ARTICLE pubs.acs.org/Langmuir
Complexation of Anionic Liposomes with Spherical Polycationic Brushes Andrey V. Sybachin,† Matthias Ballauff,‡,§ Ellina Kesselman,|| Judith Schmidt,|| Yeshayahu Talmon,|| Larisa Tsarkova,^ Fredric M. Menger,# and Alexander A. Yaroslavov*,† †
Department of Chemistry, M. V. Lomonosov Moscow State University, Leninskie Gory 1-3, 119991 Moscow, Russian Federation Department of Physics, Humboldt University Berlin, Newtonstr. 15, 12489 Berlin, Germany § Helmholtz-Zentrum Berlin f€ur Materialien und Energie, Hahn-Meitner-Platz 1, D-14109 Berlin, Germany Department of Chemical Engineering, Technion-Israel Institute of Technology, 32000 Haifa, Israel ^ DWI an der RWTH Aachen e.V., 52056 Aachen, Germany # Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States
)
‡
ABSTRACT: Spherical polycationic brushes, consisting of polystyrene particles with linear cationic macromolecules grafted onto their surfaces, were electrostatically complexed with small unilamellar anionic liposomes. Complexation was monitored using a multimethod approach that included laser electrophoresis, dynamic light scattering, fluorescence, cryogenic transmission electron microscopy, and conductivity. Liposomes adsorb onto the outer edges of the brushes rather than penetrate into their dense polycationic layer. The integrity of the liposomes remains unaltered when the liposomes reside on the polycationic brushes. The resulting complexes (roughly 40 liposomes per brush) do not dissociate into their components upon exposure to physiological solutions. The system is potentially useful in that liposomes are gathered into well-defined clusters with a high encapsulating potential. Multicomponent constructs can be easily prepared if polycationic brushes are allowed to bind to a mixture of liposomes that encapsulate different guests. This work provides an example of “systems chemistry” whereby as many as eight components, each with its own particular location and function (i.e., polystyrene core, polycationic graft, egg lecithin, cardiolipin, two fluorescent dyes, water, and buffer), collectively self-assemble.
’ INTRODUCTION Spherical bilayer lipid vesicles (liposomes) have been known ever since their preparation and description by the Bangham group over 40 years ago.1 Shortly after the first publication, liposomal containers were suggested for drug delivery. Drug encapsulation into liposomes allows an enhancement of the circulation time and bioavailability.2 Additional hydrophilization of liposomal surfaces with poly(oxyethylene) or poly(Nvinylpyrrolidone) was shown to protect liposomes against aggregation3,4 and to minimize the risk of drug uptake by the reticulo-endothelial system.5 Immobilized liposomes can act as a depot for biologically active compounds and allow their controllable leakage into the surrounding solution. However, immobilization via the adsorption of liposomes onto flat hydrophilic substrates (glass, polymer sheets, etc.) is often accompanied by spontaneous morphological rearrangements and the formation of unwanted “supported bilayer” structures arising from close interliposomal or liposome/surface contacts.6,7 Liposomes and/or surfaces that have been premodified by oligonucleotides8 or latex particles9 are among the few examples that skirt this problem. Thus, a need exists for surfaces that adsorb liposomes as strongly as do conventional hydrophilic substrates but at the same time maintain liposome integrity. In this article, we examine the complexation of anionic liposomes with spherical polycationic brushes (SPBs) consisting r 2011 American Chemical Society
of monodisperse polystyrene particles with linear cationic macromolecules grafted onto their surfaces. Because of the high grafting density and the large total charge on each grafted macromolecule, polycationic chains direct themselves outwardly away from the polystyrene core so that, metaphorically speaking, the polymer brushes resemble the head of a daisy. One might expect, therefore, that anionic liposomes adsorb on the outward flange of the cationic brushes with little or no actual penetration of liposomes into the grafted polymer layer. Special attention will be paid to the quantification of the liposome/brush complexation, to the stability of the resulting complexes in aqueous saline media, and to the integrity of the brush-bound liposomes.10
’ EXPERIMENTAL SECTION SPBs were synthesized by the graft polymerization of a cationic monomer, (trimethylaminoethylmethacrylate) ammonium chloride, on the surfaces of monodisperse polystyrene (PS) latex particles ca. 100 nm in diameter.11 According to dynamic light scattering, the mean hydrodynamic diameter of the brushes was found to be 230 nm, giving a thickness of the cationic corona (a contour length, Lc) equal to (230100)/2 = 65 nm. The SPB structure is schematically presented in Figure 1. Poly(acrylic acid) (PAA) from Aldrich with DP = 50 was Received: February 21, 2011 Published: March 30, 2011 5310
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Figure 1. Schematic representation of a spherical polycationic brush (SPB). PS, polystyrene core; Lc, thickness of the brush layer; and small negative circles, bromide counterions.
used as received. Zwitterionic phosphatidylcholine (egg lecithin, EL) (I), doubly anionic diphosphatidylglycerol (cardiolopin, CL2) (II), Nfluorescein-iso-thiocyanyldipalmitoylphosphatidylethanolamine (FITCDPPE) (III), and N-lissamine-rhodamine-sulfonyl-dioleoylglycerophosphoetanolamine ammonium (RHB-DOPE) (IV), all from Avanti, were used as received. Small unilamellar anionic liposomes of 4060 nm in diameter were prepared by the standard sonication procedure.12 Appropriate amounts of EL and CL2 solutions in methanol were mixed in a flask, after which the solvent was evaporated under vacuum. A thin lipid film was dispersed in a borate buffer (pH 9.2, 102 M) for 400 s with the aid of a 4700 ColeParmer ultrasonic homogenizer. Liposome samples thereby obtained, as separated from titanium dust by centrifugation for 5 min at 10 000 rpm, were used within 1 day. Liposomes with a molar fraction of anionic CL2 headgroups of 0.1 (i.e., ν = 2[CL2]/(2[CL2] þ [EL]) = 0.1) and with a fluorescent dye incorporated into the membrane were prepared by the same procedure except that 1 wt % FITC-DPPE or RHB-DOPE was added to the lipid mixture solution prior to methanol evaporation. The fluorescence intensity of FITC-labeled liposome suspensions was measured at λem = 525 nm (λex = 495 nm) using an F-4000 Hitachi fluorescence spectrofluorimeter. The UVvis spectra of labeled liposomes were recorded with a UV-mini 1240 Hitachi spectrophotometer. Mean hydrodynamic diameters of liposomes and their complexes with SPBs were determined by dynamic light scattering at a fixed scattering angle (90) in a thermostated cell using a Brookhaven Zeta Plus. Software provided by the manufacturer was employed to calculate diameter values. The electrophoretic mobility (EPM) of liposomes and their complexes with PEVP was measured by laser microelectrophoresis in a thermostatic cell using a Brookhaven Zeta Plus with the corresponding software. The permeability of the liposomal membranes toward a simple salt was investigated by measuring the conductivity of NaCl-loaded vesicle suspensions with a CDM83 conductometer (Radiometer) as described in ref 13. Vitrified specimens for cryo-TEM were prepared in a controlledenvironment vitrification system (CEVS) where a desirable temperature and humidity were maintained. Briefly, a ca. 5 µL drop of the liposome suspension, or SPB suspension, or a mixed SPB/liposome suspension was placed on a perforated carbon film-coated copper grid, blotted with filter paper, and plunged into liquid ethane at its freezing point. The vitrified specimens were transferred to an Oxford CT-3500 cooling
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Figure 2. Effect of poly(acrylic acid) (PAA) on the electrophoretic mobility (EPM) of SPB particles. [SPB] = 2.5 mg/mL; 102 M borate buffer; pH 9. [A] , concentration of carboxylates on the PAA. holder and observed in either a Philips CM120 or an FEI T12 transmission electron microscope at about 180 C in low-dose imaging mode to minimize electron-beam radiation damage. Images were digitally recorded with a Gatan 791 MultiScan cooled CCD camera (CM120) or with a Gatan US1000 high-resolution cooled CCD camera (T12). Details may be found elsewhere.14,15 Solutions were prepared with doubly distilled water that was additionally treated with a Milli-Q Millipore system composed of ionexchange and adsorption columns as well as a filter to remove large particles. Brush-to-liposome binding was examined at 20 C. Under these conditions, the membranes of EL/CL2 liposomes were in the fluid (liquid-crystalline) state.16
’ RESULTS AND DISCUSSION The first step was to estimate the number of cationic groups in SPBs capable of electrostatic binding to multicharged anionic species. An aqueous solution of the brushes (1.5 mL of 2.5 mg/ mL) was titrated with a 0.03 M solution of polyacrylate (PAA) in a pH 9 buffer where the PAA is totally ionized. The interaction of two oppositely charged polyions was accompanied by mutual neutralization of their charges and was detected via the electrophoretic mobility (EPM) of the resulting complex particles using laser microelectrophoresis (Figure 2). Complete neutralization of the SPB cationic charges by PAA anionic charges was achieved when the EPM reached zero. Others have shown that a linear polycationic analog, quaternized poly(dimethylaminoethylmethacrylate), forms a strong complex with polyacrylate anions.2 It is thus reasonable to assume strong complexation for the identical cationic chains grafted to the surfaces of nanosized polystyrene spheres. At EPM = 0 in Figure 2, the concentration of added acrylate subunits ([A]) from PAA equals the effective concentration of tetraalkylammonium subunits ([ Tþ]) from SPB from which we derived [Tþ] = 6.5 104 M. A solution with [Tþ] = 1 104 M in a pH 9 buffer was prepared and mixed with a suspension of anionic EL/CL2 liposomes. The SPB-to-liposome interaction was also monitored by the electrophoresis technique. As in the previous experiment, the EPM of the brushes decreased from positive values to zero when the liposomes were added, after which they became negative 5311
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Figure 3. Effect of EL/CL2 liposomes on EPM of SPB particles. EL, egg lecithin; CL2, cardiolipin; [SPB] = 0.38 mg/mL; [Tþ] = concentration of cationic nitrogens on brushes = 1.0 104 M; 102 M borate buffer; pH 9.
Figure 4. Effect of EL/CL2 liposomes on the hydrodynamic diameter of SPB particles. [SPB] = 0.38 mg/mL; [Tþ] = 1.0 104 M; 102 M borate buffer; pH 9.
(Figure 3). The size of the SPB/liposome complexes as a function of lipid concentration is given in Figure 4. It is seen that during complexation the hydrodynamic diameter of the complexes changes but only slightly until the lipid concentration exceeds 0.3 mg/mL (lower X axis) or a “charge concentration” from the anionic CL2 headgroups is 2[CL2] = 0.4 104 M (upper X axis). The size of the particles in suspension then increases and reaches a maximum at 2[CL2] = 0.8 104 M, where complete neutralization of the SPB charge by interacting liposomes (EPM = 0) had been observed (cf. Figures 3 and 4). At higher lipid concentrations, the size of SPB/liposome complex particles decreases to and levels off at 0.3 μm (300 nm), which is 70 nm larger than the diameter of the initial SPB species. In that upper lipid concentration range, stabilization (i.e., absence of aggregation) of the complex particles arises from a repulsive
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Figure 5. Dependence of the relative fluorescence of supernatants (after the separation of SPB/fluorescent liposome complexes by centrifugation) vs [lipid]. [SPB] = 0.38 mg/mL; [Tþ] = 1.0 104 M; [FITC-DPPE] = 1 wt % lipid; 102 M borate buffer; pH 9.
negative charge brought about by an excess of bound anionic liposomes. A key question concerns the integrity of liposomes while complexed with the brushes because only complexes with unaltered liposomes would be relevant to biomedical and other applications. To monitor the liposome integrity, we prepared a suspension of EL/ CL2 liposomes filled with a 1 M NaCl solution. In an initial control experiment, the suspension was treated with a 10-fold excess of Triton X-100 solution that resulted in the expected liposome damage and subsequent NaCl leakage from the liposomes with a sharp rise in conductivity. However, when the NaCl-loaded liposomes were mixed with an SPB suspension, only a negligible increase in conductivity (constant with time) was detected. Thus, the anionic EL/CL2 liposomes retain their ability to encapsulate when bound to the polycationic brushes. The exact number of liposomes capable of binding to the brushes cannot be determined from Figures 3 and 4 alone. Quantifying the binding employed the following procedure:17 A series of mixtures were prepared with a constant SPB concentration but varying concentrations of EL/CL2 liposomes labeled with 1 wt % fluorescent lipid FITC-DPPC (structure in Experimental Section). After 30 min, the suspension was centrifuged for 40 min at 18 000 rpm using a J-21 Beckman centrifuge, which was sufficient to remove all SPB/liposome complexes. The fluorescence intensity in the clear supernatants (representing unbound liposomes) was measured at λem = 525 nm (λex = 495 nm) and then recalculated to lipid concentrations (Figure 5) using a calibration plot. As follows from the Figure, no fluorescence in the supernatants was detected up to 0.65 mg/mL lipid concentration that corresponds to 2[CL2]max = 0.9 104 M. At higher liposome concentrations, where a rise in the fluorescence intensity in the supernatants was observed, only 50 nm particles (i.e., the excess fluorescent liposomes) were found in the solution. This demonstrates the complete binding of liposomes at 2[CL2] e 0.9 104 M. The number of liposomes capable of complexing with a single SPB particle (N) was estimated on the basis of the data given in Figure 5: A 0.38 g/L SPB suspension was able to adsorb all EL/CL2 liposomes 5312
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Figure 6. Effect of [Tþ] of the SPB particles on the relative fluorescence intensity of EL/CL2 liposomes labeled with 1 wt % FITC-DPPE. [Lipid]total = 1 mg/mL; 102 M borate buffer; pH 9.
from a 0.65 g/L lipid suspension. The latter could be easily converted into liposome per liter concentration (CL) according to CL ¼
Clip S1 N a 2πd2 M
ð1Þ
where Clip is the lipid concentration at saturation (0.65 g/L), S1 is the mean surface area per lipid molecule or 0.7 nm2,12 d is the diameter of liposomes, M is the mean molecular weight of lipid,12 and Na is Avogadro’s number. The number of brush particles per liter (CB) was calculated by assuming that the polystyrene core mainly contributed to the total weight of SPB particles CB ¼
6Cbrush πD3 F
ð2Þ
where Cbrush is the SPB concentration, D is the diameter of the polystyrene core, and F is its density.18 Finally, the N value was calculated as N ¼
Clip S1 N a D3 F CL ¼ CB 6Cbrush d2 M
ð3Þ
Substituting the experimental values and literature data into eq 3 gave N = 36 ( 4, which is to say that an average of ca. 40 liposomes bind to each spherical polycationic brush. In Figure 3, the EPM leveled off at 0.5 (μm/s)/(V/cm). At this EPM, a negative electrostatic barrier on the brush surface was generated that was sufficient to suppress further liposome adsorption. At EPM = 0, a liposome-to-brush charge ratio of ZEPM=0 = 2[CL2]/[Tþ] equals 0.84 whereas at saturation Zsat = 0.9. We see, therefore, that even in the saturated complex the cationic charge of the brush particles exceeds the negative charge contributed by the adsorbed liposomes. However, a reverse in the brush charge from positive to negative was observed at saturation in Figure 3. How can these apparent contradictions be reconciled? A structural model of the SPB/liposome complex can be developed on the basis of the following data. The size (hydrodynamic diameter) of the initial SPB particles is 230 nm whereas the size of the particles fully loaded with anionic liposomes is 300 nm. Because the liposome diameter is ca. 50 nm, it is reasonable to assume that liposomes electrostatically
Figure 7. Changes in the relative fluorescence intensity of EL/CL2 liposomes labeled with 1 wt % FITC-DPPE and complexed with SPB as a function of NaCl addition. [SPB] = 0.38 mg/mL; [Tþ] = 1.0 104 M; [lipid]total = 0.6 mg/mL; 102 M borate buffer; pH 9.
bind mainly with the outer cationic groups of the brushes (i.e., the liposomes adsorb on “the tips of polycation fingers”) as opposed to penetrating deep into the inner part of the brush. Literature data corroborate this deduction. As reported, one spherical polycationic brush with a core of about 100 nm in diameter typically carries about 500 covalently attached polycationic chains,11 giving a mean area per chain on the outer brush border, Sch, equal to π(230 nm)2/500 = 330 nm2. A mean distance between two neighboring chains can be roughly estimated as the square root of Sch (ca. 18 nm), which is much less than the liposome diameter of 50 nm. Thus, both light-scattering experiments and literature data indicate little or no penetration of liposomes through the dense outer layer of polycationic chains. It is not unlikely that a portion of small counteranions (e.g., the bromide ion) maintains contact with interior cationic units in the vicinity of the polystyrene core even when the brush surface is saturated with adsorbed EL/CL2 liposomes. This explains why less than a stoichiometric negative charge from absorbed CL2 units, relative to the cationic charges in the brushes, serves to impart an overall negative charge to the brushes. Liposome-to-brush complexation is sensitive to the concentration of salt in the surrounding solution. To obtain a fix on this property, we made use of the fact that cationic polymers are known as effective fluorescence quenchers.17 Thus, varying amounts of polycation brush suspension were added to a suspension of fluorescently labeled EL/CL 2 liposomes (1 mg/mL), and a decrease in fluorescence due to the complexation was observed (Figure 6). The injection of NaCl solution into the SPB/liposome complex suspension led to the recovery of the fluorescence intensity, ultimately to its initial level (Figure 7), indicating total complex dissociation. The salt-sensitive electrostatic nature of the brush-to-liposome complexation was thereby proved. Additional insight into the morphology of the SPB/liposome complexes was obtained by cryo-TEM. Figure 8a displays a typical cryo-TEM micrograph of SPB particles in a watersalt buffer solution. Polycationic chains, extending from the polystyrene “nuclei”, are visible without the addition of any contrast-enhancing material. An electron micrograph of EL/CL2 liposomes (Figure 8b) corroborates their unilamellar structure with an 5313
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Figure 8. Cryogenic transmission electron microscopy images of (a) a 1.5 mg/mL SPB suspension, (b) a 0.1 mg/mL EL/CL2 liposome suspension, and (c) an SPB/liposome complex prepared by mixing a 1.5 mg/mL SPB suspension and a 0.1 mg/mL liposome suspension.
Table 1. Complexation of Polycationic Brushes with Labeled Liposome Mixturesa Qi
Qcomp
1
1.01 ( 0.05
2
2.0 ( 0.1
3
2.99 ( 0.24
4
3.7 ( 0.5
a
Qi is the ratio of FITC-labeled liposomes to RHB-labeled liposomes prior to mixture with the SPB particles. Qcomp is the same ratio on the surface of the SPB particles.
Figure 9. Spectra of (1) FITC-DPPE-labeled EL/CL2 liposomes and (2) RHB-DOPE-labeled EL/CL2 liposomes. [Lipid]total = 1 mg/mL.
average liposome size in good agreement with that measured by dynamic light scattering. SPB/liposome particles are shown in Figure 8c. Each particle consists of one SPB sphere covered with several liposomes. The micrograph clearly shows that the liposomes retain their spherical shape after complexation with the cationic brushes. A micrograph taken 2 h after complex formation (not shown) demonstrated a similar picture of intact, unmodified liposomes. The described methodology allows surface modification either by a uniform group of liposomes or by mixtures of liposomes with different contents. In the latter case, a mixture can be composed of liposomes with an identical lipid composition but loaded with different hydrophilic (water-soluble) or/and hydrophobic (membrane-soluble) substances. Because internalized liposomal guests should not affect binding processes, it is reasonable to expect that equally charged but differently filled liposomes should manifest similar affinities toward the brushes. Therefore, ratios of diverse liposomes in a mixture prior to binding should be identical to the liposomal ratios as attached to the brushes. To test the preceding hypothesis experimentally, we prepared two types of anionic EL/CL2 liposomes. FITC-DPPE was incorporated into the membranes of the first class of liposomes, and another fluorescent label, RHB-DOPE, was placed in the membranes of the second. The labeled lipids carried anionic groups, but because of their negligible molar content, the total
surface charges of liposomes were mainly contributed by negative charges of the 10% molar fraction of the embedded CL2 headgroups. In other words, the anionic charge was essentially the same for both types of labeled liposomes. Electronic spectra for initial labeled liposomes are represented in Figure 9 with extinction coefficients of ε500 = 0.078 mL mg1 cm1 at 500 nm for FITC-labeled liposomes and ε572 = 0.126 mL mg1 cm1 at 572 nm for RHB-labeled liposomes. Suspensions of the two liposomes were mixed so that the FITC-labeled liposome to RHB-labeled liposomes ratio (Qi) was varied from 1 to 4. The mixtures were then added to a cationic brush suspension such that the total liposome concentration was always twice as high as required for saturation of the brush surfaces. After separation of the SPB/liposome complexes by centrifugation, liposome concentrations in the supernatants were determined spectrophotometrically, which thereby gave the concentrations of each liposome type bound to the SPB particles. On the basis of these data, the Q ratio for SPB-complexed labeled liposomes (Qcomp) was obtained. As follows from Table 1, Qcomp = Qi for all studied liposome mixtures. Thus, the ratio between FITC-labeled liposomes and RHB-labeled liposomes on the surfaces of SPB particles were always equal to that in the initial liposome mixtures.
’ CONCLUSIONS Small unilamellar anionic liposomes effectively bind to spherical polycationic brushes. Liposomes adsorb onto the outer borders of the brushes but do not penetrate the dense polycationic layer. Each brush particle can bind up to 40 liposomes. The size of a complex particle depends on the liposome to brush ratio. The integrity of liposomes remains unchanged when binding to the brushes. The resulting brushliposome complexes do not dissociate to their components in a physiological solution. Mixing anionic 5314
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liposomes with different contents and complexing the resulting mixtures with polycationic brushes seems to be a promising way to prepare multicomponent drug-delivery systems.
’ ACKNOWLEDGMENT The work was supported by the Russian Foundation for Fundamental Research and the German Science Foundation and previously by the National Institutes of Health. The cryoTEM work was performed at Technion’s Laboratory of Electron Microscopy of Soft Materials, which is supported by the Technion Russell Berrie Nanotechnology Institute (RBNI). ’ REFERENCES (1) Bangham, A. D.; Horne, R. W. J. Mol. Biol. 1964, 8, 660. (2) Ishida, T.; Takanashi, Y.; Doi, H.; Yamamoto, I.; Kiwada, H. Int. J. Pharm. 2002, 232, 59. (3) Kenworthy, A. K.; Simon, S. A.; McIntosh, T. J. Biophys. J. 1995, 68, 1903. (4) Torchilin, V. P.; Shtilman, M. I.; Trubetskoy, V. S.; Whiteman, K.; Milstein, A. M. Biochim. Biophys. Acta 1994, 1195, 181. (5) Gabizon, A.; Catane, R.; Uziely, B.; Kaufman, B.; Safra, T.; Cohen, R.; Martin, F.; Huang, A.; Barenholz, Y. Cancer Res. 1994, 54, 987. (6) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159. (7) Richter, R. P.; Berat, R.; Brisson, A. R. Langmuir 2006, 22, 3497. (8) Yoshina-Ishii, C.; Boxer, S. G. J. Am. Chem. Soc. 2003, 125, 3696. (9) Zhang, L.; Hong, L.; Yu, Y.; Bae, S. C.; Granick, S. J. Am. Chem. Soc. 2006, 128, 9026. (10) A preliminary report of this work can be found inYaroslavov, A. A.; Sybachin, V.; Schrinner, M.; Ballauff, M.; Tsarkova, L.; Kesselman, E.; Schmidt, J.; Talmon, Y.; Menger, F. M. J. Am. Chem. Soc. 2010, 132, 5948. (11) Mei, Y.; Lu, Y.; Polzer, F.; Ballauff, M.; Drechsler, M. Chem. Mater. 2007, 19, 1062. (12) New, R. R. C., Ed. Liposomes: A Practical Approach; IRL Press: Oxford, U.K., 1990. (13) Kozlova, N. O.; Bruskovskaya, I. B.; Okuneva, I. B.; MelikNubarov, N. S.; Yaroslavov, A. A.; Kabanov, V. A.; Menger, F. M. Biochim. Biophys. Acta 2001, 1514, 139. (14) Bellare, J. R.; Davis, H. T.; Scriven, L. E.; Talmon, Y. J. Electron Microsc. Technol. 1988, 10, 87. (15) Talmon, Y. In Giant Micelles; Zana, R., Kaler, E. A., Eds.; CRC Press: New York, 2007; pp 163178. (16) Yaroslavov, A. A.; Efimova, A. A.; Lobyshev, V. I.; Kabanov, V. A. Biochim. Biophys. Acta 2002, 1560, 14. (17) Raghuraman, H.; Chattopadhyay, A. Biophys. J. 2004, 87, 2419. (18) Kahler, H.; Lloyd, B. J., Jr. Science 1951, 114, 34.
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