0306.3Olh/79/0701-1061/$02.00/0
l Radiation
Sensitivity:
Facts and Models
RADIOLYSIS
OF HETEROGENEOUS JANOS
Department
of Chemistry,
Texas Agricultural
H.
FENDLER,
& Mechanical
INANIMATE
SYSTEMS
Ph.D. University,
College Station, TX 77843, U.S.A.
Aqueous micelles, reversed micelles, liposomes and surfactant vesicles have been utilized in recent years to model the more complex microenvironment of biological systems and energy deposition therein. Micelles are dynamically formed from surfactants at their critical micelle concentration either in water (aqueous micelles) or in nonpolar solvents (reversed micelles). Conversely, surfactant vesicles and liposomes are thermodynamically stable. Brief physical chemical descriptions of these models are given. The available data on the interaction of the hydrated electron, the hydroxyl radical and the hydrogen atom with the models themselves are summarized. Attention is focused on recent investigations of radical and excited state processes in the micellar environments. The results obtained are related to problems of radiobiological importance. Radiolysis,
Aqueous micelles, Reversed micelles, Liposomes, INTRODUCTION
e& + .H + .OH + Hz + H202 + H30+
(1)
whose yields are: GeIq = 2.8 t 0.1, GH = 0.6 ? 0.1, GoH = 2.420.3, GH2 = 0.45 and GH,o, = 0.71. With gamma rays the primary products givkn in reaction (1) are formed inhomogeneously in small, widely spaced clusters, which have been known as spurs, within a time scale of lo-” to 10m8sec. The direct visualization by pulse radiolysis, of the hydrated electron and of the transients formed subsequently to energy deposition has provided major momentum for’ radiation chemical studies. A large body of information has been accumulated on the reactions of the simplest nucleophile, the hydrated electron, and the hydroxyl radical, in the past 15 years;3’ rate constants have been determined for the reaction of these species with thousands of substrates.’ Radiation chemistry has, in fact, been applied to mechanistic organic and inorganic chemistry.‘0,26,29 Radiation chemical investigations in aqueous media have been rationalized Acknowledgements-Support Foundation and the Robert
of the A. Welch
vesicles.
by invoking their relevance to radiobiological processes; such biomolecules as amino acids, nucleotides, quinones, porphyrins, etc., have been examined. Until fairly recently, all of these investigations had been carried out in dilute solutions. Real radiobiology, however, does not occur in dilute aqueous solutions. Although the body contains more than 75% water, most of it is bound to, and interacts with, proteins, membranes and other macromolecule assemblies. It is widely recognized that the properties of water at biological interfaces differ considerably from that of aqueous solutions containing only low molecular weight electrolytes and nonelectrolytes.” The effective viscosity of water at biological interfaces is very much higher while the effective polarity is very much lower than those of pure water. At the same time, the proteins themselves associate, form complexes with other molecules, and are involved in secondary and tertiary structures. In addition to hydrogen bonding, hydrophobic forces become quite important. It may be fully expected, therefore, that energy deposition and its consequences are different in biological fluids and in dilute aqueous solutions. These differences have been recognized to some extent by radiation chemists who have extended their investigations to enzymes and proteins. Most of this work had inevitably been restricted to the in-
radiation chemists have During the past 2 decades obtained a detailed understanding of the processes involved when high energy is deposited in water.2.3’.32 The primary chemical result of water irradiation is the formation of the following species: HZ0 -
Surfactant
knowledged.
National Science Foundation is ac1061
1062
Radiation
Oncology
0
Biology
0
Physics
vestigation of enzyme denaturations by steady state radiolysis, or to the first few steps of e&, .H, and .OH attacks. At present, quantitative investigations of all the steps involved in the radiation chemistry of such large molecules is not yet feasible. The need to bridge the gap between detailed radiation chemical investigations of the individual steps following energy deposition in dilute aqueous solutions and in the body of living organisms has been recognized for some time. Studies have been carried out on various model systems. The purpose of the present paper is to summarize the results obtained with model systems which mimic heterogenity. Micelles and vesicles have been found to be useful in many respects as models for mimicking secondary and tertiary associations, changes in microenvironments and for providing compartments which separate reactive species. Results of photochemistry and radiation chemistry will be highlighted following a brief description of the physical chemical properties of these systems; we will focus attention on recent results, primarily based on work carried out in our own laboratories. DESCRIPTION
OF MODELS
Aqueous micelles Amphiphatic molecules, surfactants, dynamically associate above a certain concentration, known as the critical micelle concentration (CMC), to large aggregates known as micelles.‘4 Figure 1 is a schematic illustration of a micelle. The surfactant can be neutral or charged. Typically micelles contain 60-100 monomers. To a first approximation, aqueous micelles are spherical having rather liquid hydrocarbon-like interiors. The colloid chemistry of micellization and the properties of aqueous micelles are well understood. Analogies had been drawn between the structure of spherical micelles and a globular protein, as well as between substrate-micelle and substrate-enzyme reactions and interactions. Nonpolar molecules are readily solubilized by aqueous micelles. Solubilization is also a dynamic process. The predominant substrate solubilization site is at the water-micelle interface, the Stern layer, or in the immediate vicinity of the surfactant headgroups. Substrate intercalation into the deep micellar interior has not been demonstrated. Electrostatic and hydrophobic interactions determine the extent and site of substrate solubilization. Information is required on the reactivity and photochemistry of micelle forming surfactants themselves prior to investigating the reactions that take place therein. Reversed micelles Surfactants also associate in nonpolar solvents.“.‘4 In the absence of any other additives their association
July
1979,
Volume
5, No. 7
kStern layer up to (I feu; 8 Gouy-Chapman double layer, up to0 several hundred A
Fig. 1. Schematic
representation
of an aqueous micelle.
is of the monomer e dimer $ trimer $ . . . n-mer type. The size of the aggregates is dependent on the concentration of the surfactant. Typically, for alkylammonium carboxylates, the average aggregation number is in the order of 8-10. Addition of a third component, such as water, dramatically increases the size of the aggregates. Figure 2 illustrates the structure of a swollen reversed micelle. Surfactant aggregates in nonpolar solvents solubilize copious amounts of water and polar molecules in their interior. Solubilized molecules are held in the interior of reversed micelles more strongly than those in aqueous micelles. Favorable and concerted proton transfer becomes feasible in the environment of surfactantentrapped water pools. One can change microscopic polarities and viscosities at will by altering the amount of added water. Indeed it is quite feasible to obtain polarities as low as that of benzene in the interior of reversed micelles.” Since the substrates in the surfactant-entrapped water pool is separated from the bulk water by half a hydrocarbon bilayer, reversed micelles can be considered to be very primitive membrane models. Liposomes There is a very important difference between micelles and vesicles. Micelles are formed dynamically from monomeric surfactants, and the substratemicelle interaction is also dynamic. On the other hand, liposomes or surfactant vesicles, once formed, are stable for several weeks; the diffusion of substrates entrapped therein can only occur slowly, or
Radiolysis
of heterogeneous
inanimate
systems 0 J. H. FENIII ER
1063
of dodecylammonium propionate aggregate in benzene in the presence of solubilized model for the ultrafast proton transfer at the hydration shell of the surfactant headgroups in reversed micellar DAP in benzene. Since the concentration of surfactant is in a very large excess over the probe, proton transfer must occur from dodecylammonium propionic acid to pyrene I-carboxylate. For the sake of clarity, 2 pyrene moieties are drawn in the aggregate shown. In reality, there is much less than 1 probe per aggregate. Fig. 2. An illustration
water. Also, a proposed
they can be liberated by the destruction of the vesicle. Liposomes, smectic mesophases of phospholipid bilayers, have been utilized as membrane models since 1953.3 There are several ways to form spherical liposomes. The most commonly used one is to disperse in water a thin phospholipid film which had been deposited on the wall of a flask by evaporating a chloroform solution of the phospholipid and cholesterol. The aqueous dispersal leads to the formation of multicompartment liposomes whose sizes are not uniform. Sonication above their phase transition temperature gives fairly uniform single compartment bilayer vesicles, which on the average have 250 A diameters. Several molecules have been entrapped in the interior of liposomes. They can be released only if the liposome is destroyed by lysing with alcohol or with a detergent. Figure 3 illustrates the solubilization sites of polar, amphiphatic and apolar molecules in multicompartment liposomes. Small ions permeate the vesicle relatively freely, however. Liposomes undergo osmotic shrinkage and swelling if they are placed into appropriate electrolyte solutions. They
also undergo fusion to larger entities. The biophysics of liposomes has been extensively investigated in the past 2 decades.3,25 Surfactant
vesicles
The formation of spherical bilayers from completely synthetic surfactants has been demonstrated reCent~y.7.27.28"4 Such surfactant vesicles were formed from dialkyldimethylammonium halides7.27.34 and dicetylphosphate” upon sonication of their aqueous dispersions. If the sonication is carried out for a short time, multicompartment vesicles are formed. On the other hand, sonication for a longer time results in single compartment bilayer vesicles whose diameter ranges between 800-1500 A. Figure 4 is a naive presentation of a surfactant vesicle. Both the dialkyldimethylammonium halides and the dicetylphosphate surfactant vesicles are formed only if the sonication is carried out above their phase transition temperatures. Once these vesicles are formed, they remain stable for months. Within a few electrolytes they are osmotically active. In the absence of other additives, the dialkyldimethyl-
Radiation
1064
Oncology
POLAR SOLUTE
0
Biology
IN AOUEOUS
l
July
Physics
1979,
Volume
5, No. 7
PHASE
LIPOSOME
Fig. 3. Available
solubilization
sites for polar, nonpolar and amphiphatic liposomes.
ammonium halide surfactant vesicles are relatively permeable to protons and to small electrolytes. Addition of cholesterol, however, firms up these vesicles. The most useful properties of surfactant vesicles are that they can entrap and retain large molecules in their interior for some time. Since they are chemically simple and do not degrade, one can visualize extensive utilization of surfactant vesicles both in basic and applied ._ studies.
molecules
in multicompartment
REACTIVITY OF RADIATION SPECIES WITH THE MODEL
INDUCED SYSTEMS
As expected, simple surfactants which have no aromatic moiety are relatively unreactive towards hydrated ions.16 Rate constants for the reaction of the hydrated electron with hexadecyltrimethylammonium bromide, sodium dodecyl sulphate or polyoxyethylene(l5)nonylphenol are in the order of 10’ to IO6 M-’ secm’.18 The hydrated electron is also unreactive Cl-
DODAC 2C,,iJ2C,CI
n
Fig. 4. Formation
of completely
synthetic
dioctadecylammonium
chloride surfactant
vesicles.
Radiolysis
Table 1. Rate constants
of heterogeneous
inanimate
systems
0 J. H. FENDLER
for the reactions of e&, .OH and .H with surfactants and vesiclest
1065
(S), micelles
k, M-’ set-’
e,+S Hexadecyltrimethylammonium bromide [S] < CMC [S] > CMC Sodium dodecyl sulfate [S] < CMC [S] > CMC PoIyoxyethylene(6)nonylphenol [S] < CMC [S] >CMC Spherical bilayer /3-y-distereaoyl-L-a-phosphatidylcholine vesicle*
.OH+S
.H+S
NA
l Radiation
Sensitivity:
Facts and Models
RADIOLYSIS
OF HETEROGENEOUS JANOS
Department
of Chemistry,
Texas Agricultural
H.
FENDLER,
& Mechanical
INANIMATE
SYSTEMS
Ph.D. University,
College Station, TX 77843, U.S.A.
Aqueous micelles, reversed micelles, liposomes and surfactant vesicles have been utilized in recent years to model the more complex microenvironment of biological systems and energy deposition therein. Micelles are dynamically formed from surfactants at their critical micelle concentration either in water (aqueous micelles) or in nonpolar solvents (reversed micelles). Conversely, surfactant vesicles and liposomes are thermodynamically stable. Brief physical chemical descriptions of these models are given. The available data on the interaction of the hydrated electron, the hydroxyl radical and the hydrogen atom with the models themselves are summarized. Attention is focused on recent investigations of radical and excited state processes in the micellar environments. The results obtained are related to problems of radiobiological importance. Radiolysis,
Aqueous micelles, Reversed micelles, Liposomes, INTRODUCTION
e& + .H + .OH + Hz + H202 + H30+
(1)
whose yields are: GeIq = 2.8 t 0.1, GH = 0.6 ? 0.1, GoH = 2.420.3, GH2 = 0.45 and GH,o, = 0.71. With gamma rays the primary products givkn in reaction (1) are formed inhomogeneously in small, widely spaced clusters, which have been known as spurs, within a time scale of lo-” to 10m8sec. The direct visualization by pulse radiolysis, of the hydrated electron and of the transients formed subsequently to energy deposition has provided major momentum for’ radiation chemical studies. A large body of information has been accumulated on the reactions of the simplest nucleophile, the hydrated electron, and the hydroxyl radical, in the past 15 years;3’ rate constants have been determined for the reaction of these species with thousands of substrates.’ Radiation chemistry has, in fact, been applied to mechanistic organic and inorganic chemistry.‘0,26,29 Radiation chemical investigations in aqueous media have been rationalized Acknowledgements-Support Foundation and the Robert
of the A. Welch
vesicles.
by invoking their relevance to radiobiological processes; such biomolecules as amino acids, nucleotides, quinones, porphyrins, etc., have been examined. Until fairly recently, all of these investigations had been carried out in dilute solutions. Real radiobiology, however, does not occur in dilute aqueous solutions. Although the body contains more than 75% water, most of it is bound to, and interacts with, proteins, membranes and other macromolecule assemblies. It is widely recognized that the properties of water at biological interfaces differ considerably from that of aqueous solutions containing only low molecular weight electrolytes and nonelectrolytes.” The effective viscosity of water at biological interfaces is very much higher while the effective polarity is very much lower than those of pure water. At the same time, the proteins themselves associate, form complexes with other molecules, and are involved in secondary and tertiary structures. In addition to hydrogen bonding, hydrophobic forces become quite important. It may be fully expected, therefore, that energy deposition and its consequences are different in biological fluids and in dilute aqueous solutions. These differences have been recognized to some extent by radiation chemists who have extended their investigations to enzymes and proteins. Most of this work had inevitably been restricted to the in-
radiation chemists have During the past 2 decades obtained a detailed understanding of the processes involved when high energy is deposited in water.2.3’.32 The primary chemical result of water irradiation is the formation of the following species: HZ0 -
Surfactant
knowledged.
National Science Foundation is ac1061
1062
Radiation
Oncology
0
Biology
0
Physics
vestigation of enzyme denaturations by steady state radiolysis, or to the first few steps of e&, .H, and .OH attacks. At present, quantitative investigations of all the steps involved in the radiation chemistry of such large molecules is not yet feasible. The need to bridge the gap between detailed radiation chemical investigations of the individual steps following energy deposition in dilute aqueous solutions and in the body of living organisms has been recognized for some time. Studies have been carried out on various model systems. The purpose of the present paper is to summarize the results obtained with model systems which mimic heterogenity. Micelles and vesicles have been found to be useful in many respects as models for mimicking secondary and tertiary associations, changes in microenvironments and for providing compartments which separate reactive species. Results of photochemistry and radiation chemistry will be highlighted following a brief description of the physical chemical properties of these systems; we will focus attention on recent results, primarily based on work carried out in our own laboratories. DESCRIPTION
OF MODELS
Aqueous micelles Amphiphatic molecules, surfactants, dynamically associate above a certain concentration, known as the critical micelle concentration (CMC), to large aggregates known as micelles.‘4 Figure 1 is a schematic illustration of a micelle. The surfactant can be neutral or charged. Typically micelles contain 60-100 monomers. To a first approximation, aqueous micelles are spherical having rather liquid hydrocarbon-like interiors. The colloid chemistry of micellization and the properties of aqueous micelles are well understood. Analogies had been drawn between the structure of spherical micelles and a globular protein, as well as between substrate-micelle and substrate-enzyme reactions and interactions. Nonpolar molecules are readily solubilized by aqueous micelles. Solubilization is also a dynamic process. The predominant substrate solubilization site is at the water-micelle interface, the Stern layer, or in the immediate vicinity of the surfactant headgroups. Substrate intercalation into the deep micellar interior has not been demonstrated. Electrostatic and hydrophobic interactions determine the extent and site of substrate solubilization. Information is required on the reactivity and photochemistry of micelle forming surfactants themselves prior to investigating the reactions that take place therein. Reversed micelles Surfactants also associate in nonpolar solvents.“.‘4 In the absence of any other additives their association
July
1979,
Volume
5, No. 7
kStern layer up to (I feu; 8 Gouy-Chapman double layer, up to0 several hundred A
Fig. 1. Schematic
representation
of an aqueous micelle.
is of the monomer e dimer $ trimer $ . . . n-mer type. The size of the aggregates is dependent on the concentration of the surfactant. Typically, for alkylammonium carboxylates, the average aggregation number is in the order of 8-10. Addition of a third component, such as water, dramatically increases the size of the aggregates. Figure 2 illustrates the structure of a swollen reversed micelle. Surfactant aggregates in nonpolar solvents solubilize copious amounts of water and polar molecules in their interior. Solubilized molecules are held in the interior of reversed micelles more strongly than those in aqueous micelles. Favorable and concerted proton transfer becomes feasible in the environment of surfactantentrapped water pools. One can change microscopic polarities and viscosities at will by altering the amount of added water. Indeed it is quite feasible to obtain polarities as low as that of benzene in the interior of reversed micelles.” Since the substrates in the surfactant-entrapped water pool is separated from the bulk water by half a hydrocarbon bilayer, reversed micelles can be considered to be very primitive membrane models. Liposomes There is a very important difference between micelles and vesicles. Micelles are formed dynamically from monomeric surfactants, and the substratemicelle interaction is also dynamic. On the other hand, liposomes or surfactant vesicles, once formed, are stable for several weeks; the diffusion of substrates entrapped therein can only occur slowly, or
Radiolysis
of heterogeneous
inanimate
systems 0 J. H. FENIII ER
1063
of dodecylammonium propionate aggregate in benzene in the presence of solubilized model for the ultrafast proton transfer at the hydration shell of the surfactant headgroups in reversed micellar DAP in benzene. Since the concentration of surfactant is in a very large excess over the probe, proton transfer must occur from dodecylammonium propionic acid to pyrene I-carboxylate. For the sake of clarity, 2 pyrene moieties are drawn in the aggregate shown. In reality, there is much less than 1 probe per aggregate. Fig. 2. An illustration
water. Also, a proposed
they can be liberated by the destruction of the vesicle. Liposomes, smectic mesophases of phospholipid bilayers, have been utilized as membrane models since 1953.3 There are several ways to form spherical liposomes. The most commonly used one is to disperse in water a thin phospholipid film which had been deposited on the wall of a flask by evaporating a chloroform solution of the phospholipid and cholesterol. The aqueous dispersal leads to the formation of multicompartment liposomes whose sizes are not uniform. Sonication above their phase transition temperature gives fairly uniform single compartment bilayer vesicles, which on the average have 250 A diameters. Several molecules have been entrapped in the interior of liposomes. They can be released only if the liposome is destroyed by lysing with alcohol or with a detergent. Figure 3 illustrates the solubilization sites of polar, amphiphatic and apolar molecules in multicompartment liposomes. Small ions permeate the vesicle relatively freely, however. Liposomes undergo osmotic shrinkage and swelling if they are placed into appropriate electrolyte solutions. They
also undergo fusion to larger entities. The biophysics of liposomes has been extensively investigated in the past 2 decades.3,25 Surfactant
vesicles
The formation of spherical bilayers from completely synthetic surfactants has been demonstrated reCent~y.7.27.28"4 Such surfactant vesicles were formed from dialkyldimethylammonium halides7.27.34 and dicetylphosphate” upon sonication of their aqueous dispersions. If the sonication is carried out for a short time, multicompartment vesicles are formed. On the other hand, sonication for a longer time results in single compartment bilayer vesicles whose diameter ranges between 800-1500 A. Figure 4 is a naive presentation of a surfactant vesicle. Both the dialkyldimethylammonium halides and the dicetylphosphate surfactant vesicles are formed only if the sonication is carried out above their phase transition temperatures. Once these vesicles are formed, they remain stable for months. Within a few electrolytes they are osmotically active. In the absence of other additives, the dialkyldimethyl-
Radiation
1064
Oncology
POLAR SOLUTE
0
Biology
IN AOUEOUS
l
July
Physics
1979,
Volume
5, No. 7
PHASE
LIPOSOME
Fig. 3. Available
solubilization
sites for polar, nonpolar and amphiphatic liposomes.
ammonium halide surfactant vesicles are relatively permeable to protons and to small electrolytes. Addition of cholesterol, however, firms up these vesicles. The most useful properties of surfactant vesicles are that they can entrap and retain large molecules in their interior for some time. Since they are chemically simple and do not degrade, one can visualize extensive utilization of surfactant vesicles both in basic and applied ._ studies.
molecules
in multicompartment
REACTIVITY OF RADIATION SPECIES WITH THE MODEL
INDUCED SYSTEMS
As expected, simple surfactants which have no aromatic moiety are relatively unreactive towards hydrated ions.16 Rate constants for the reaction of the hydrated electron with hexadecyltrimethylammonium bromide, sodium dodecyl sulphate or polyoxyethylene(l5)nonylphenol are in the order of 10’ to IO6 M-’ secm’.18 The hydrated electron is also unreactive Cl-
DODAC 2C,,iJ2C,CI
n
Fig. 4. Formation
of completely
synthetic
dioctadecylammonium
chloride surfactant
vesicles.
Radiolysis
Table 1. Rate constants
of heterogeneous
inanimate
systems
0 J. H. FENDLER
for the reactions of e&, .OH and .H with surfactants and vesiclest
1065
(S), micelles
k, M-’ set-’
e,+S Hexadecyltrimethylammonium bromide [S] < CMC [S] > CMC Sodium dodecyl sulfate [S] < CMC [S] > CMC PoIyoxyethylene(6)nonylphenol [S] < CMC [S] >CMC Spherical bilayer /3-y-distereaoyl-L-a-phosphatidylcholine vesicle*
.OH+S
.H+S
NA
