Proc. Nati. Acad. Sci. USAVol. 91, pp. 3029-3033, April 1994Medical Sciences

Na3[B20H17NH3]: Synthesis and liposomal delivery to murine tumors(bone/poyheda/boron neutron cpre thapy)

DEBRA A. FEAKES, KENNETH SHELLY, CAROLYN B. KNOBLER, AND M. FREDERICK HAWTHORNE*Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90024

Contributed by M. Frederick Hawthorne, December 15, 1993

ABSTRACT The polyhedral borane ion [n-B2,HlaJs re-acts with liquid ammonia in the presence of a suitable base toproduce an apical-equatorial (ae) Isomer of the [B,.H17NH3J-ion, [1-(2'-BeH,)-2-NHIE3BIep3. The structure of this prod-uct has been confire by "B NMR c opy and x-rayc lgraphy. This spe undergoes acid-catalyzed rear-

rangement to an apical-apical (ae) isomer, [1-(1'-B1JHI)-2-NHI3BJhs]P-, whose structure has been determined by "1BNMR spectroscopy. The sodi salts of both the ao and the a2isomers of [B2,H17NH3J- have been encaited within smallunilamelar liposomes, composed of distearoyl phosphatidyl-choline/chlAesterol (1:1), and investigated as boron-deliveryagents for boron neutron capture therapy (BNCT) of cancer.The blodistribution ofboron was determined after the hijectionof liposomal sensions into BALB/c mice brwing EMT6tumors. Both [Bn,,Hl7NH3J- isomers exhibited excellent tumoruptake and selectivity at very low injected doses, achievingpeak tumor boron concentrations of30-40 jAg ofB/g of tissueand tumor/blood boron ratios of -5. The enhanced retentionof the [B.Hl7NH3]3- isomers by EMT6 tumors may be attrib-uted to their facile intracellular oxidation to an extremelyreactive NH3-substituted [n-B2,.H18f- ion, the electrophilic[B2,Hl7NH3J- ion. Both isomers of [B2,Hl7NH3]3- are at least0.5 V more easily oxidized than other previously investigatedspe containing 20 boron atoms. In another experiment,[ae-B2.Hl7NH3p3 was encapsuiated in liposomes preparedwith 5% PEG-2000-dstearoyl phosphatidylethanolame inthe liposome membrane. As expected, these liposomes exhib-ited a longer circulation lifetime in the biodistribution exper-iment, resulting in the continued accumulation of boron in thetumor over the entire 48-hr experiment and reaching a max-imum of 47 mg of B/g of tumor.

Boron neutron capture therapy (BNCT) is a binary cancertherapy that requires the selective deposition of significantquantities (>15 pug of B/g of tumor) of the boron-10 isotopein cancer cells (1). Irradiation of the localized 10B nuclei withthermal neutrons results in neutron capture and nuclearfission leading to the production of the highly energeticspecies 7Li and 4He. Each of these fission products has aneffective range of 410 ;Lm in tissue. This short range limitsthe effects of the individual fission events to a single cell orits immediate neighbors. Therefore, the selective concentra-tion of the 10B nuclei within tumor cells, followed by theircapture of thermal neutrons, should result in localized de-struction of the malignant cells in the presence of normalneighboring cells.

Successful application of BNCT depends on the identifi-cation of boron compounds, such as boron-conjugated por-phyrins (2), that possess a natural mechanism for tumor cellaccretion or, alternatively, a tumor-specific delivery modal-ity through which compounds rich in boron may be specifi-cally delivered to tumor cells. The use of tumor-specific

delivery modalities, such as tumor-targeted monoclonal an-tibody conjugates, has thus far been limited by the highlycompetitive loss of conjugate to liver once therapeuticallyeffective amounts of boron have been conjugated to theantibody (3).The ideal delivery mechanism should be able to incorpo-

rate large quantities of boron without affecting its selectivedelivery of boron to the tumor. Small unilamellar liposomesencapsulating concentrated aqueous solutions of polyhedralborane salts and injected intravenously have been shown todeliver therapeutic quantities of boron selectively to tumorsin vivo (4).The advantages of liposomal delivery of boron to tumor

cells for application in BNCT are numerous. Primarily,unilamellar liposomes selectively deliver their contents to avariety of tumors in a manner that is essentially independentof their contents. Liposomes have also been shown totransport their contents to the interior of tumor cells (5).However, in order to deliver therapeutic quantities of boronto the tumor cells, concentrated aqueous solutions of poly-hedral borane salts must be encapsulated. This requires theproduction of liposomes that are under significant osmoticstress.The composition ofthe phospholipid bilayer used in vesicle

fabrication was chosen to maximize the in vivo stability oftheliposomes even under the osmotic stress imposed by thehypertonic borane salt solutions used. Although attempts todetermine the serum stability of liposomes of this type havebeen complicated by nonspecific binding of the borane saltsto serum proteins, indirect evidence of their stability wasevident in the biodistribution results, since high blood boronlevels are observed for several hours during each time-courseexperiment. Release of the liposome contents due to insta-bility of the bilayer would result in rapid reduction of theblood boron concentration, as observed following the admin-istration of unencapsulated species (4).Although the liposomal delivery of boron compounds to

the tumor in vivo has been successful, only hydrophilic boroncompounds possessing the potential to form covalent bondswith intracellular protein moieties were retained by the tumor(4). Compounds that lack this chemical reactivity were rap-idly cleared from all tissues, including tumor.The polyhedral borane anions of current interest for lipo-

somal encapsulation are centered around the so-called nor-mal isomer of [B20H,8]2-, designated [n-B20H,8]2-. Initially,the [n-B2oHI8]2- anion was chosen for investigation becauseof its high boron content per unit charge and because of itsknown reactivity with nucleophiles (6, 7). Additional advan-tages include the fact that the compound is easily synthesizedin high yield, the synthesis is amenable to scale-up with

Abbreviations: BNCT, boron neutron capture therapy; [n-B2oHls]2,normal form of [B2oHg]2-; [i-B20His]2-, photoisomer of [B2oHls]2-;[ae-B2oHl7NH3]3-, [142'-B1oH9)-2-NH3B1oHS]3-; [a2-B2oHl7NH3]3-,[114'-BloH9)-2-NH3BloH8]3-; [e2-B2oHl7OH4, [2-(2'-BloH9)-6-OH-BloHBsI-; DSPC, distearoyl phosphatidylcholine; DSPE, distearoylphosphatidylethanolamine; i.d., injected dose.*To whom reprint requests should be addressed.

3029

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3030 Medical Sciences: Feakes et al.

'OB-enriched precursors, and the sodium salts of [n-B20H18]2-and its derivatives are extremely water soluble.The two [B1oH9]- cage fragments present in [n-B2oHi8]2-

are linked by a pair of three-center two-electron bondsbetween boron atoms (8). These bonds are relatively electrondeficient and therefore susceptible to nucleophilic attack infacile reactions with both hydroxide (7) and methoxide ion(6), as shown below:

2- 4

-HH20

where o = BH and . = B. Another useful reaction displayedby [n-B20H18]2- is its photoisomerization to an isomer des-ignated [i-B2oH18]2- (9). This photoisomer is also susceptibleto nucleophilic attack. Although both the liposome-encap-sulated n- and i- isomers are retained by tumor in tumor-bearing mice, the product obtained from the hydrolysis ofthephotoisomer, [2-(2'-B1oH9)-6-OH-BioH8]4, designated [e2-B20H170H]4-, is rapidly cleared from all tissues includingtumor (4). In an effort to examine the effects of charge andsubstituent on in vivo tumor retention, the sodium salts oftwo(B~oHi7NH3]3- isomers obtained from [n-B2oHi]2 wereencapsulated in liposomes for murine biodistribution exper-iments.

MATERIALS AND METHODSMaterials. Synthetic reactions were performed under an

atmosphere of dry N2. The polyhedral borane starting mate-rials were prepared by published methods (10, 11). Acetoni-trile was distilled from CaIH2 prior to use. Sodium acetylide(Aldrich, 18% slurry in xylene/light mineral oil) and anhy-drous NH3 (Matheson) were used without further purification.Distearoyl phosphatidylcholine (DSPC) and PEG 2000-conjugated distearoyl phosphatidylethanolamine (PEG-DSPE) were from Avanti Polar Lipids, and cholesterol wassupplied by either Calbiochem or Sigma.

hysical Measurements. The "1B Fourier transform NMRspectra were obtained with a Bruker AM-500 instrument at 160MHz. Boron chemical shifts were externally referenced toBF3*Et2O in C62H6; peaks upfield of the reference are desig-nated as negative. Cyclic voltammograms were obtained witha Bioanalytical Systems CV-27 voltammograph usingEt4NPF6/CH3CN as the supporting electrolyte vs. Ag/AgCl.

(Et4N)3[1-(2'-B1J1g)-2-NH3BH], Dsnted (EtN3[a-B2.H17NH3]. Anhydrous ammonia (2000 ml) was condensedonto dried Na2[n-B20Hls] (36.7 mmol) using both a dry ice/acetone bath and a dry ice/acetone condenser. A slurry ofsodium acetylide (28.0 ml, 115 mmol) was added to the stirredsolution via syringe. The cold bath was removed, the dryice/acetone condenser was maintained for 6 hr, and then thereaction vessel was allowed to warm to room temperature.The remini traces of NH3 and xylene were removed invacuo, and the residue was dissolved in 500 ml of absoluteethainol and filtered. The product was isolated by precipitationusing a saturated solution of Et4NJBr in absolute EtOH. Theoff-white solid was dried in vacuo to give 19.3 g of [Et4NI3[ae-B2oH17NH3] (30 mmol, 82% yield). The anion could also beisolated by precipitation with an ethanolic solution ofMe4NClto obtain (Me4N)3[ae-B20H17NH3]. 11Be1H} NMR (ppm, H20,relative areas in parentheses): 9.7 (1), 2.7 (1), -2.3 (1), -8.4(1), -15.6 (1), -25.7, -26.9, -29.8, -31.9. The compoundwas ion exchanged to the sodium form in aqueous solution

using Bio-Rad AG50W-X8, 50- to 100-mesh cation exchangeresin.

(Et4N)3[1-(l'-BlI,)-2-NH3BoHJ, Desgnatd (Et4N)3[a2-B25H17N113]. Trifluoroacetic acid (6.5 ml, 84 mmol) was addedslowly to a solution of 13.6 mmol of Na3[ae-B20H17NH3] in130 ml of CH3CN. The mixture was stirred overnight underan atmosphere of dry N2. The apparent pH was increased to-12 by the addition of 3 M NaOH. The solvent was removed

in vacuo and the resulting residue was dissolved in 120 ml ofabsolute ethanol and filtered. The product was isolated as theEt4N+ salt by precipitation using a saturated solution ofEt4NBr in absolute ethanol. The off-white product wasisolated by filtration and dried in vacuo to obtain 4.9 g of[Et4Nb3[a2-B2oHi7NH3] (7.6 mmol, 56% yield). '1B{'H} NMR(ppm, H20, relative areas in parentheses): 9.7 (2), -6.0 (1),-7.8 (1), -15.3 (1), -25.5 (2), -26.3 (2), -29.6 (10), -31.3(1). The compound was ion exchanged to the sodium form inaqueous solution using Bio-Rad AG50W-X8, 50- to 100-meshcation exchange resin.

Vesicle Preparation. Liposome suspensions were preparedby probe sonication of a 300-mg dried film composed ofequimolar amounts ofDSPC and cholesterol with the hydrat-ing solution (typically 6 ml, 200 mM in the boron-containingsodium salt) at 650C for 15-30 min. The composition of thelipid film used for the PEG-containing liposomes was DSPC/cholesterol/PEG-DSPE, 1:1:0.1. Vesicles were separatedfrom the remaining free borane salt by elution through acolumn of Sephadex G-25 (medium) with isotonic phosphate-buffered lactose (PBL) buffer. Liposomal preparations werediluted with the PBL buffer to a lipid concentration of 23-24mg/ml and sterilized by filtration through a 0.22-pm Milliporemembrane. The integrity of the encapsulated borane salt wasconfirmed by 11B NMR at 160 MHz. The volume-weightedmean vesicle diameter of the liposomes reported here (bydynamic light scattering) ranged from 70 to 115 nm for the fiveexperiments. Encapsulation efficiencies for these experi-ments were z3%, a normal value for the preparation ofliposomes by probe sonication. The unencapsulated boranesalts were routinely recovered by aqueous elution from thecolumn.Murine Studies. Murine biodistribution studies utilized

female BALB/c mice (16-20 g), with EMT6 tumors im-planted in the right flank 7-10 days prior to the experiment.Tumor mass at the time of sacrifice was 125-350 mg. Injec-tions of liposome suspensions (200 0l) were made in the tailvein. Details of the murine experiments have been reported(4). Boron analyses of tissues and of liposome suspensionswere performed by inductively coupled plasma-atomic emis-sion spectroscopy at the Idaho National Engineering Labo-ratory.

RESULTSThe reaction ofNH2, generated in situ, with [n-B2oHis]2- inliquid NH3 produces [1-(2'-B1IH9)-2-NH3BoHS]3-, an api-cal-equatorial species designated [ae-B20H17NH3P, asshown below:

NH3 + NaC=CH - NaNH2 + HC=CH

%12-NH2- 71 3-

NH2NH3

where o = 13H and . = B. The 11B NMR spectra of thisspecies are consistent with this structural assignment. Three

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Nadl. Acad. Sci. USA 91 (1994) 3031

apical boron signals, at 2.7, -2.3, and -8.4 ppm, appear assinglets in the proton-decoupled spectrum and as doublets inthe proton-coupled spectrum. A broad singlet at 18.8 ppm isassigned to the apical boron atom of the boron-boron bondconnecting the two 10-boron atom cages. The signal for theequatorial boron atom of the boron-boron bond is beneaththe upfield peaks. The boron atom attached to the nitrogen ofthe ammonio group exhibits a broad singlet at -15.6 ppm.The remaining signals, all upfield relative to the apical boronand substituted boron signals, are due to the remainingequatorial boron atoms in the two cages and appear asdoublets in the proton-coupled spectrum.A single crystal of [Me4Nb3[ae-B2oHI7NH3] was grown

from an aqueous solution by slow evaporation of the solvent.The crystal was characterized by x-ray crystallography andthe structure of the anion is shown below:

BI

B7'

Although disorder in the crystal structure prevented a com-pletely satisfactory refinement, the gross connectivity of thecompound was clearly determined. The two 10-boron atomcages and the boron-boron distance connecting the cages aresimilar to those observed in the [a2-B2oHi8si' and [e2-B2oH,8]4- ions (12). Thl only unusual characteristic in the[ae-B2oH17NH3P anion is the nonlinearity of theB(10-B(1)-B(2') array [169.1(8)0]. Since this structurerepresents, so far as we know, the first apical-equatorial20-boron atom species to be characterized crystallographi-cally, there is no basis for comparison to other structures.

Acidification of Na3[ae-B20H17NH3] with trifluoroaceticacid in acetonitrile yields a species believed to beNa2B20Hi8NH3, the ammonio derivative of [B2oH19]3- (13).Basification of this solution with aqueous NaOH yields[1-(1'-BioH9)-2-NH3BIoH8]3- the apical-apical isomer of[B2oH17NH3]3- designated [a2-B2OH17NH3]3, as shown be-low:

3-

a) TFA/CH3CNb) NaOH

3-

NH3

isomer to the a2 isomer is consistent with that observed withboth the analogous hydroxy, [B20H170H]4-, derivatives (10)and the unsubstituted, [B20H18]4-, series (14).The biodistribution of liposomes loaded with Na3[ae-

B20H17NH3] is shown in Fig. 1A [injected dose (i.d.), 198 Pgof B; =11 mg of boron/kg of body weight]. The initialmeasured tumor boron concentration (27.0 Mug of B/g oftumor) represented 14% i.d./g oftissue. Over a period of -30hr, the tumor boron concentration increased to the observedmaximum (32.3 Mg of B/g of tumor). Although the tumorboron concentration then began to decrease, the 48-hr con-centration (25.4 Mug of B/g of tumor) was still 94% of the 6-hrconcentration and a tumor/blood boron ratio of 5.3 had beenachieved.The biodistribution of unencapsulated ("free") Na3[ae-

B2oHI7NH3J is shown in Fig. 1B (i.d., 175 Mg of B; =10 mgof B/kg of body weight). Similar to other borane anionsstudied in vivo (4), no significant accumulation of boron wasobserved in either the spleen or the blood. The liver boronconcentration had a 6-hr value of 13.4 Mg of B/g of liver,which decreased to 7.7 Mg of B/g of liver at the 48-hr timepoint. The initial measured tumor boron concentration wasrather high (7.7 Mug of B/g of tumor) compared to valuescommonly observed with other free borane salts, achieving amaximum of 8.4 Mg of B/g oftumor at 16 hr. After 16 hr, thetumor boron decreased to a value of 4.4 Mg of B/g of tumorand a tumor/blood boron ratio of 5.0 was observed.The biodistribution of the known ammonio derivative of

Na2[B10H10], Na[2-NH3BloHgJ (15), encapsulated in lipo-somes is shown in Fig. 1C (i.d., 146 Mg of B; -8 mg of B/kgofbody weight). The 6-hr tumor boron concentration (21.1 MgofB/g oftumor) quickly decreased, resulting in a 48-hr tumorboron concentration of 4.1 Mg of B/g of tumor and a tumor/blood boron ratio of 2.4. No significant accumulation ofboron in any ofthe other tissues examined was observed overthe 48-hr time period.

60

01 ,i,0w

0 0

.=

00

Q

20 '

lo~0

O0lac)

30 .*,r:

20._20

10 o00

where o = BH, . = B, and TFA = trifluoroacetic acid. Theprotonated species can also be prepared in aqueous mediumby acidifying a water solution of [Me4Nb3[ae-B2oHI7NH3] toproduce a precipitate of [Me4N12B2oHj8NH3. However, thispathway does not proceed as cleanly as does the nonaqueousroute. The 11B NMR spectra of the a2 isomer are consistentwith its assigned structure. Two apical boron signals, at -6.0and -7.8 ppm, are singlets in the proton-decoupled spectrumand doublets in the proton-coupled spectrum. Each signalrepresents a single boron atom. A broad boron-boron signalat 9.7 ppm is assigned to the two apical boron atoms con-necting the two 10-boron atom cages. The boron atomattached to the nitrogen atom of the amino group exhibits abroad singlet at -15.3 ppm. Four upfield signals occur at-25.5, -26.3, -29.6, and -31.3 ppm, with a relative inten-sity of 2:2:10:1. The acid-catalyzed rearrangement of the ae

Time, h

0 10 20 30 40 50Time, h

FIG. 1. Murine tissue boron concentrations. (A) Na3[ae-B20H17NH3], 198 pg of B (=11 mg of B/kg of body weight). (B)Unencapsulated Na3[ae-B20H17NH3], 175 pg of B (=10 mg of B/kgof body weight). (C) NaBjOH9NH3, 146 pg of B (-8 mg of B/kg ofbody weight). (D) Na3[a2-B20H17NH3], 288 pg of B (=16 mg of B/kgof body weight). (E) Na3[ae-B2OHj7NH3] in liposomes containingPEG-DSPE, 398 pg ofB (-22 mg ofB/kg ofbody weight). *, Blood;*, tumor; o, liver; A, spleen. Each data point represents the averageof five mice. For clarity, error bars are not shown in the graphicaldata; standard deviations were typically 5-15% of the averagevalues.

Medical Sciences: Feakes et aL

D

3032 Medical Sciences: Feakes et al.

Table 1. Half-peak oxidation potentials (Ep/2) vs. Ag/AgCl forborane species in acetonitrile using Et4NPF6 as thesupporting electrolyte

Borane anion Ep/2, mV[a2-B2oHl7NH3]3- -60[ae-B20Hl7NH3]3 -130[e2-B2oHl7OH]4- +460[B2oHi9]3- +590[2-NH3BioHg]- +830

The biodistribution of encapsulated Na3[a2-B20H17NH3] isshown in Fig. 1D (i.d., 288 pg of B; =16 mg of B/kg ofbodyweight). The initial measured tumor boron concentration(28.4 pg of B/g of tumor) represented 9.9%o i.d./g of tissue.The tumor boron concentration increased to a plateau at 16hr (40.0 pg of B/g of tumor). This value was essentiallyconstant over the remaining time period, resulting in a 48-hrtumor boron concentration of 37.0 pg of B/g of tumor and atumor/blood boron ratio of 4.8.The biodistribution of encapsulated Na3[ae-B20H17NH3] in

liposomes containing 5 mol% PEG-DSPE in the membranebilayer is shown in Fig. 1E (i.d., 398 pg ofB; =22 mg ofB/kgof body weight). The tumor boron concentrations increasedcontinuously from a 6-hr value of 27.4 pg of B/g of tumor toa 48-hr value of 46.7 pg of B/g of tumor. The blood boronconcentrations fell from a 6-hr value of 87.2 pg of B/g ofblood to a 48-hr value of 19.3 pg of B/g of blood. Therelatively hi 48-hr blood boron concentration resulted in alow tumor/blood boron ratio of 2.4.The half-peak oxidation potentials of several polyhedral

borane species in acetonitrile were determined by cyclicvoltammetry vs. Ag/AgCl (Table 1) using Et4NPF6 as thesupporting electrolyte. Both of the ammonio species, [ae-B2oH17NH3P3-and [a2-B20Hl7NH3]3, oxidized >0.5 V moreeasily than the hydroxy derivative, [e2-B20H170H]4-, theunsubstituted species, [B2oH19]3-, and the 10-boron atomammonio derivative, [2-NH3B1oH9]-.

DISCUSSION

Nucleophilic attack ofNH-, formed in situ by the reaction ofsodium acetylide with liquid NH3, on the electron-deficientintercage bonding region of [n-B20H18]2 forms, directly andin high yield, the ae isomer of [B2oH17NH3]3-. Althoughalternative bases, such as NaH, or direct reaction withsodium amide (NaNH2) yield the identical product, thesereactions proceed slowly and often do not reach completion.These observations are most likely due to the high solubilityof sodium acetylide in liquid NH3 as compared to either NaHor NaNH2. No direct reaction between [n-B20H18]2- and NH3is observed when the base is omitted. The photoisomer,[i-B2oH18]2-, also reacts with [NH2]- to form an additionalseries of [B20H17NH3]3- isomers, which are the subject ofanother study.The single-crystal x-ray diffraction study of (Me4N)3[ae-

B2oH17NH3J confirms the gross structure and connectivity ofthis species and is, so far as we know, the first structuralcharacterization of an anion containing two [B1oH9]- cagesbound together at apical-equatorial boron atoms. The mostunusual feature of the anion is the B(10)-B(1-B(2') angleof 169.1(8)0. Although it is possible that this deviation fromlinearity is due to the presence of the NH3 ligand and apossible hydrogen-bonding interaction of this substituent andthe B(1'-B(2') edge ofthe cage, no hydrogen atom has beenlocated in a position suitable for hydrogen-bond formation.The distance between N and B(1') is 3.27(2) A and theN-B(2') separation is 3.31(2) A. The bond distances and

bond angles within the 20-boron atom skeleton are analogousto those observed for the a2 and e2 isomers of [B2oH1S]4- (12).The nonaqueous protonation of [ae-B20H17NH3]3- in

CH3CN and subsequent neutralization with aqueous NaOHyields the a2 isomer of [B20H17NH3]3-. The structure ofthisspecies has been unambiguously confirmed by 11B NMR. Arearrangement of this type carried out in aqueous solu-tion yields unidentified by-products. The rearrangementis believed to proceed through a protonated species,[B2oH18NH3]2-, analogous to the reported [B20H19]3- ofunknown structure (14).The a2 configuration of both the unsubstituted 20-boron

atom species, [B2oH18]4-, and the hydroxy-substituted com-pounds, [B2oH170H]4-, is the thermodynamically stableproduct (7). Conversion of the ae isomer to the a2 isomer of[B2oH17OH]4- is readily achieved in aqueous solution. Al-though the [a2-B20H17NH3]3- species is more difficult toobtain than the [a2-B2oH17OH]4 species, it is also the ther-modynamically more stable isomer. Conversion of the [ae-B2oH17NH3]3- isomer to the [a2-B20H17NH3]3 isomer willoccur during prolonged storage in aqueous solution at roomtemperature.When encapsulated in liposomes, Na3[ae-B20H17NH3] is

accreted by tumor over a period of =30 hr and demonstratestherapeutic levels (>15 pg ofB/g oftumor) at all points ofthetime course experiment. This prolonged retention enables theother tissues (liver, spleen, and blood) to clear boron, ulti-mately resulting in a tumor/blood ratio of 5.3.The biodistribution ofunencapsulated Na3[ae-B2oH17NH3]

was determined to establish the advantage of liposomaldelivery. Although the liver, spleen, and blood curves aresimilar to those ofother unencapsulated borane salts, such asNa2[i-B2oH18] (4), the tumor values clearly indicate somedegree of selective tumor binding. The 6-hr tumor boronconcentration of 7.7 pg of B/g of tumor is maintained over aperiod of -30 hr before decreasing to 4.4 pg ofB/g oftumor.A tumor/blood boron ratio of 9.4 was found at 16 hr. Incomparison, the [i-B2oHi8]2- species, which also displayspromising biodistribution results when encapsulated in lipo-somes, does not achieve tumor boron concentrations of >2.0pg of B/g of tumor when not encapsulated in liposomes anddemonstrates a tumor/blood ratio of only 1.0 at 24 hr (4).The therapeutic concentrations obtained in the biodistri-

bution of liposomal Na3[ae-B20H17NH3] are not the result ofanionic charge or the presence of a substituted 20-boron atomcage. This is evidenced by the low boron concentrationsobtained in the biodistributions ofliposomal Na3[B20H19] andNa4[e2-B20H170H] (4). In an effort to determine whether theammonio functionality of the [B20H17NH3]3- isomers is re-sponsible for their in vivo tumor retention, the known am-monio derivative of [BioHo,]2- [2-NH3B1oH9]- (15), wassynthesized and encapsulated in liposomes for in vivo exper-iments. Although the initial measured tumor boron concen-tration is quite high for a 10-boron atom species (21.1 pg ofB/g of tumor), all tissues, including tumor, rapidly clearboron. Therefore, it is unlikely that the tumor retentionobserved for Na3[B2oHl7NH3] is a direct result of the ammo-nio substituent.When the injected dose is taken into account, the basic

features of the biodistribution of the encapsulated a2 isomerof [B2oH17NH3]3- are not signiicantly different from those ofthe corresponding ae isomer. The tumor boron concentra-tions observed in the a2 isomer biodistribution experiment donot decrease significantly after reaching a plateau at 16 hr.Therapeutic values of boron are maintained in tumor whilethe boron concentrations in other tissues decrease.The circulation lifetime of liposomes can be increased by

appending PEG moieties to the bilayer (16). The PEG groupscoat the surface of the liposome, preventing opsonins fromadhering to the liposome and thus labeling the liposome as a

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 3033

foreign particle. This prevents the clearance of circulatingliposomes by fixed macrophages of the liver and spleen.

Introduction of the PEG (PEG-DSPE) coating to the lipo-somes containing [ae-B2OHj7NH3]3- increases their circula-tion lifetime, as shown by the relatively high blood boronconcentration retained at 48 hr (19.3 j&g ofB/g of blood). Theincreased in vivo lifetime of these liposomes enables thetumor to accrete boron throughout the entire 48-hr timecourse experiment, resulting in a final tumor boron concen-tration of 47 gg of B/g of tumor. The relatively high 48-hrblood boron concentration reduces the tumor/blood boronratio to a value of 2.4. Extending the duration of the biodis-tribution experiment may raise the tumor/blood boron ratiowhile maintaining a high therapeutic tumor boron level.The biodistribution analyses discussed above have shown

that the superior in vivo tumor retention of both isomers ofNa3[B20Hl7NH3] is not a direct result of the charge of theanion, the fact that it is a substituted 20-boron atom cage, orthe fact that it is an ammonio-substituted polyhedral boranespecies. Comparison of the half-peak oxidation potentials of[ae-B2oH17NH3j3P, [a2-B2OHl7NH3]3-, [e2-B20oH70H]41,[B2oH,9]3-, and [2-BjoH9NH31F suggests the most directevidence pertaining to the possible mechanism of in vivo, andpresumably intracellular, retention. Both of the isomers of[B2oHl7NH3]3- investigated are oxidized >0.5 V more easilythan any of the other species studied. The hypotheticaltwo-electron oxidation product of [B2oHl7NH3]3- would be[B20Hl7NH3]- (as shown below), a species that should beeven more susceptible to nucleophilic attack than [B2oH,8]2-due to both its reduced negative charge and the presence ofan electron-withdrawing substituent.

3- 1-

Attempts to synthesize [B2oHl7NH3]4 directly by chemicaloxidation have been thwarted by the extreme instability ofthe reaction product, which reacts further to form reducedspecies. The latter appear by analysis of their "1B NMRspectra to have more than one substituent per 20-boron cage.

CONCLUSIONSAt this time, liposomal encapsulation of boron-rich com-pounds provides the most generally attractive delivery mo-dality for application to BNCT. Liposomes have been con-sistently shown to selectively deliver therapeutic amounts(>15 pg of B/g of tumor) of boron to tumor in vivo,presumably through intracellular uptake. Due to the inherenttumor selectivity of liposomes (4), only small amounts ofboron, as evidenced by the low injected doses used in theseexperiments (8-22 mg of B/kg of body weight), are requiredto achieve therapeutic amounts of boron in tumor. Otherboron-containing compounds and delivery techniques oftenrequire injected doses of 50 mg of B/kg of body weight ormore to achieve therapeutic tumor concentrations. Once theencapsulated species is delivered by the liposomes to theinterior of the tumor cell, the characteristics of the boron-containing species establish whether it will be retained by thecell or simply eliminated. Therefore, guidelines now exist for

the selection of appropriate candidate species for synthesis,liposomal encapsulation, and in vivo evaluation.The reduced ammonio derivatives of [n-B2oH,8]2-, [ae-

B20H17NH3]3- and [a2-B2oHj7NH3]3, are currently the mostpromising water-soluble compounds known for liposome-mediated BNCT. The biodistributions of both of these com-pounds are characterized by high initial tumor uptake fol-lowed by a further increase in tumor boron concentration.The observed tumor boron concentrations maintain thera-peutic values over the entire 48-hr time period, providing anopportunity for the boron concentrations of other tissues,particularly blood, to decrease.Based on the data collected thus far, it is believed that the

ability of the two known isomers of [B20H17NH3]3- to beretained by the tumor in vivo is due to their facile intracellularoxidation to produce [B2oHl7NH3]-. This oxidized species,like [n-B2oH18]2-, is expected to be extremely susceptible tonucleophilic attack and thus amenable for reaction withintracellular protein moieties, as shown below:

1- 3-

where o = BH, . = B, and (D = intracellular protein.

We thank Teresa A. Krisch of Vestar, Inc., for assisting with themurine biodistribution experiments. We are indebted to Dr. WilliamBauer of the Idaho National Engineering Laboratories for theinductively coupled plasma-atomic emission spectroscopic boronanalyses. This research was conducted as part of the BNCT programof the Idaho National Engineering Laboratory and was performedunder the auspices of the Office of Energy Research, U.S. Depart-ment of Energy (DOE) under DOE Field Office, Idaho, ContractDE-AC07-761D01570.

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Medical Sciences: Feakes et al.