eight cycles of freezing and thawing. Final downsizing and conversion to unilamellar vesicles was achieved as follows: (i) for LCOB by extrusion through 100 nm polycarbonate membranes with the LiposoFast apparatus (Avestin, Ottawa, CA) and (ii) for H2PzCOB by five cycles of sonication of 20 s at 70% power level, using a Bandelin Electronic Sonoplus HD2070 (Bandelin

Electronic UW2070 tip) sonicator. In all formulations the final lipid content was 28 mM (∼20 mg/mL). Different molar ratios with respect to total lipid concentration were investigated for each boron compound. These were in the range 15-50% for LCOB and 0.09-1.8% for H2PzCOB. It has been established by neutron absorption30 that lactosyl carborane is totally incorporated into liposomes under our working conditions, whereas incorporation of carboporphyrazine is only partial, and for purposes of quantification, it has to be measured a posteriori for each liposome formulation.28 Nevertheless, the high boron content of H2PzCOB (80 atoms per molecule), secondary to the use of 10B enriched carboranes in the synthesis, provided sufficient intensity for boron quantification in cell cultures by the R particle method (see below). The H2PzCOB incorporation rate was measured spectrophotometrically after liposome disruption. This was achieved by diluting 300 μL of the liposome suspension with water (1:10) and by adding 5 mL of CHCl3. Freezing the sample at 253 K for 10-12 h followed by quick thawing resulted in separation of the porphyrazine from the H2PzCOB/lipid complex. Repeated extractions with CHCl3 (3-4 times with 5 mL each) allowed the isolation of the porphyrazine, which is practically insoluble in water, from the lipids used in the liposome formulation. This process was carried out until no trace of the band at 712 nm was visible in the electronic spectrum. The drug was quantified from

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the intensity of this same band at 712 nm, using the molar absorption coefficient ε = 2.8  104 M-1 cm-1. ζ Potential. Surface charge measurements were performed with a Coulter DELSA 440 SX (Coulter Corporation). The ζ value was calculated from the electrophoretic mobility by means of the Helmholtz-Smoluchowski relationship. More details are reported in a previous paper.33 All samples were diluted 20 times to meet instrumental sensitivity requirements. Size Measurements. The liposome size was measured by dynamic light scattering (DLS) with a Coulter submicrometer particle analyzer, model N4SD, equipped with a 4 mW helium-neon laser (632.8 nm) and 90 detector. The autocorrelation function of the scattered light was analyzed by unimodal analysis, which assumes a log Gaussian distribution of the vesicle size. This allowed us to obtain the mean diameter and the polydispersity index (PI) of plain and loaded liposomes. Biological Experiments. DHD/K12/TRb Cell Line. DHD/ K12/TRb (DHD) is an established cell line from colon adenocarcinoma chemically induced in BD-IX rats by oral administration of 1,2-dimethylhydrazine (no. 90062901, ECACC, Salisbury, Wiltshire, U.K.).34,35 DHD cells were grown as a monolayer in a medium composed of a 1:1 (v/v) mixture of Ham’s F10 and DMEM (Euroclone, Italy), with a low glucose concentration (1 g/L), and containing 40 μg/mL gentamicin and 10% fetal bovine serum. B16-F10 Cell Line. B16-F10 cells were grown in Dulbecco’s modified Eagle medium containing 4500 mg/L glucose (DMEM) (GIBCO, Life Technologies, Italy) supplemented with 10% fetal bovine serum (GIBCO, Life Technologies, Italy) at 37 C in a 10% CO2 humidified atmosphere. Cells (5.0  105) were seeded in 100 mm Falcon dishes and propagated every 3 days by incubation for 1 min with a 0.25% trypsin solution (SIGMA, Italy). Cultures were periodically tested for Mycoplasma contamination using Chen’s fluorochrome method.36 Cells (3.0  105) were seeded in 60 mm Falcon dishes, and after 48 h each dish was incubated with the different preparations of liposomes and LCOB. Intracellular 10B Uptake. For boron uptake measurements, cells were cultured in medium supplemented with 10B-loaded liposomes and LCOB solutions. Liposomes were diluted 100 times with respect to the starting 28 mM lipid concentration. After a 4 h treatment, the medium was removed and the cells were washed three times in PBS, trypsinized, and resuspended in boron-free medium. Cells were then counted and split into two aliquots, one intended for the cytotoxicity test and the other for the analysis of the intracellular boron concentration. Control cells were processed in the same way as the boron-treated samples. Cells, enriched by centrifugation (10 min, 300g), were finally layered on two or three Mylar disks at a concentration of 4.0  106 cells/disk. Cytotoxicity Tests. For both cell lines the cytotoxicity of the checked boronated compounds was evaluated by means of the plating efficiency test. Diluted cells were plated at three different concentrations (50, 100, 250 cells/plate) in five replicate Petri plates for each of them. They were allowed to grow at 37 C for about 10 days. Colonies were fixed, stained, and counted for the estimation of the fraction of cells surviving after the treatment. No relevant toxic effect was observed for any liposome samples or LCOB solutions. Boron Concentration Measurements. Samples for 10B quantification with thermal neutrons were prepared by depositing a known amount of cells incubated with boronated compounds on Mylar disks. These cells were left on the same support for the drying process. All samples were weighed before and after drying to evaluate the weighing factor to correct for the concentration obtained in dry specimens. The Mylar supports covered by dry cells were positioned in a rotating holder, facing a thin silicon detector. The holder and the detector were placed

Altieri et al.

Scheme 4. Sketch Showing the Principle of the Boron Concentration Measurement by R Spectrometrya

a Cells loaded with boron compounds and a solid state silicon detector are shown during the phase of neutron irradiation. Samples and detector are positioned in a vacuum chamber inside the thermal column of the Triga Mark II reactor.

inside a vacuum chamber, and the entire system was irradiated inside the thermal column of the TRIGA Mark II reactor at the University of Pavia, Italy. At the irradiation position, the thermal neutron flux was about 2  109 cm-2 s-1. The rotating device allowed the irradiation and analysis of 10 samples without the need for shutting down the reactor and opening the column. A sketch of this home-built apparatus is shown in Scheme 4. Each sample was irradiated for 10 min. The spectra of all the charged particles emitted from the sample (i.e., R particles and 7 Li ions from the reaction of boron, and protons from the reaction of nitrogen) were collected electronically. The portion of spectra due to the R component only was used to calculate the boron content in the dry cells. The concentration of the original sample was then obtained using the correction factor measured before. This technique is innovative compared to the standard methods used in BNCT and offers a number of advantages. In particular, for tissues taken from a tumor zone, it allows the separation of the contributions coming from the tumor, the normal tissue, necrotic tissue, and all other kinds of tissues that may coexist in a biopsy. Separation is accomplished by correcting the result of the spectroscopy with neutronigraphic images of a subsequent cut of sample and with the histology of a third cut. By use of this method, it is possible to determine the concentration of 10B in the tumor cells with higher precision compared to conventional methods that allow only an average estimation of the concentration in the entire sample.

Acknowledgment. The authors are indebted to the researchers whose contribution was important for the extensive liposome characterization previously done and, in particular, to Dr. Julian Oberdisse (University of Montpellier II, France), Dr. Olivier Spalla (CEA, Saclay, France), and Dr Anna Salvati (University College, Dublin, U.K.). Dr. Luca Calamai (Department of Soil Science and Plant Nutrition, University of Florence, Italy) is acknowledged for assistance in ζ potential measurements. D.P. and G.R. gratefully acknowledge financial support by MIUR (Grants PRIN2007 and 2007XWBRR4_02), and Dr. Maria Pia Donzello, (University “La Sapienza”, Rome, Italy), for elemental analyses data. Supporting Information Available: Details of the synthesis and analytic data for 10B-enriched LCOB and H2PzCOB and

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their precursors. This material is available free of charge via the Internet at http://pubs.acs.org.

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