Biologically engineered microstructures: controlled release applications

Journal of Controlled Release, 28 ( 1994) 3-l 3 Elsevier Science B.V.

3

SSDZ0168-3659(93)E0127-2

COREL 00942

Biologically engineered microstructures: applications Joel. M. Schnuf,

controlled release

Ronald Price, Alan S. Rudolph

Centerfor BioMolecular Science and Engineering, Code 6900 Naval Research Laboratory, Washington, DC 20375-5000, USA (Received 26 February 1993; accepted in revised form 3 September 1993)

The area of self-assembled ultrafine particulate-based composites (nano composites) has been a major thrust in advanced material development. In this paper we report on the application of biologically derived, self-assembled cylindrical microstructures to form advanced composite materials for controlled release applications. These microstructures (we call them tubules) have many applications in the material sciences. This paper will focus on the potential for rationally controlling the fabrication of submicron microstructures for controlled release applications. Key words: Biological engineering;

Microstructure;

Introduction This paper will focus on initial assessments for controlled release applications of a lipid-based, hollow microcylindrical, soda-straw like microstructure, called a tubule [ 1 ] (Fig. 1) [ 21. By using these structures as templates, the structure can be made more durable and the functionality can be modified. For example, tubules have been developed as templates for metallization. Suitably conducting hollow metal cylinders provide opportunities for the development of advanced high dielectric materials [ 3-5 1. Copper coated tubules have also been used for controlled release applications. Lipids, the basic building block of biological membranes, have polar or charged head groups and non-polar tails. This leads to two different interactions with solvents at the different ends of the molecule. As a result of this property, lipids *Corresponding

author

Controlled

release application

self-assemble to form several different microstructures depending on solvent and temperature conditions. The nature of the microstructure is controlled by the size, charge and polarity of the head group, the volume occupied by the non-polar acyl groups that comprise the tail of the molecule and the specific geometry of the molecule, as well as by the nature of the solvent. Figure 2 depicts a lipid molecule and several of the microstructures it can form [ 61. One common assembly of lipids are liposomes. Liposomes are spherical structures composed of a lipid bilayer that encloses an aqueous volume. A number of important applications, most involving targeted release, have been identified for liposomes [ 7 1. In our laboratory we have pioneered the development of an artificial blood surrogate that utilizes liposomes as an ‘artificial cell’ to encapsulate hemoglobin (Fig. 3 ) for oxygen carrying fluid applications [ 8- 12 1. Oxygen has been shown to freely move across the liposomal bilayer while the hemoglobin remains en-

Microstructure geometry and controlled release

Fig. 1. Electron microgaph of a lipid tubule. The dimater is about 0.5, the length about 30 and the wall thickness is about 300 A.

capsulated as the liposome circulate. The kinetics of oxygen release from liposome encapsulated hemoglobin is a surface to volume effect with smaller liposomes showing more rapid oxygen exchange than large liposomes. Liposomes have also been used to release antibiotics, tumoricidal agents, antifungals, growth factors and fragrances. The ability to target liposomes in vivo has met with limited success as these microstructures are recognized and removed by the mononuclear phagocyte system [ 7 1. The release protiles of water soluble solutes entrapped within liposomes can also based on a ‘all or none’ approach with release observed as a result of a compromise in the liposomal bilayer when the lipid bilayer is impermeable (or almost impermeable) to the entrapped solute. More lipophilic materials can diffuse more easily across the liposome bilayer. While the spherical geometry has numerous applications, other geometries can also be of interest.

In order to understand the potential benefits of tubule-based controlled release, we will review the effect of topology on controlled release. When a pot-us material is placed between two solutions of different concentrations of a chemical species, the species will move from the solution of high concentration, through the porus media, into the solution of lower concentration. The rate of flow of these chemical species controlled by two factors: (1)capillary or pore diffusion and (2) permeation. Porus diffusion rates are independent of the chemical properties of the media and are affected primarily by the nature of the porosity. Permeation is strongly affected by the compatibility of the matrix and the permeating material. It represents the ability of a chemical species to ‘dwell’ for a time at an ‘active site’ of the matrix polymer and then move on to another site. A matrix which strongly adsorbs the chemical species would exhibit, for example, slower diffusion release rates then one that did not. We will now consider the situation of differing geometries. Flat porus media For the case of a flat porus media placed between two solutions the diffusion rate will be proportional to the difference in concentration as well as the surface area of the system, As the concentration difference decreases, so does the rate. In this case the surface area is a constant. Let us call the path of diffusion from one solution to the other (for the case of bulk diffusion ), L. Remember that there is a restraining matrix between the two solutions. Thus the path that the chemical species must actually follow to get to the other side is considerably longer than the simple length, L. Following this logic leads immediately to the result that the ‘real’ diffusion rate is considerably lower than it would be if there were no matrix. The nature of the chemical or physical interactions between the diffusing chemical species

5

r

Bilayer

*

Tubule

Mic :elle

DMPC I

10

I 1,000

I

100

I 10,000

LOG SCALE (ANGSTROMS) Fig. 2. Schematic represetation

of a lipid molecule and some of the microstrcutures (prepared by B. Gaber and R. Light).

and the matrix will affect the permeation factor. In this case the chemical species moves from one ‘active site’ to another within the material, and not through the channels within the matrix. One very coarse measure of the strength of this effect is the solubility of the chemical species in the matrix material. Thus the release rate for a flat media will be controlled by the solubility of the chemical species, the porosity of the separating media and the concentration gradient between the two solutions. Spheres By putting the high concentration solution ‘inside’ the separating matrix, we can begin to de-

that self-assemble

from lipid molecules

sign a system for sustained release. Typically the chemical species is placed in a hollow polymer sphere. Since the concentration in the sphere is 100% and the concentration in the media is initially zero, there is a very high concentration gradient. For non-porus polymeric shells this leads to a very high osmotic pressure, e.g., the osmotic pressure is directly proportional to the concentration difference across an interface. The result is an increased water flux to the interior of the hollow polymer sphere. If the osmotic pressure increases above the deformability (or tensile strength) of the thin polymer shell, the shell will break, resulting in the release of the chemical species to the media. Soon the concentration in the media rises and the osmotic pressure on the walls of the remaining spheres decreases. As the

6

Fig. 3. Liposome-encapsulated hemoglobin. Scanning electron micrograph of liposome-encapsulated hemoglobin and human red blood cells. Sample was fixed in gluteraldehyde and post fixed using osmium tetroxide prior to dehydration and sputter coating with gold. Scale bar = 20.

solute passes from the general polymer matrix to the outside world (e.g., in the laboratory this would be a stirred solution) the osmotic pressure raises again leading to further ruptures and the process is repeated. This approach leads to numerous ‘discrete’ release events and to a nonlinear release rate. A second situation can arise if the encapsulated shell is semi-impermeable to the solute. Now the induced osmotic pressure will not be not great enough to rupture the thin polymeric sphere. This case is discussed below. This approach has been successfully used in many applications [ 13,141. Because of the distribution

and frequency of the discrete ‘rupture’ events, the release rates are not linear, but are dependent on the ‘bursting’ of the shells. This approach can be used for relatively long sustained release [ 15 1. We return to the case of diffusion of the solute across the semipermeable shell. The degree to which the diffusional transport across the thin polymeric shell will occur again depends on the porosity, permeation, concentration gradient and the surface area of the shell. The surface area of the sphere is controlled in large part by the osmotic pressure placed on the sphere due to the concentration gradient. As the encapsulant leaves

w H20

Osmotic Pressure Induces swelling encapsulant may be released depending on porosity

Fig. 4. Release

from

spheres (prepared Stratton).

Polymer shell bursts releasing encapsulant

by Stehpanie

the sphere, enters the media, then enters the outside environment. The osmotic pressure decreases and the surface area decreases. The relationship between the surface area of the sphere and the concentration gradient is non-linear. This is due to the linear relationship between the osmotic pressure and the diameter of the sphere, i.e., the radius of encapsulating sphere expands as the pressure increases. The surface area of the sphere expands as the square of the radius. Thus diffusion through such a system will be a ‘nonlinear’ function of the concentration gradient, leading to non-linear release rates as a function of time. In the case of liposomes, the release of hydrophilic encapsulants is largely dominated by the concentration gradient and partition coefficient (analogous to permeation). Release rates are frequently ‘all or none’ phenomena, largely driven by the osmotic rupture events described previously. Release of hydrophobic agents are driven by large partition coefficients and often parallel the concentration gradient created by solute loading provided the agent moves to another hydrophobic site. The situations described above are shown in Fig. 4. Rigid hollow microcylinders Now consider the case of small rigid hollow cylinders with impermeable walls placed in the media. In this case the diffusion of the encapsulant to the media is affected by the diameter of the cylinders, (since this is the area available for

diffusion), the aspect ratio of the cylinder and the concentration gradient. In this case, the effective porosity of the cylinder is reflected in the cylinder inner diameter. This is especially true as long as the walls of the cylinder are relatively impermeable to solutes. If the encapsulant is mixed with a polymeric carrier (P 1) (different from that of the coating P2 ), then factors affecting the diffusion of the encapsulant into the coating polymer (P2) would be: (i) the concentration gradient between PI and P2; (ii) the diameter (effective pore area) of the cylinder and associated capillary action; and (iii) the porosity of the media (P2). The diffusion rates of species out of this system are clearly quite different from the two cases described previously. Because the cylinder is rigid and the walls impermeable, the diffusional surface area size stays constant, and their are no nonlinearities caused by a changing concentration gradient. The factors affecting diffusion between the media and the environment would be the same as before: (i) the permeation of the encapsulant in the carrier polymer P 1; (ii) the porosity of the carrier polymer P 1; (iii) the porosity of the exterior polymer coating P2; (iv) the permeation of the encapsulant in the polymeric coating P2; and (v) the concentration gradient between the polymeric coating P2 and external environment. This system is shown in Fig. 5. Ultra small hollow cylinders appear to be very attractive for sustained controlled release applications because it offers the opportunity for controlling release rates by varying P2 and P 1, as well as size and number of the cylinders. The self-assembled microstructures described in the first section provide an attractive opportunity to demonstrate the potential of hollow cylinders for the rationally controlled release. Our first experiments in this area have focused on the development of a controlled release system for antifouling applications using metallized lipid tubules [ 16 1. For metal thicknesses over about 500 A, the walls become very impermeable (at least to typical solutes) for antifouling suggesting they are good candidates for controlled release applications [ 17,181.

increase in the economic costs associated with biofouling. Marine biofouling growth on underwater ship hulls increases hydrodynamic drag and hull weight due to the increasing biomass. If reasonable service speeds for the ship are to be maintained, power output must be increased, resulting in higher consumption of fuel and increased wear on the machinery. Increases in fuel consumption exceeding 10% are common, as are decreases in operational hull speeds of up to 16%

[191*

External Media Fig. 5. Release from hollow cylinders (prepared by Stephanie Stratton).

Controlled release from microcylinders Antifouling applications An early solution to the problem of barnacle growth on ships was copper sheathing chosen to protect wooden hulls against the ravages of the teredo worm. Today the modem equivalent of copper sheathing is a polymeric ablative or selfpolishing paint, using copper powder or cuprous oxide as the primary toxicant. Although copper, as an antifoulant, has stood the test of time, it is often more effective against animal species than plant growth. In order to improve on the performance of copper, the addition of many other metal species have been employed, such as mercury, arsenic, cadmium, lead and tin. Currently, in more enlightened times, these heavy metals have been abandoned as persistent toxicants that have adverse effect on the environment and more directly on man’s health as a consumer of fish and shellfish. In addition to environmental concerns, fuels have become more expensive and fossil fuel reserves have begun to shrink. This is leading to an

In an effort to ease the economic pressures that continue to mount on governments, industry and individuals, service cycles are being increased. The US Navy has lengthened operational cycles to 5 years, with 7-lo-year cycles under consideration to further reduce maintenance costs. When antifouling paints fail early on in the service cycle, it is not always possible to haul and paint the ship ahead of schedule due to lack of funds or facilities. This situation has led to higher fuel costs and the decreased performance associated with a fouled hull. In order to achieve the goal of less polluting yet effective antifouling paints, strict control of the release rates in copper-based paints in addition to the entrapment and controlled release of alternate compounds must be accomplished. These alternate compounds are often active at levels far lower than those needed for copper. Thus proper control of release rates is necessary to prevent these alternate compounds from being discharged from the coating in excess of actual requirements. Conservative, i.e., low release rates led directly to long service lifetimes for the paints. Not only must an antifouling paint offer performance in service; it must meet increasing demands from governmental regulators conceming water pollution standards ( < 18 pg/l maximum), air quality standards for volatile organic solvents (VOC ) and the occupational and health regulations governing the application and disposal of antifouling coatings. As discussed before suitable metallized tubules may offer significant opportunities for control of long-term control of release. By controlling the physical properties of the polymeric

9

Release of Z-Methoxynaphthalene from Paint (P2)Coating (P2a)-Acrylic (PZb)-PkC Vinyl (PI) 110 100 j

J

90

$80

Carrier

(Pla)‘Epoxy

monomer

(Plb)-Epoxy

Polymer

-;

,

0

/.

‘!I PZb,Pla * PPa,Pla PZb,Plb +

PZa,Plb

100 200

. .

300 400

500 600

700

800

900

TIME IN HOURS

Fig. 6. Percentage of 2-methoxynaphthalene as a function of time, from polymeric coatings, consisting of either an acrylic coating (P2a) or a PVC vinyl (P2b) coating and an viscous epoxy monomer carrier (PI a), or a crosslinked epoxy carrier (Plb). Showing that (P 1) the polymeric carrier has a large effect on the release rates demonstrated for each of the coating polymer types (P2).

carrier (P 1)) the diameter of the cylinder and the ‘permeability’ of the paint coating (P2 ) , significant variations in the total percentage of encapsulant released as a function of time has been demonstrated. Results from experiments performed by Price et al. support this hypothesis [ 20,2 11. This is graphically shown in Fig. 6. Microcylinders are also able to isolate the encapsulant from the environment thus providing a mechanism for enhanced chemical stability of the encapsulants. These ‘tubules’ are quite compatible with existing paints and may even offer some improvements to the ultimate mechanical properties of the composite [ I I- 14 1. Recent experiments have suggested that tubule-based controlled release paints may indeed be a viable approach for antifouling applications. Methods for environmental exposure testing Price [ 23 ] has performed several studies using metallic microcylinders that average 0.5 pm in diameter and range from 10 to 250 pm in

length with interior diameters ranging from 0.25 to 0.4 pm. The metallized tubules were dried so that they would act as microcapillary tubes that would then entrap and retain a number of liquid materials upon insertion to a liquid medium. Reported release rates are exponentials with longterm time constants that approximate linear release rates for very long periods of time and depend on the encapsulant and the molecular weight and cross linking of the carrier vehicle. Price et al. reports on the use of this system for antifouling. In one case [ 201 twenty, 1 l-cm liberglass rods, which had been cut from 0.35 mm diameter stock, were coated by dipping with the desired formulation of antifouling coating. Following air drying for at least 48 h, the rods were mounted in a floating array consisting of a rectangular float of PVC pipe to which a diamond polypropylene fish impound netting was attached. The rods were attached to the net with rubber grommets and the entire array was attached to a raft in the field at Coconut Island, Hawaii, or Taylors Creek, Beaufort, North Carolina. At the first sign of fouling, rods were withdrawn and examined to determine the composition and relative percentage cover of fouling organisms. Tetracycline was used in these studies of release from coatings and microstructures as it is easily quantified by spectrophotometric analysis in water. In addition, tetracycline is a registered antifouling agent and therefore was considered safe in this application. Initial findings indicated tetracycline could be released at exponential release rates with very long release rates that approximated linear rates over long periods of time from both epoxy resin films and from vinyl-based paints. It is interesting to note in Fig. 7 that, when not encapsulated, the tetracycline is found to release in a matter of a few hours from VYHH coatings; however, when in an encapsulated sample, tetracycline continues to release after 500 days of use. In order to explore the possibility of lowering the need for antibiotics or persistent copper toxicants, two further approaches were tested. First was the use of isothiazolone which is an experi-

10

VINYL PAINT FILMS CONTAINING TETRACYCLINE FREE ASSOCIATED MICROTUBULE ENCAPSULATED

0

-

TETRACYCLINE

-

MICROTUBULE TETRACYCLINE TO IDENTICAL

TIME

IN VINYL FAINT ENCAPSULATED ADDED COATING

20

10 IN

AND

30

DAYS

Fig. 7. Figure showing release rates of tetracycline from tubules in a (P 1) carrier polymer compared to release rates of tetracycline in a single (P2) polymer matrix.

mental antifouling agent that has been shown to degrade rapidly in the marine environment (Harrington, 1989 >. When encapsulated in both copper and iron microcylinders and added to a vinyl matrix this additive was shown to be effective at repelling fouling marine species in testing. Another approach to the development of nontoxic or biodegradable coatings pursued by Price was the use of extracts from the sea pansy (Renilla ~e~~~~~~.~),and structural analogs of these compounds, which have been shown to reduce biofouling. Figure 8 illustrates a pair of test rods which were taken from a sample exposed at Coconut Island, Hawaii for 6 months. It can be clearly seen that the experimental rod has successfully repelled fouling during the test period with concentrations of 2% by weight active agent. The results described above clearly show that a controlled release system based upon metallized tubules has promise as a means of providing a release mechanisms for antifouling paints with significant advantages over existing formulations.

Fig. 8. This figure illustrates a pair of test rods which were taken from a sample exposed at Coconut Island, Hawaii for 6 months. Comparison of effectiveness of tubule based encapsulation vs non-ecapsulated sysem in Navy paint C-92 11.

Controlled release of proteins from lipid mi~r~ylinders We have also explored the use of cylindrical lipid microstructures for the controlled release of a class of proteins known as biological response modifiers [ 24,25 1. Biological response modifiers are defined as a large and growing number of growth factors and cytokines which are expressed by a wide variety of cell types [ 26 1. These proteins regulate cellular growth and differentiation and are important in homeostasis as well as pathogenic processes. The applications for the release of such agents are wide, and the action and mechanism of these classes of biological

agents are being defined for a host of important cellular events. The driving force for controlling the release of growth factors and cytokines is to understand how these agents control such events and perhaps exert some control in cellular response. In addition, using a semi-crystalline microcylinder for controlled release might afford additional advantages as a structural matrix for cellular response to occur. The controlled release from the lipid microcylinders of growth factors and cytokines important in the regeneration of soft tissue following a wounding event has also been investigated. The lifetime of growth factors such as TGF-j? at a wound site is short [ 24 1, and repeated exogenous dosing of such factors has been demonstrated to have positive results in the acceleration of wound healing [25,26], through the stimulation of fibronectin and matrix remodelling at the wound site. Our initial efforts to develop lipid microcylinders as release vehicles for wound repair have focused on issues of toxicity of the vehicle itself and the demonstration of controlled release of TGF-P in vitro. The interaction of the microcylinders with cells important to wound healing events had to be defined both in vitro and in vivo in order to demonstrate that the microcylinders themselves do not illicit an immune response that would be deleterious to their application in wound healing [ 27,28 1. The release profiles of ‘251-labelled TGF-P release from microcylinders is seen in Fig. 9. The release of TGF-j? (a 2%kDa, relatively hydrophobic protein) is slow with an initial release of 5-10 ng over the course of a day, followed by a slow 1 ng/day release. This profile is consistent regardless of the initial concentration of TGF-P loaded into the microcylinders. It is also a typical profile observed for most of the proteins which we have examined. The profile can be altered by temperature (Fig. 9 ) , as approaching the transition temperature of the lipid (42°C) results in a slight increase in released protein, while exceeding the transition temperature results in the conversion of the microcylinder into a liposome and the subsequent release of protein. Similar results have been obtained with the cytokine,

0’ 0

10

JO

20 T,me

40

50

(hours)

Fig. 9. Temperature-dependent release of “‘I-transforming growth factor (TGF) from DC8,9PC tubules. Temperature ranges from biological at 37°C (closed circle) to the aqueous T, of DC8,9PC at 43°C (closed triangle).

interleukin-2. It should be pointed out that many proteins will associate with the lipid microcylinder surface depending on the ‘hydrophobicity’ of the protein. In these cases, the desorption of the protein from the surface will be an additional factor in modeling the release from the microcylinders. The partition coefficient of the protein in the wall of the microcylinder will define the equilibrium concentration of protein that will locate there. The desorption of the protein from the surface, and the concentration gradient created at the surface, will dictate the concentration of the protein release to the P2 matrix. Efforts to define the bioactivity of the released and encapsulated proteins are underway. The interaction of lipid microcylinders with Tcells and macrophages from peripheral human blood, two important mediators of the immune response during wound healing, in culture has been studied [ 2 5 1. Macrophages are observed to adhere and stretch along the long axis of the microcylinders [ 241. There is no evidence that the microcylinders are phagocytized by macrophages, probably because of their length or aspect ratio. The degree of association can be altered by modifying the microcylinder surface with the inclusion of ganglioside GM1 which reduces the number of macrophages which are ob-

60

12

served to adhere to the microcylinders. We are currently examining the functional response of macrophages to the lipid microcylinders. Initial studies which investigate the T-cell response to tetanus toxoid in the presence of lipid microcylinders suggests that the macrophage ability to present antigen may be impaired as the T-cell response to the toxoid was inhibited in a concentration dependent manner by the presence of the microcylinders [ 26 1. T-cell function was not altered as the microcylinders do not alter the ability of T-cells to mount a response to normal mitogens such as PMA. Our effort to understand the ‘safety’ of using microcylinders in vivo applications has been directed at two in vivo animal models. We have applied lipid microcylinders in hydrogel composites which have been implanted into healthy mice and applied in a rat full thickness skin wound model.

Summary and Conclusion Several issues must be resolved before the ultimate utility of this approach can be determined. The variable controlling release rate must be quantified and optimized for each particular application. Important variables are the molecular weight of the antifouling agent, diameter and length of the tubules, permeability of the incorporating polymer as well as the matrix paint. While we have been able to make gallon quantities of test paints in our laboratory, the question of scale up has still to be seriously addressed. Cost is always an important issue. We had thought that the lipid costs (up to $4000 per pound) would be a serious problem for technology transfer. However, we have had a recent success at recovering the lipid for metallized tubules [ 12 ] and recycling that lipid to make more tubules which were subsequently metallized. Thus lipid costs should not be barrier to use. Costs of the metal, antifouling agent, and matrix, coupled with the processing costs will determine the commercial viability of this approach.

Acknowledgements

search, Office of Naval Technology, Naval Medical Research and Development Command, Defense Advanced Research Projects Agency and the National Science Foundation for partial support of the research described above. This work could not have done without the work of the entire Center for Bio/Molecular Science and Engineering tubule project research team and their contributions are gratefully acknowledged. We also wish to thank Professor R. Shashidhar, Dr. Barry Spargo, Professor B. Ratna, Dr. Bhakta Rath, Dr. William Tolles, Dr. Dick Rein, Dr. Ira Skurnick, Captain Steve Snyder and Dr. B. Gaber for many helpful discussions.

References 1

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We gratefully acknowledge the support of the Naval Research Laboratory, Office of Naval Re-

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the International Conference on Biofouling, Bangalore, India, American Institute of Biology Press, M.F. Thompson (Ed.), 1992, pp. 321-333 R.R. Price and M. Patchan, Elimination of rosin from vinyl antifouling coatings by the use of novel cylindrical channel forming microtubules. Polymers in the marine environment, Trans. Inst. Marine Eng., London, 199 1, pp. 171-177. R. Price and M. Patchan, Entrapment and release characteristics of 2-methoxynaphthalene from cylindrical microstructures formed from phospholipids, J. Microencapsulation, 8, (3) (1991) 301-306. A.S. Rudolph, G.E. Stillwell, R.O. Cliff, B. Kahn, B.J. Spargo, F.M. Rollwagen and R.L. Monroy, Biocompatability of lipid microstructures: effect on cell growth and antigen presentation in culture, Biomaterials, 13 ( 1992) 1085-1092. B.J. Spargo, GE. Stillwell, R.O. Cliff, R.L. Monroy, F.M. Rollwagen and A.S. Rudolph, Tecnological development of lipid microtubules: biocompatibility and controlled release, tissue inducing biomaterials, Materials Research Society Symposium Proc., 252 ( 1992) 285291. P. Dijke and K.K. Iwata, Biotechnology, 7 ( 1989) 793798. A. Buckley, J.M. Davidson, C.D. Kamerath, T.B. Wolt and S.C. Woodward, Proc. Natl. Acad. Sci., 82, (1995) 7340-7344. E.R. Edelman, E. Mathiowitz, R. Langer and M. Klagsbrun, Controlled and modulated release of tibroblast growth factor, Biomaterials, 12 ( 199 1) 6 19-626.