- Email: [email protected]
Using brine shrimp as a drug carrier for therapeutic applications in aquaculture
Aquacultural Engineering 13 (1994) 301-309 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0144-8609/94/$7.00 ELSEVIER
Using Brine Shrimp as a Drug Carrier for Therapeutic Applications in Aquaculture A. Aguilar-Aguila, A. Tejeda Mansir & A. Ruiz Manr/quez Especialidaden Biotecnologia,Departamento de Ingenier/aQuimicay Metalurgia, Universidadde Sonora, Rosalesy BoulevardLuis Encinas J. Hermosillo,Sonora, M6xico (Received31 March 1993; accepted 7 August 1993) ABSTRACT A method is described to introduce water-insoluble drugs into brine shrimp nauplii (BSN). The uptake of drug by brine shrimp may be modeled as a batch adsorption process; in this way, one can calculate the dosage required for a given population of BSN. The results of this study show that a drug can be loaded into BSN at concentrations up to 9 lug per nauplius. Such concentration is high enough to fulfill the recommended dosage for an adequate treatment of BSN-feeding fish or shrimp larvae. Results are also presented on the loss of drug upon the transfer of larvae from medicated water to drug-free water.
INTRODUCTION The successful shrimp larval culture depends very largely on the effective prevention and control of diseases, such as bacterial infections. The usual way to treat them is by adding prophylactic or therapeutic quantities of agents to the culture water (Sindermann, 1977; Lightner, 1984). One of the most widely employed feeding organisms in the larval culture of crustacean and fish, is the brine shrimp, Artemia sp. This crustacean is widely used as a food in aquaculture, especially in the early stages of crustacean and fish culture (Sorgeloos, 1973). The most common way to use Artemia is by placing cysts in salt water; after several hours, the first larval stage emerges, named nauplius (plural nauplii). These nauplii are used directly as a food. 301
302
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
In recent years, the world supply and practical utilization of the brine shrimp nauplius (BSN) have been notably improved as a result of more than a decade of intensive research, and studies are mainly focused on analyzing its nutritional value and use as food for aquaculture (Sandoval, 1993). Mohney et al. (1989) have proposed a bioencapsulation method as an innovative way of drug delivery to the shrimp larvae. The method consists of feeding BSN with the water-insoluble agent Romet-30, and using these nauplii to feed shrimp larvae. Thus, the nauplii act as drug delivery agents. However, the work of Mohney et al. does not describe the way to calculate the dosage requirement for BSN, and the process is somewhat empirical. The objective of this work is to model the uptake of drug by the BSN, in order to estimate the adequate quantities to be used in a therapeutic or prophylactic treatment.
THEORETICAL CONSIDERATIONS In order to make a quantitative approach to the uptake of the drug by BSN, we must first consider the BSN as an 'empty space' that can be filled with drug particles, i.e. a biocapsule. When the BSN uptakes enough particles, it becomes a 'full space' and cannot uptake more particles. This can be expressed as a chemical reaction: Particles + BSN ~ Biocapsules
( 1)
where 'Particles" refers to the amount of the drug Romet-30 that will be uptaken; 'BSN' is the nauplii population that will feed on the particles; and 'Biocapsules' is the brine shrimp population containing an amount of the drug. Equation (1) assumes that uptake behaves like a first-order chemical reaction, in which the nauplius initially consumes the drug very fast (high uptake rate). As the BSN becomes saturated (a 'full space'), the rate of consumption decreases gradually. Finally, the BSN stops feeding, when a complete saturation is attained. This type of kinetic is very similar to saturation kinetics of the type of the Langmuir isotherm for the adsorption process, described by Belter et al. (1988). Brine shrimp is a filter-feeding organism, which means it will take up any particle of the proper diameter. Assigning the BSN feeding rate to a measure of how fast the BSN can filter any kind of particle, except the drug, and the bioencapsulation rate to a measure particular to the consumption of the drug the maximum specific uptake rate [ Vmax]of the
Brineshrimpas therapeuticagent
303
BSN can now be defined as: [ Vmax]= [Brine shrimp feeding rate] + [Bioencapsulation rate]
(2)
On the other hand, the Langmuir model assumes that the adsorption process is a reversible reaction, described by an equilibrium constant, like any chemical reaction. A modified saturation constant K for the bioencapsulation process, based on rates instead of concentration values, can be defined as: K = [Particle concentration][BSN feeding rate] [Bioencapsulation rate]
(3)
and combining the eqns (2) and (3) one obtains: [Bioencapsulation rate] =
[ Vinci[Particle concentration] K + [Particle concentration]
(4)
or expressed using conventional nomenclature: q = q0____~Y K+y
(5)
where q is the specific drug uptake rate (/~g/BSN-h); q0 is the maximum specific drug uptake rate (/~g/BSN-h); y is the concentration of particles or dosage (mg/ml); and K is the saturation constant (mg/ml). Both q0 and K may be determined experimentally calculating the specific drug uptake rate (q) for different dosages (y), and using a plot of 1/q versus 1/y. The slope of this plot is K/qo, and the intercept is 1/qo, as in the Lineweaver-Burk plot used for enzyme kinetics (Lehninger, 1981). Defining the drug content on the BSN population as C (~g/BSN), for a batch drug uptake process, mass balances of the drug inside and outside the BSN yield the respective differential equations: dC =q dt dy= dt
(6)
q F
(7)
where F is a conversion factor that relates microgram per BSN to milligram per liter.
304
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
Equations (6) and (7) can be solved simultaneously to calculate the concentration of drug inside and outside the BSN over time.
MATERIALS AND METHODS The bioencapsulation test was performed by harvesting and placing approximately 100 BSN, Anemia franciscana (San Francisco Bay Brand), per milliliter of sterile seawater in conical-bottom glass flasks provided with an airstone. Dosages of 0, 2.5, 5, 7.5 and 10 mg/ml of Romet-30 (Hoffman LaRoche) were added to each flask, and were then suspended for uptake periods of 2, 4, 6 and 8 h, in the presence of aeration and light. Each dosage and time period treatment was made in triplicate to observe the reproducibility of the results. After this period the BSN were collected with a 100 mesh screen and rinsed with sterile seawater. The BSN were concentrated on a known volume between ] and 3 ml, and the total BSN in the sample were counted, using appropriated dilution. The BSN were then disrupted on a mortar, and three 0.02 ml samples were taken and spread on filter paper discs for determination of drug concentration as described below. The concentration of the drug on BSN was measured using the Kirby-Bauer test (Jones et al., 1986; Cappuccino & Sherman, 1987), which consists of placing a disk impregnated with antibiotic or any drug in a Petri dish containing a solid culture medium, Muller-Hinton agar and the bacteria to be tested. For this experiment a Romet-30-sensitive Vibrio sp. strain was used. The strain was isolated from diseased blue shrimp Penaeus stylirostris on an aquaculture facility near Guaymas, Mexico (BIOTECMAR S.C.P.A.). Once the disks are placed on the dish, they are incubated for 24 h at 37°C. After incubation, the bacteria grow over the plate, except in a zone around the disk. The diameter of this inhibition zone is proportional to the concentration of the drug. To estimate the drug concentration on the brine shrimp nauplii, a plot of drug concentration versus inhibition diameter was constructed by using disks impregnated with different drug concentrations. The values obtained on this plot were fitted using linear regression, obtaining the following equation to correlate the inhibition diameter to the drug concentration: d = 1.141 + 3.3"Cj where d is the diameter of inhibition; and Cd is the concentration of drug in the disk. The fitting gave a correlation coefficient r 2 of 0.884; this relative low value for the correlation can be explained because the
Brine shrimp as therapeutic agent
305
diffusion of the drug from the disk to the medium is not linear, although for the range on which the samples were measured, the estimated values have a good approximation. As an additional experiment to determine the loss of drug in the BSN after the bioencapsulation period, BSN previously treated with the drug were placed in sterile seawater, and the drug concentration was determined after four different time intervals. No experiments on biocapsule feeding to fish or shrimp larvae were performed.
RESULTS AND DISCUSSION In the tests of the BSN that was not treated, a contamination area around the disks was seen, probably caused by bacteria naturally occurring in brine shrimp cysts, which were incubated in the seawater while the cysts were hatching. These samples were considered to have a drug concentration of zero. The results of the bioencapsulation tests ranged from 0 to 9 ~g per BSN, depending on the dosage and the time period allowed for drug uptake. The mean for each treatment is shown in Table 1. Mohney et al. (1989) report an uptake of 0.1/~g/BSN for a drug dosage of 3 mg/ ml and an uptake time of 4 h. These lower values could be due to the use of live organisms instead of minced nauplii in the drug concentration test. Figure 1 is the plot of the active compound uptake (microgram per nauplius) versus uptake residence time using different drug dosage (mg/ ml). The profiles of these plots are very similar to the Langrnuir isotherms described earlier, and a tendency to saturation when the uptake residence time increases can be observed.
TABLE 1 Mean Results (/zg/BNS) of the Bioencapsulation Test on Different Drug Dosage and Time Periods
Time fh)
0 2 4 6 8
Drug dosage (mglml) 2"5
5
7"5
I0
0 1.331 2.146 2"760 3'076
0 2.214 3.571 4"181 4-900
0 2"701 4-286 6"123 7"164
0 3"608 5"854 8'601 9"007
306
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez 10 •
Z
•
8
?
•
~
4
~
2
[]
i
i
l
l
2
4
6
8
10
Time, hours
Fig. 1.
Active compound uptake at different dosages (mg/liter): o, 2.5; o, 5; n, 7.5; m, 10. Solid lines show numerical simulations using q0 = 1.831 and K = 3.353.
The kinetic constants q0 and K were determinated using the plot of 1/q versus 1/y in Fig. 2, as explained in the Theoretical Considerations section. The values obtained were of 1.831/~g/BSN-h and 3-353 mg/ml, respectively; the value of F was calculated by dividing the microgram to milligram equivalence by the BSN density used on the experiments, to give a value of F= 10 ~g-ml/ml-BSN. These constants were used for the numerical simulation of the model. Equations (6) and (7) were solved simultaneously using a fourth-order Runge-Kutta algorithm (Kopal, 1955). The drug concentration values (y) used for the simulation were the dosage for each experiment. The dynamic behavior of the drug uptake is presented as solid lines on Fig. 1. It can be seen that this mathematical model (eqns (5)-(7)) describes well the time profile of uptake for each of the dosages used. For the highest dosage used (10 mg/ml) the simulated profile is slightly lower than the measured data; in this case, an adherence of tiny particles of the drug to the body of the BSN was noted by microscopic observations. This phenomenon is related to the very high drug concentration, and explains the difference between the predicted and observed data. In the other lower drug dosages the adherence was not so evident. The results of the experiment to determine the loss of Romet-30 as a function of time when the biocapsules are placed in seawater without the drug are presented in Fig. 3. With this plot the points represent the mean values and the bars are standard deviation. These results show that eight
307
Brine shrimp as therapeutic agent 3
i z r~
ea
0
0.0
i
i
r
i
0.1
0.2
0.3
0.4
0.5
1 / y, liter / mg
Fig. 2.
Lineweaver-Burk plot to estimate uptake kinetics of drug particles in BSN, q0 = 1.831/~g/BNS-h; K = 3.353 rag/liter. 5.
Z 23.
4.
3,
1
2,
1-
0 0
!
!
i
2
4
6
8
Residence Time, Hours
Fig. 3.
Loss of Romet-30 on the biocapsules when placed in drug-free sterile seawater.
hours after placing the nauplii in seawater, the concentration of the drug is reduced by half, and it may not have an effective therapeutic effect on shrimp or fish larvae. Drug loss can be explained as either a leaching out from the BSN or a degradation by the metabolism of the BSN. In the first case, eqn (1) will be a reversible process; if the drug is degraded by nauplii, eqn (1) will not
308
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
be a reversible process. The agreement of experimental data with the model suggests that the process is really reversible. More evidence can be found in the work of Mohney et al. (1989); as these authors use live organisms to make the drug concentration tests, the inhibition zone may be due only to the drug leaching out. The loss of the drug in the nauplii can be compensated as recommended by Szyper (1989), if its effect is quantified and sufficient prey organisms are supplied. In this case, the nauplii must be calculated for its consumption before eight hours. Another way to resolve this is raising the treatment frequency, although this is not a very recommendable strategy, due to the collateral effects that could be caused on the shrimp larvae or the bacteria present in the culture water (Jones et al., 1986).
CONCLUSIONS The model used on this paper describes very well the kinetics of drug uptake, making possible a more quantitative approach for the bioencapsulation process, and giving a way to estimate the appropriate dosage for treatment. The constants obtained in this paper are specific for the BSN strain and process conditions used here like temperature, illumination, salinity, etc. Differences could be observed for other strains or even lots used, and for different conditions, so the best way to make sure of the results is to calculate the constants in the place where the bioencapsulation will be done. Although no experiments were carried out with shrimp larvae, the Mohney experiments led us to conceive that BSN can be used as a vector for directing an insoluble particle to shrimp larvae. This makes the treatment more effective than suspending the particles in the shrimp larval culture water, because the BSN can be maintained at very high population densities (up to 100 000 organisms per liter of seawater) during the drug uptake. Therefore, this method is very attractive for the new highcost medicaments. Also, the bioencapsulation process could be used with other particle compounds, like nutrients, hormones or vaccines.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Juan A. Dworak and the aquaculture facilities at Instituto Tecnologico del Mar, Guaymas. Technical Support by Ernestina Almada and Carlos Flores at BIOTECMAR and revision and discussion by Octavio T. Ram/rez are kindly appreciated.
Brine shrimp as therapeuticagent
309
REFERENCES Belter, E A., Cussler, E. L. & Hu, W. (1988). Bioseparations. John Wiley, New York, Ch. 6, pp. 145-79. Cappuccino, J. G. & Sherman, N. (1987). Experiment 43. In Microbiology, A Laboratory Manual. The Benjamin Cummings Publishing Co., Menlo Park, CA, pp. 247-54. Jones, J. G., Gardener, S., Simon, B. M. & Pickup, R. W. (1986). Factors affecting the measurement of antibiotic resistance in bacteria isolated from lake water. J. Appl. BacterioL, 60, 455-62. Kopal, Z. (1955). Numerical Analysis (lst edn). John Wiley, New York, pp. 195-213. Lehninger, A. L. (1981 ). Biochemistry (2nd edn). Worth Publishers, New York, Ch. 8, pp. 183-216. Lightner, D. V. (1984). A review of the diseases of cultured penaeid shrimp and prawn with emphasis on recent discoveries and development. In Proc. 1st Int. Conf. on the Culture of Penaeid Shrimps, Aquaculture Department, Southeast Asia Fisheries Center, Iloilo, Philippines, pp. 79-102. Mohney, L. L., Lightner, D. V. & Bauerlein, M. (1989). Bioencapsulation of therapeutic quantities of the antibacterial Romet-30 in nauplii of the brine shrimp Artemia and in the nematode Panagrellus redivivus. J. World Aquaculture Soc., 21 (3), 186-90. Sandoval, F. C., Ramirez, L. E B. & Lobina, D. V. (1993). The biochemical composition of the cysts of some Mexican populations of Artemiafranciscana Kellogg. Comparative Biochem. Physiol., 1114, 163-7. Sindermann, C. J. (1971). Disease diagnosis and control in North American marine aquaculture and fisher sciences. Aquaculture, 1, 9-29. Sorgeloos, E (1973). High density culture of the brine shrimp Artemia salina. Aquaculture, 1,385-91. Szyper, J. P. (1989). Nutritional depletion of the aquaculture feed organisms Euterpina acutifrons, Artemia sp. and Brachionus plicatilis during starvation. J. World Aquaculture Soc., 211, 162-9.
Using Brine Shrimp as a Drug Carrier for Therapeutic Applications in Aquaculture A. Aguilar-Aguila, A. Tejeda Mansir & A. Ruiz Manr/quez Especialidaden Biotecnologia,Departamento de Ingenier/aQuimicay Metalurgia, Universidadde Sonora, Rosalesy BoulevardLuis Encinas J. Hermosillo,Sonora, M6xico (Received31 March 1993; accepted 7 August 1993) ABSTRACT A method is described to introduce water-insoluble drugs into brine shrimp nauplii (BSN). The uptake of drug by brine shrimp may be modeled as a batch adsorption process; in this way, one can calculate the dosage required for a given population of BSN. The results of this study show that a drug can be loaded into BSN at concentrations up to 9 lug per nauplius. Such concentration is high enough to fulfill the recommended dosage for an adequate treatment of BSN-feeding fish or shrimp larvae. Results are also presented on the loss of drug upon the transfer of larvae from medicated water to drug-free water.
INTRODUCTION The successful shrimp larval culture depends very largely on the effective prevention and control of diseases, such as bacterial infections. The usual way to treat them is by adding prophylactic or therapeutic quantities of agents to the culture water (Sindermann, 1977; Lightner, 1984). One of the most widely employed feeding organisms in the larval culture of crustacean and fish, is the brine shrimp, Artemia sp. This crustacean is widely used as a food in aquaculture, especially in the early stages of crustacean and fish culture (Sorgeloos, 1973). The most common way to use Artemia is by placing cysts in salt water; after several hours, the first larval stage emerges, named nauplius (plural nauplii). These nauplii are used directly as a food. 301
302
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
In recent years, the world supply and practical utilization of the brine shrimp nauplius (BSN) have been notably improved as a result of more than a decade of intensive research, and studies are mainly focused on analyzing its nutritional value and use as food for aquaculture (Sandoval, 1993). Mohney et al. (1989) have proposed a bioencapsulation method as an innovative way of drug delivery to the shrimp larvae. The method consists of feeding BSN with the water-insoluble agent Romet-30, and using these nauplii to feed shrimp larvae. Thus, the nauplii act as drug delivery agents. However, the work of Mohney et al. does not describe the way to calculate the dosage requirement for BSN, and the process is somewhat empirical. The objective of this work is to model the uptake of drug by the BSN, in order to estimate the adequate quantities to be used in a therapeutic or prophylactic treatment.
THEORETICAL CONSIDERATIONS In order to make a quantitative approach to the uptake of the drug by BSN, we must first consider the BSN as an 'empty space' that can be filled with drug particles, i.e. a biocapsule. When the BSN uptakes enough particles, it becomes a 'full space' and cannot uptake more particles. This can be expressed as a chemical reaction: Particles + BSN ~ Biocapsules
( 1)
where 'Particles" refers to the amount of the drug Romet-30 that will be uptaken; 'BSN' is the nauplii population that will feed on the particles; and 'Biocapsules' is the brine shrimp population containing an amount of the drug. Equation (1) assumes that uptake behaves like a first-order chemical reaction, in which the nauplius initially consumes the drug very fast (high uptake rate). As the BSN becomes saturated (a 'full space'), the rate of consumption decreases gradually. Finally, the BSN stops feeding, when a complete saturation is attained. This type of kinetic is very similar to saturation kinetics of the type of the Langmuir isotherm for the adsorption process, described by Belter et al. (1988). Brine shrimp is a filter-feeding organism, which means it will take up any particle of the proper diameter. Assigning the BSN feeding rate to a measure of how fast the BSN can filter any kind of particle, except the drug, and the bioencapsulation rate to a measure particular to the consumption of the drug the maximum specific uptake rate [ Vmax]of the
Brineshrimpas therapeuticagent
303
BSN can now be defined as: [ Vmax]= [Brine shrimp feeding rate] + [Bioencapsulation rate]
(2)
On the other hand, the Langmuir model assumes that the adsorption process is a reversible reaction, described by an equilibrium constant, like any chemical reaction. A modified saturation constant K for the bioencapsulation process, based on rates instead of concentration values, can be defined as: K = [Particle concentration][BSN feeding rate] [Bioencapsulation rate]
(3)
and combining the eqns (2) and (3) one obtains: [Bioencapsulation rate] =
[ Vinci[Particle concentration] K + [Particle concentration]
(4)
or expressed using conventional nomenclature: q = q0____~Y K+y
(5)
where q is the specific drug uptake rate (/~g/BSN-h); q0 is the maximum specific drug uptake rate (/~g/BSN-h); y is the concentration of particles or dosage (mg/ml); and K is the saturation constant (mg/ml). Both q0 and K may be determined experimentally calculating the specific drug uptake rate (q) for different dosages (y), and using a plot of 1/q versus 1/y. The slope of this plot is K/qo, and the intercept is 1/qo, as in the Lineweaver-Burk plot used for enzyme kinetics (Lehninger, 1981). Defining the drug content on the BSN population as C (~g/BSN), for a batch drug uptake process, mass balances of the drug inside and outside the BSN yield the respective differential equations: dC =q dt dy= dt
(6)
q F
(7)
where F is a conversion factor that relates microgram per BSN to milligram per liter.
304
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
Equations (6) and (7) can be solved simultaneously to calculate the concentration of drug inside and outside the BSN over time.
MATERIALS AND METHODS The bioencapsulation test was performed by harvesting and placing approximately 100 BSN, Anemia franciscana (San Francisco Bay Brand), per milliliter of sterile seawater in conical-bottom glass flasks provided with an airstone. Dosages of 0, 2.5, 5, 7.5 and 10 mg/ml of Romet-30 (Hoffman LaRoche) were added to each flask, and were then suspended for uptake periods of 2, 4, 6 and 8 h, in the presence of aeration and light. Each dosage and time period treatment was made in triplicate to observe the reproducibility of the results. After this period the BSN were collected with a 100 mesh screen and rinsed with sterile seawater. The BSN were concentrated on a known volume between ] and 3 ml, and the total BSN in the sample were counted, using appropriated dilution. The BSN were then disrupted on a mortar, and three 0.02 ml samples were taken and spread on filter paper discs for determination of drug concentration as described below. The concentration of the drug on BSN was measured using the Kirby-Bauer test (Jones et al., 1986; Cappuccino & Sherman, 1987), which consists of placing a disk impregnated with antibiotic or any drug in a Petri dish containing a solid culture medium, Muller-Hinton agar and the bacteria to be tested. For this experiment a Romet-30-sensitive Vibrio sp. strain was used. The strain was isolated from diseased blue shrimp Penaeus stylirostris on an aquaculture facility near Guaymas, Mexico (BIOTECMAR S.C.P.A.). Once the disks are placed on the dish, they are incubated for 24 h at 37°C. After incubation, the bacteria grow over the plate, except in a zone around the disk. The diameter of this inhibition zone is proportional to the concentration of the drug. To estimate the drug concentration on the brine shrimp nauplii, a plot of drug concentration versus inhibition diameter was constructed by using disks impregnated with different drug concentrations. The values obtained on this plot were fitted using linear regression, obtaining the following equation to correlate the inhibition diameter to the drug concentration: d = 1.141 + 3.3"Cj where d is the diameter of inhibition; and Cd is the concentration of drug in the disk. The fitting gave a correlation coefficient r 2 of 0.884; this relative low value for the correlation can be explained because the
Brine shrimp as therapeutic agent
305
diffusion of the drug from the disk to the medium is not linear, although for the range on which the samples were measured, the estimated values have a good approximation. As an additional experiment to determine the loss of drug in the BSN after the bioencapsulation period, BSN previously treated with the drug were placed in sterile seawater, and the drug concentration was determined after four different time intervals. No experiments on biocapsule feeding to fish or shrimp larvae were performed.
RESULTS AND DISCUSSION In the tests of the BSN that was not treated, a contamination area around the disks was seen, probably caused by bacteria naturally occurring in brine shrimp cysts, which were incubated in the seawater while the cysts were hatching. These samples were considered to have a drug concentration of zero. The results of the bioencapsulation tests ranged from 0 to 9 ~g per BSN, depending on the dosage and the time period allowed for drug uptake. The mean for each treatment is shown in Table 1. Mohney et al. (1989) report an uptake of 0.1/~g/BSN for a drug dosage of 3 mg/ ml and an uptake time of 4 h. These lower values could be due to the use of live organisms instead of minced nauplii in the drug concentration test. Figure 1 is the plot of the active compound uptake (microgram per nauplius) versus uptake residence time using different drug dosage (mg/ ml). The profiles of these plots are very similar to the Langrnuir isotherms described earlier, and a tendency to saturation when the uptake residence time increases can be observed.
TABLE 1 Mean Results (/zg/BNS) of the Bioencapsulation Test on Different Drug Dosage and Time Periods
Time fh)
0 2 4 6 8
Drug dosage (mglml) 2"5
5
7"5
I0
0 1.331 2.146 2"760 3'076
0 2.214 3.571 4"181 4-900
0 2"701 4-286 6"123 7"164
0 3"608 5"854 8'601 9"007
306
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez 10 •
Z
•
8
?
•
~
4
~
2
[]
i
i
l
l
2
4
6
8
10
Time, hours
Fig. 1.
Active compound uptake at different dosages (mg/liter): o, 2.5; o, 5; n, 7.5; m, 10. Solid lines show numerical simulations using q0 = 1.831 and K = 3.353.
The kinetic constants q0 and K were determinated using the plot of 1/q versus 1/y in Fig. 2, as explained in the Theoretical Considerations section. The values obtained were of 1.831/~g/BSN-h and 3-353 mg/ml, respectively; the value of F was calculated by dividing the microgram to milligram equivalence by the BSN density used on the experiments, to give a value of F= 10 ~g-ml/ml-BSN. These constants were used for the numerical simulation of the model. Equations (6) and (7) were solved simultaneously using a fourth-order Runge-Kutta algorithm (Kopal, 1955). The drug concentration values (y) used for the simulation were the dosage for each experiment. The dynamic behavior of the drug uptake is presented as solid lines on Fig. 1. It can be seen that this mathematical model (eqns (5)-(7)) describes well the time profile of uptake for each of the dosages used. For the highest dosage used (10 mg/ml) the simulated profile is slightly lower than the measured data; in this case, an adherence of tiny particles of the drug to the body of the BSN was noted by microscopic observations. This phenomenon is related to the very high drug concentration, and explains the difference between the predicted and observed data. In the other lower drug dosages the adherence was not so evident. The results of the experiment to determine the loss of Romet-30 as a function of time when the biocapsules are placed in seawater without the drug are presented in Fig. 3. With this plot the points represent the mean values and the bars are standard deviation. These results show that eight
307
Brine shrimp as therapeutic agent 3
i z r~
ea
0
0.0
i
i
r
i
0.1
0.2
0.3
0.4
0.5
1 / y, liter / mg
Fig. 2.
Lineweaver-Burk plot to estimate uptake kinetics of drug particles in BSN, q0 = 1.831/~g/BNS-h; K = 3.353 rag/liter. 5.
Z 23.
4.
3,
1
2,
1-
0 0
!
!
i
2
4
6
8
Residence Time, Hours
Fig. 3.
Loss of Romet-30 on the biocapsules when placed in drug-free sterile seawater.
hours after placing the nauplii in seawater, the concentration of the drug is reduced by half, and it may not have an effective therapeutic effect on shrimp or fish larvae. Drug loss can be explained as either a leaching out from the BSN or a degradation by the metabolism of the BSN. In the first case, eqn (1) will be a reversible process; if the drug is degraded by nauplii, eqn (1) will not
308
A. Aguilar-Aguila, A. T. Mansir, A. R. Manriquez
be a reversible process. The agreement of experimental data with the model suggests that the process is really reversible. More evidence can be found in the work of Mohney et al. (1989); as these authors use live organisms to make the drug concentration tests, the inhibition zone may be due only to the drug leaching out. The loss of the drug in the nauplii can be compensated as recommended by Szyper (1989), if its effect is quantified and sufficient prey organisms are supplied. In this case, the nauplii must be calculated for its consumption before eight hours. Another way to resolve this is raising the treatment frequency, although this is not a very recommendable strategy, due to the collateral effects that could be caused on the shrimp larvae or the bacteria present in the culture water (Jones et al., 1986).
CONCLUSIONS The model used on this paper describes very well the kinetics of drug uptake, making possible a more quantitative approach for the bioencapsulation process, and giving a way to estimate the appropriate dosage for treatment. The constants obtained in this paper are specific for the BSN strain and process conditions used here like temperature, illumination, salinity, etc. Differences could be observed for other strains or even lots used, and for different conditions, so the best way to make sure of the results is to calculate the constants in the place where the bioencapsulation will be done. Although no experiments were carried out with shrimp larvae, the Mohney experiments led us to conceive that BSN can be used as a vector for directing an insoluble particle to shrimp larvae. This makes the treatment more effective than suspending the particles in the shrimp larval culture water, because the BSN can be maintained at very high population densities (up to 100 000 organisms per liter of seawater) during the drug uptake. Therefore, this method is very attractive for the new highcost medicaments. Also, the bioencapsulation process could be used with other particle compounds, like nutrients, hormones or vaccines.
ACKNOWLEDGEMENTS The authors gratefully acknowledge Juan A. Dworak and the aquaculture facilities at Instituto Tecnologico del Mar, Guaymas. Technical Support by Ernestina Almada and Carlos Flores at BIOTECMAR and revision and discussion by Octavio T. Ram/rez are kindly appreciated.
Brine shrimp as therapeuticagent
309
REFERENCES Belter, E A., Cussler, E. L. & Hu, W. (1988). Bioseparations. John Wiley, New York, Ch. 6, pp. 145-79. Cappuccino, J. G. & Sherman, N. (1987). Experiment 43. In Microbiology, A Laboratory Manual. The Benjamin Cummings Publishing Co., Menlo Park, CA, pp. 247-54. Jones, J. G., Gardener, S., Simon, B. M. & Pickup, R. W. (1986). Factors affecting the measurement of antibiotic resistance in bacteria isolated from lake water. J. Appl. BacterioL, 60, 455-62. Kopal, Z. (1955). Numerical Analysis (lst edn). John Wiley, New York, pp. 195-213. Lehninger, A. L. (1981 ). Biochemistry (2nd edn). Worth Publishers, New York, Ch. 8, pp. 183-216. Lightner, D. V. (1984). A review of the diseases of cultured penaeid shrimp and prawn with emphasis on recent discoveries and development. In Proc. 1st Int. Conf. on the Culture of Penaeid Shrimps, Aquaculture Department, Southeast Asia Fisheries Center, Iloilo, Philippines, pp. 79-102. Mohney, L. L., Lightner, D. V. & Bauerlein, M. (1989). Bioencapsulation of therapeutic quantities of the antibacterial Romet-30 in nauplii of the brine shrimp Artemia and in the nematode Panagrellus redivivus. J. World Aquaculture Soc., 21 (3), 186-90. Sandoval, F. C., Ramirez, L. E B. & Lobina, D. V. (1993). The biochemical composition of the cysts of some Mexican populations of Artemiafranciscana Kellogg. Comparative Biochem. Physiol., 1114, 163-7. Sindermann, C. J. (1971). Disease diagnosis and control in North American marine aquaculture and fisher sciences. Aquaculture, 1, 9-29. Sorgeloos, E (1973). High density culture of the brine shrimp Artemia salina. Aquaculture, 1,385-91. Szyper, J. P. (1989). Nutritional depletion of the aquaculture feed organisms Euterpina acutifrons, Artemia sp. and Brachionus plicatilis during starvation. J. World Aquaculture Soc., 211, 162-9.