Chapter 9
Methodologies and Models of Cross-Linking Polymerization in Supramolecular Assemblies Thomas M . Sisson, Henry G. Lamparski, Silvia Kölchens, Tina Peterson, Anissa Elayadi, and David F. O'Brien 1
Carl S. Marvel Laboratories, Department of Chemistry, University of Arizona, Tucson, AZ 85721
Polymerization of monomeric lipids in an assembly proceeds in a linear- or crosslinking manner depending on the number of polymerizable groups per monomer. The gel point for polymerizations in two-dimensional lipid assemblies was determined by correlation of the onset of significant changes in the bilayer properties with bilayer composition. The experimental methods determined changes either in lipid lateral diffusion, polymer solubility, or assembly stability to a nonionic surfactant. Each method used indicated a substantial mole fraction (0.30±0.05) of the bis-substituted lipid was necessary for crosslinking. Two models rationalizing the inefficient crosslinking observed in organized media were proposed and further tested. The experimental data are consistent with an intramolecular macrocyclization of the bis-lipid as a principal pathway responsible for the crosslinking inefficiency. The solubility methodology was employed to study the nature of polymerizations in inverted hexagonal and bicontinuous inverted cubic phases. Polymerization of supramolecular assemblies is an effective means to modify their chemical and physical properties (7). The polymerization of monomelic lipids in an assembly proceeds in a linear or crosslinking manner depending on the number of polymerizable groups per monomeric lipid. Lipids that contain a single reactive moiety in either of the hydrophobic tails or associated with the hydrophilic head group yield linear polymers. Polymerization of lipids with reactive groups in each hydrophobic tail generally yields crosslinked polymers. Linear and crosslinked polymeric assemblies exhibit significant differences in physical properties, e.g. permeability (2-4), chemical stability (5,6), solubility (5,7), lateral diffusion of components (8), among others. The formation of linear polymer chains in bilayer vesicles causes a moderate reduction in permeability of water soluble solutes, e.g. glucose or sucrose, relative to that of unpolymerized bilayers. In contrast the formation of crosslinked poly(lipid) vesicles is reported to decrease the permeability by two orders of magnitude (2,3). The stability of vesicles to surfactant solubilization increases from unpolymerized vesicles, to linear poly(lipid) vesicles, and finally to crosslinked ilayer vesicles (5). The increased chemical stability can 1
Corresponding author.
©1998 American Chemical Society
119
120 be attributed to the crosslinking of the lipids into a covalently linked monodomain polymeric vesicle. In this report we review three approaches to the characterization of the gel point for polymerizations constrained by the two-dimensional nature of lipid bilayers, and examine the relative effectiveness of unsymmetrical and symmetrical crosslinking agents on the efficiency of crosslinking in organized assemblies. The observations obtained with lamellar assemblies may also be applied to characterize crosslinking in nonlamellar phases, i.e. bicontinuous cubic (Qn) and inverted hexagonal (Hn) phases. Results and Discussion Fluorescence photobleaching recovery (FPR) has been utilized to determine the lateral diffusion coefficient (D) of a fluorescent probe within the bilayer following the procedures previously developed for unpolymerized lipid assemblies (8). Lateral diffusion coefficients for NBD-PE in linear or crosslinked polymeric hydrated lipid bilayers were measured, where the mole fraction of NBD-PE was 1 0 (9,10). The D for the probe decreased moderately as the degree of polymerization of linear poly(lipid) increased. The effect of the bilayer molar ratio of mono-AcrylPC and bis-AcrylPC (Scheme 1) was determined. The relative reactivities of acryloyl group mono-AcrylPC and both acryloyl groups in bis-AcrylPC are expected to be similar since they are chemically equivalent. The positional differences between the sn-l and sn-2 chains of the bis-AcrylPC should not significantly effect the relative reactivity of the two groups. At low mole fractions of the crosslinker lipid, bisAcrylPC, in the bilayer the D was relatively unaffected. However at mole fractions of bis-AcrylPC greater than 0.3 the diffusion coefficient for N B D - P E was significantly reduced. The FPR measurements show a significant decrease in the dynamic motion of the NBD-PE probe at a bilayer composition of ca. 2:1 mono- and bis-AcrylPC. This inhibition of small molecule mobility within the bilayer signals a change in the polymerized bilayer structure, which was ascribed to gelation (crosslinking) (8,11). 3
ο. .σ .p. ο ο
ο. .σ R ο ο
Mono-AcrylPC
Bis-AcrylPC
Mono-SorbPC
Bis-SorbPC
Scheme 1.
Poly(lipid) solubility was measured by attempting to dissolve the dehydrated lipid polymer, removing any insoluble material and weighing the recovered soluble polymer after removal of the solvent. The drastic change in solubility between
121
sample compositions that are below and above the gel point has been usefully employed in isotropic polymerizations as an indication of crosslinking (72). The bilayer composition was controlled by varying the molar ratio of monosubstituted and bis-substituted lipids and examining the change in solubility associated with the onset of crosslinking. Because gelation is dependent on the percent conversion to polymer, these experiments were performed at high conversion (>85%) (75). A preliminary examination of the effectiveness of various solvents for polymerized zwitterionic PC lipids indicated that 1,1,1,3,3,3-hexafluoro2-propanol (HHP) was the most effective solvent for these polymers. HFIP is an excellent hydrogen-bond donor, therefore it can interact strongly with the carbonyl functionalities of the polymeric lipids (14-16). Furthermore its low self-association makes it a good solvent for zwitterionic polymers (77). The solubility of linearly polymerized phospholipids in HFIP is great enough to permit the effective use of poly(lipid) solubility to estimate the mole fraction of the bis-substituted lipid required for effective crosslinking of the bilayer. The weight percent of the lipid sample that was soluble in HFIP is shown in Figure 1 as a function of mole fraction of bis-AcrylPC in the bilayer at the time of the polymerization.
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Mole Fraction bis-AcrylPC Figure 1. Weight percent solubility of polymerized mono-AcrylPC/bis-AcrylPC bilayers as a function of the mole percent of bis-AcrylPC. Initiation by decomposition of A I B N at 60±2 °C; [M]/[I] = 5. The polymerized lipid solubility is drastically different if the mole fraction of bisAcrylPC is 0.25 (soluble) as opposed to 0.38 (insoluble). This physical change suggests the formation of crosslinked polymers when the mono-AcrylPC to bisAcrylPC ratio was ca. 2:1, whereas at a molar ratio of ca. 3:1 or higher the polymerized lipids do not appear to be crosslinked. The data in Figure 1 also show that even when the bilayer composition has insufficient bis-AcrylPC to effectively crosslink the bilayer, the resulting polymer chains were not completely solubilized by the HFIP. The poly(AcrylPC) chains are relatively large and the reported polydispersity indicates that some of the polymer chains have molecular weights approaching a million (18,19). High molecular weight chains, such as the larger poly(AcrylPC) chains, can become entangled thereby preventing complete solubilization even in HFIP. Data for poly(SorbPC) bilayer compositions also
122 indicate a change in solubility at ca. 2:1 ratio of mono-SorbPC to bis-SorbPC (77). Radical polymerization of mono-SorbPC yields poly(lipid) with molecular weights one quarter that of mono-AcrylPC. Consequently the solubility of noncrosslinked poly(SorbPC) in HFIP was greater than that of poly(AcrylPC) (18-20). Ease of solubilization can be usefully employed to characterize the chemical stability of lipid bilayer vesicles in aqueous suspension. Although organic solvents are not especially effective for such studies, surfactants are ideal as long as the polymer chains are short enough to be incorporated into a mixed micelle. It has long been known that surfactant micelles are effective at solubilizing lipids and other biomembrane components. The kinetics of solubilization and interaction of nonionic surfactants with phospholipid vesicles has been reviewed (21-23). The effective solubilization of lipids into micelles requires that the surfactant concentration be greater than its critical micelle concentration (CMC) and that the surfactant to lipid molar ratio be ca. 3 to 5. Regen and coworkers reported that stability of vesicles to surfactant treatment increases in the following order: unpolymerized < linearly polymerized < crosslinked (5). Surfactant solubilization (lysis) of bilayers initially involves the incorporation of surfactant into the bilayer. When the surfactant concentration predominates, the lipids no longer exist in a vesicle but are incorporated into a mixed micelle of lipid and surfactant. If the lipids are crosslinked in a polymerized vesicle or are incorporated into quite large linear polymers they cannot be readily solubilized by the surfactant. The chemical stabilities of polymerized vesicles composed of both monoand bis-SorbPC were examined by surfactant lysis monitored by QELS (Figure 2). 1.6
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Mole Fraction bis-SorbPC Figure 2. Average mean diameter of polymerized mono-Sorb/bis-SorbPC L U V after addition of 15 equivalents of Triton X-100 as a function of mole fraction bisSorbPC. Polymerization by direct photoirradiation. The average particle size after the addition of 15 equivalents of Triton X-100 (TX100) to the polymerized bilayer samplevaries as a function of the mole fraction of bis-SorbPC in the bilayer prior to polymerization. A change in vesicle stability to the addition of TX-100 occurs for vesicle compositions with greater than a mole fraction of 0.34 bis-SorbPC. The resistance to solubilization of the polymerized
123 vesicles composed of high mole fractions of bis-SorbPC is attributed to effective 2Dgelation of the vesicles during the polymerization, because crosslinking of the lipids prevents the extraction of lipid molecules from the vesicle and into mixed micelles. Investigation of Crosslinking Inefficiency. These studies indicate that effective bilayer crosslinking requires a high (0.3±0.05) mole fraction of the appropriate bisPC. This contrasts with the quite low mole fractions of bis-monomers that are usually necessary for crosslinking polymerizations in isotropic media (1324). Inefficiencies in crosslinking reactions could result from low conversions to polymer, significant differences in chemical reactivity, and/or small degrees of polymerization. The first two possibilities appear unlikely because the experimental data reported here were all obtained at high conversions of monomers to polymer, and as noted earlier the relative reactivities of the groups in the mono- and bis-lipids should be quite similar. Moreover differences in the degree of polymerization of AcrylPC monomers (Χ =233±29) and SorbPC monomers (Χ =7±3) only resulted in modest differences in the observed crosslinking efficiency (19,20). Thus other potential explanations for the crosslinking inefficiency were examined. A reasonable possibility is intramolecular macrocyclization of die sn-l and sn-2 groups in the same lipid (Scheme 2). Such a macrocyclization would yield a ring within a linear polymeric backbone. Although the formation of such a large macrocycle is statistically unfavored in an isotropic media, the two-dimensional constrained environment of the bilayer reduces the number of conformations available to the lipid tails and backbone, thereby enhancing the possibility of intramolecular Λ
Λ
Scheme 2. macrocyclization. The drawing in Scheme 2 implies that the two reactive groups in a bis-PC are positionally inequivalent in a relatively static bilayer (25). This suggests an A B type crosslinking with sn-l tail of bis-PC and mono-PC preferentially reacting with each other in a topological fashion rather than with the sn-2 tail of bis-PC. In this case a crosslink between polymer chains occurs when a reactive group on the sn-2 tail of a bis-PC lipid in one polymer chain reacts with a similar group in a neighboring polymer chain. This mode of crosslinking appears to require reactive sn-2 tails to form dimeric links between poly (lipid) chains. A competitive process is the reaction of the groups in sn-2 tails with others in the same polymer chain. Such an intrachain reaction modulates the polymer motion without linking polymer chains together and therefore requires a larger mole fraction of the bis-PC to cause gelation. A dynamic bilayer such as that found at temperatures above the main phase transition temperature, T m , provides a more favorable environment for the reaction of a reactive group in sn-l tails with each other as well as with reactive groups in sn-2 tails. Such a process leads to an irregular polymer backbone with an occasional bis-PC bridge between two polymer chains via a sn-l group in one polymer chain and a sn-2 group in the second chain. Recent
124 molecular simulations of bilayers in the L a phase (>Tm) reveal a dynamic mixture of lipid tail conformations for the portion of the lipid tail distal to the water interface (26^7). Further examination of the crosslinking efficiency utilized a symmetrical crosslinking agent, SorbNXL8 (4). The synthesis of SorbNXL8 was accomplished using the efficient reactions described by Figure 3 (28). The synthesis of the starting acid 9-(sorbyloxy)nonanoic acid (1) was accomplished by the methods previously reported (29,30). The stability of poly(lipid) vesicles obtained from polymerization of various bilayer compositions of mono-SorbPC and SorbNXL8 were examined by surfactant lysis. The relative reactivity of mono-SorbPC and SorbNXL8 should be similar since the reactive group is identical. The monoSorbPC appears to have an equal opportunity to react with either chain of SorbNXL8, since the symmetrical afunctional monomer places both reactive sorbyl
Figure 3. Synthesis of symmetrical crosslinking agent SorbNXL8: a) oxalyl chloride, benzene; b) methyl diethanolamine, DMAP, THF; c) CH3Br, acetone. moieties at the same depth within a bilayer (31). Reaction of one SorbNXL8 acyl chain may reduce the reactivity of the second group (24). Note that the high overall conversion insures the reaction of both sorbyl groups on the symmetrical crosslinker. The change in average mean diameter of the vesicle particles after the addition of 8 equivalents of TX-100 to the polymerized bilayer sample is shown in Figure 4 as a function of the mole fraction of SorbNXL8 in the bilayer prior to polymerization. A change in vesicle stability to TX-100 occurs for vesicle samples having between 0.35 and 0.53 mole fraction SorbNXL8. This behavior is similar to that observed for polymerizations of bilayer vesicles composed of mono-SorbPC and bis-SorbPC (Figure 2). If bis-SorbPC was predominantly an A B type of crosslinker, then the crosslinking efficiency of SorbNXL8 and bis-SorbPC should be dissimilar. Since that was not observed these data suggest that intramolecular macrocyclization is an important reaction path in bilayers, that may contribute to the observed inefficiency of crosslinking in bilayer polymerization with bis-substituted lipids. Crosslinking Polymerizations in Nonlamellar Phases. A polymerizable bicontinuous cubic phase with Pn3m symmetry is formed by a 3:1 mixture of the PE:PC lipids shown in Scheme 3 (32). The structure before and after polymerization was confirmed by P NMR, X-ray diffraction and transmission electron microscopy (TEM). The bis-PC shown not only acts to facilitate formation of the cubic phase but serves as a crosslinking agent in the polymerization because it 3 1
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Figure 4. Average mean diameter of polymerized mono-Sorb/SorbNXL8 L U V after addition of 8 equivalents of Triton X-100 as a function of mole fraction SorbNXL8. Polymerization by direct photoirradiation. is a bifunctional monomer. Since the mole fraction of the bis-PC (0.25) may be sufficient to crosslink this assembly, we examined the solubility of the polymerized Qn phase. Note that previous studies in the lamellar phase for SorbPC lipids revealed the onset of gelation at a lower mole fraction of bis-SorbPC with radical polymerizations compared to U V irradiation (77). After polymerization the sample was dehydrated and found to be insoluble in HFIP, thereby indicating the assembly was crosslinked. The assembly retained its architecture after dehydration and in the presence of organic solvent as observed by T E M . Whether the effective crosslinking observed at a 0.25 mole fraction of bis-PC was favored by the placement of the reactive groups near the lipid glycerol backbone and/or due to the nature of the nonlamellar phase still remains to be examined.
MonoPE
Scheme 3.
BisPC
The solubility methodology was also employed to study the nature of polymerizations in the Hn phase for the lipid shown in Scheme 4. The bis-PE lipid formed a Hn phase before and after polymerization at temperatures from 5 to 80 °C 31
as determined by variable temperature P N M R and X-ray diffraction (35). This polymerization in the Hn phase yielded an assembly which was completely
126 insoluble in all solvents including HFIP. The mole fraction of bis-lipid was unity and in this case a crosslinked polymeric assembly was easily achieved. The insolubility and retention of architecture in the presence of organic media greatly expands the possible utility of these novel nanoporous materials.
Scheme 4. General Discussion The solubility and surfactant lysis data for the AcrylPC bilayers agrees well with the lateral diffusion measurements obtained by FRP, thereby providing a high degree of confidence in both procedures since the errors in each are dissimilar. Both methodologies are insensitive to the molecular weights of the polymeric lipids. The HFIP solubility protocol is most effective with poly(lipid) chains that have a degree of polymerization less than ca. 10 . As long as this possible limitation is given proper consideration, the HFIP procedure appears to have broad applicability for the determination of gelation of polymerized assemblies, i.e. bilayers, L B multilayers, etc. The solubility of cubic and hexagonal phases has been used to provide evidence for polymerization yielding crosslinked nonlamellar assemblies. The solubility procedure does not require the sophisticated instrumentation necessary for FRP studies, and the amount of lipid required is only dependent on the sensitivity of an available balance. The effect of polymer size limits the applicability of surfactant lysis, but not necessarily the type of assembly since the addition of TX-100 could be expanded into nonlamellar phases with an appropriate change in monitoring of dissolution. For example the dissolution of Η π or lamellar phases formed by 3
31
phospholipids could be monitored by P NMR (34). Experimental Solvents and Reagents. The lipids were purified using silicic acid specifically treated for lipid chromatography (Biosil-A 200-400 mesh; Biorad). Lipid purity was evaluated by thin-layer chromatography (TLC) with chloroform/methanol/water (65:25:4 by volume) and visualized by a U V lamp. Ion exchange resin A G 501-X8 was obtained from Bio-Rad (Richmond, CA). Flash chromatography silica gel (200425 mesh) was obtained from Fischer Scientific. Azobis(isobutyronitrile), A I B N , was purified by recrystallization three times from methanol. 4-(N,NDimethylamino)pyridine (DMAP) was obtained from Aldrich Chemical Co. (Milwaukee, WI) and was purified by recrystallization from chloroform/diethyl ether (1:1, v/v) three times. Potassium persulfate, sodium bisulfite, Triton X-100 (TX-
127 100), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Aldrich Chemicals and used as received. The lipids were hydrated in M i l l i - Q water, Millipore Inc.. Benzene and tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl. Chloroform was distilled from calcium hydride. N , N Dimethylformamide (DMF) was vacuum distilled and stored over molecular sieves (5A). A l l other reagents were used as received or purified by standard methods. Methods. Compounds containing UV-sensitive groups were handled under yellow lights. H N M R spectra were acquired on a Varian-Unity 300 spectrometer in chloroform-d with tetramethylsilane as an internal reference. U V - V i s absorption spectra were recorded on a Varian DMS 200 spectrophotometer. Quasi-elastic light scattering (QELS) was performed with a BI8000-autocorrelator from Brookhaven Instruments Corp. and particle size was calculated with the accompanying software. The polymerizable lipids, i.e. mono-SorbPC, bis-SorbPC, mono-AcrylPC, and bisAcrylPC, were synthesized as described previously (19,20). l
9-(Sorbyloxy)nonanoic Acid (1). 9-(Sorbyloxy)nonan-l-ol (29,30) (4.2 g, 16.4 mmol) in DMF (20 mL) was added dropwise to pyridinium dichromate (PDC) (21.5 g, 57.3 mmol) in DMF (30 mL) at 0 °C. The reaction was allowed to warm slowly to room temperature and stirred under a positive argon atmosphere for 1 day. The mixture was filtered through silica gel and this process repeated until no trace of PDC was observed. The solution was then concentrated and the crude aldehyde purified by column chromatography using Hex/EtOAc/HCOOH (60/40/1), affording 8-(sorbyloxy)octanoic acid. Yield 2.8 g (64%); T L C : R f = 0.51 (Hex/EtOAc/HCOOH, 60/40/1); (l)H-NMR ( C D C I 3 ) d 1.20-1.40 ppm (br s, 8H, C H 2 ) , 1.52-1.70 ppm (m, 4H, C H 2 C H 2 R ) , 1.78-1.86 ppm (d, 3H, C = C H C H 3 ) , 2.282.37 ppm (t, 2H, Œ 2 C O 2 H ) , 4.05-4.15 ppm (t, 2H, C O 2 C H 2 ) , 5.70-5.79 ppm (d,
1H, C=CHC02), 6.05-6.22 ppm (m, 2H, =CH-CH=), 7.15-7.27 ppm (m, 1H, CH=CHCH3).
9-(Sorbyloxy)nonanoyl Chloride (2). 9-(Sorbyloxy)nonanoic acid (0.66 g, 2.6 mmol) in dry benzene (10 ml) was reacted with oxalyl chloride (0.47 g, 2.9 mmol) by dropwise addition. The solution was stirred at room temperature for 30 min beyond gas evolution and then excess oxalyl chloride and solvent were removed by rotary evaporation. Recrystallization from benzene (2x) afforded the acid chloride as colorless needles that were used directly in the esterification. Yield 0.70 g (100%). Bis-9-(sorbyIoxy)nonanoyl Methyl Diethanolamine (3). 9-(Sorbyloxy) nonanoyl chloride (0.70, 2.6 mmol) in THF (10 ml) was added dropwise to methyl diethanolamine (0.15g, 1.2mmol) and 4-dimethylaminopyridine (0.46 g, 3.7 mmol) in T H F (20 ml). The reaction was stirred overnight under argon. 4Dimethylaminopyridinium hydrochloride was filtered off and the filtrate concentrated by rotary evaporation resulting in a viscous oil which was further purified by flash silica gel chromatography using Hex/EtOAc/TEA, 100/100/1. Yield 0.44 g, (59%): T L C R f = 0.25 (Hex/EtOAc/TEA, 100/100/1); * H - N M R ( C D C I 3 ) d 1.20-1.40 ppm (br s, 16H, C H 2 ) , 1.52-1.70 ppm (m, 8H, C H 2 C H 2 R ) , 1.82-1.88 ppm (d, 6H, C = C H C H 3 ) , 2.12-2.21 ppm (t, 4H, C H 2 C O 2 R ) , 2.22 ppm (s, 3H, N Q I 3 ) , 4.08-4.18 ppm (m, 8H, N C H 2 C H 2 , C O 2 C H 2 ) , 4.59-4.63 ppm (m, 4H,
N C H 2 ) , 5.70-5.79 ppm (d, 2H, C=CHC02), 6.05-6.22 ppm (m, 4H, =CHCH=), 7.15-7.27 ppm (m, 2H, C H = C H C H 3 ) .
128
Bis-9-(sorbyIoxy)nonanoyI Dimethyl Diethanolamine (4). Bromomethane in diethylether (0.75 ml, 1.5 mmol) was added to 3 (0.44 g, 0.70 mmol) in dry acetone (10 ml) at 0 °C. The reaction was stirred overnight and a precipitate formed. The solvent and excess bromomethane were removed by rotary evaporation resulting in a white solid. Yield 0.43 g (96%); *H-NMR ( C D C I 3 ) d 1.20-1.40 ppm (br s, 16H, C H 2 ) , 1.52-1.70 ppm (m, 8H, C H 2 C H 2 R ) , 1.82-1.88 ppm (d, 6H, C=CHCii3), 2.12-2.21 ppm (t, 4H, C H 2 C O 2 R ) , 3.58 ppm (s, 6H, N C H 3 ) , 4.08-4.15 ppm (t, 4H, N C H 2 C E 2 ) , 4.14-4.18 ppm (m, 4H, C O 2 C H 2 ) , 4.59-4.63 ppm (m, 4H,
N C H 2 ) , 5.70-5.79 ppm (d, 2H, C=CHC02), 6.05-6.22 ppm (m, 4H, =CHCH=), 7.15-7.27 ppm (m, 2H, CH=CHCH3). U V (λmax = 262 nm C H 3 O H , ε = 44,715 L/cm mol) Thermal AIBN Polymerizations. Large multilamellar vesicles of polymerizable lipid were prepared as follows: approximately 2.5 to 15 mg of lipid was evaporated from stock solutions (10 mg/ml in benzene) by passing a stream of argon over the sample and drying under high vacuum (0.4 mm Hg) for a minimum of 4 h. Each lipid was dried and weighed separately for mixtures of mono- and bis-PC. The lipid weight was determined and each lipid film redissolved in 2 ml of benzene, then the lipids were combined. The appropriate amount of initiator from a freshly prepared A I B N stock solution (1-1.5 mg/ml in benzene) was added to yield a monomer to initiator ratio of 5, where [M] represents the concentration of polymerizable groups. The solvent was evaporated as above and dried under high vacuum for 2 h. A l l steps involving lipid and A I B N were performed in subdued light. The dried lipid/AIBN film was hydrated with deoxygenated MilliQ water to a final concentration of 7 mM. Samples were vortexed to uniformity and subjected to ten freeze-thaw-vortex cycles (-77°C-*T +5 °C) cycles. Samples were placed in an ampoule sealed with a septum and flushed with argon for 0.5 h. Polymerizations were performed at 60±2 °C in a water circulating bath under a positive argon pressure. A l l polymerizations were carried out in the absence of light. Polymerizations were monitored by U V absorption spectroscopy of aliquots diluted with Milli-Q water to ca. 80 μ Μ m
Redox Polymerizations. Unilamellar vesicles (LUV) of SorbPC were prepared as above in the absence of AIBN. The lipid suspension was extruded ten times through two stacked Nuclepore polycarbonate filters (0.1 mm diameter) at 37 °C using a stainless steel extruder from Lipex Biomembranes. The redox initiator was prepared from K 2 S 2 O 8 (300 mg, 1.1 mmol) and NaHS03 (115 mg, 1.1 mmol) weighed into a 10 ml volumetric flask and diluted. An aliquot (200-300 ml) was pipetted into the vesicle suspension. The sample was placed into an ampoule and sealed with a septum and flushed with argon for 0.5 h. Polymerizations were performed at 60±2 °C in a water circulating bath under a positive argon pressure for 18 h. A l l polymerizations were carried out in the absence of light. Polymerizations were monitored by U V absorption spectroscopy of aliquots diluted with Milli-Q water to ca. 80 μΜ. Direct Photoirradiation. L U V were placed into a 3 ml quartz cuvette equipped with a magnetic stirbar and placed 1 cm from a low pressure Hg vapor Pen Lamp. Samples with a lipid concentration of 100-300 μΜ were irradiated for 45 min at a temperature of 40 °C. Polymerizations were monitored by U V absorption spectroscopy as above.
129 Weight Percent Solubility. Only samples with greater than 85% monomer conversion as determined by UV-Vis spectroscopy were used in the solubilty studies. Samples were lyophilized after polymerization. The lipid was weighed and HFIP added until the concentration was 5 mgJml. The samples were shaken for 2 min and allowed to stand for 5 min. The solution was filtered into a dry test tube through a pipet with glass wool to remove any solid particles. The pipet was then rinsed with 0.5 ml of HFIP. The solvent was removed by passing a stream of argon over the sample then drying under high vacuum for a minimum of 4 h leaving the soluble polymer, which was weighed and used to calculate the percent solubilty. Surfactant Dissolution of Vesicles. L U V were prepared as described above. After polymerization the L U V were characterized by QELS for 2 ml sample with a lipid concentration of 100-300 μΜ. An aliquot of 45 m M TX-100 solution was added. Each aliquot was equal to 2 equivalents of lipid. The light scattering intensities were determined again by QELS. TX-100 was added in 2 equivalent increments up to 15 total equivalents. Measurements at each concentration of T X 100 were performed a minimum of three times. The average mean diameter of vesiclesJparticles was calculated by multiple mathematical procedures, i.e. C U M U L A N T , exponential sampling, non-negatively constrained least square, and CONTIN. With the exception of exponential sampling, consistent results were obtained. Acknowledgment. This research was supported by the National Science Foundation. Literature Cited (1) O'Brien, D. F., Trends Polym. Sci. 1994, 2, 183-188. (2) Dorn, K.; Klingbiel, R. T.; Specht, D. P.; Tyminski, P. N.; Ringsdorf, H.; O'Brien, D. F., J. Am. Chem. Soc. 1984, 106, 1627-1633. (3) Stefely, J.; Markowitz, Μ. Α.; Regen, S. L., J. Am. Chem. Soc. 1988, 110, 7463-7469. (4) Ohno, H.; Takeoka, S.; Hayashi, N.; Tsuchida, E., Makromol. Chem., Rapid Comm. 1987, 8, 215-218. (5) Regen, S. L.; Singh, Α.; Oehme, G.; Singh, M., J. Amer. Chem. Soc. 1982, 104, 791-795. (6) Tsuchida, E.; Hasegawa, E.; Kimura, N.; Hatashita, M.; Makino, C., Macromolecules 1992, 25, 207-212. (7) Ohno, H.; Takeoka, S.; Iwai, H.; Tsuchida, E., Macromolecules 1988, 27, 319-322. (8) Kölchens, S.; Lamparski, H.; O'Brien, D. F., Macromolecules 1993, 26, 398400. (9) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elson, E. L.; Webb, W. W., Biophys. J. 1976, 16, 1055-1069. (10) Koppel, D. E.; Axelrod, D.; Schlessinger, J.; Elson, E. L.; Webb, W. W., Biophys. J. 1976, 16, 1315-1329. (11) Sisson, T. M.; Lamparski, H. G.; Kölchens, S.; Elayadi, Α.; O'Brien, D. F., Macromolecules 1996, 29, 8321-8329. (12) Ulbrich, K.; Ilavsky, M.; Dusek, K.; Kopecek, J., Europ. Polym. J. 1977, 13, 579-585 (13) Storey, B., J. Polym. Sci. 1965, A3, 265-282. (14) Hamori, E.; Prusinowski, L. R.; Sparks, P. G.; Hugues, R. E., J. Phys. Chem. 1965, 69, 1101-1105. (15) Montroy Soto, V. M.; Galin, J. C., Polymer 1984, 25, 254-262. (16) Pujol-Fortin, M.; Galin, J.-C., Polymer 1994, 34, 1462-1472.
130 (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34)
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