Patent Publication Number: US-H2119-H

Title: Acoustic fusion of aquatic animal tissue cells with biological agents

Description:
The present invention relates in general to the perforation of living cells during delivery thereto of biological agents for various purposes including tagging and DNA infusion. 
     BACKGROUND OF THE INVENTION 
     A process for transfer of chemical agents within some surrounding medium through living tissue referred to as poration, is well known in the art as disclosed for example in U.S. Pat. No. 5,128,257 to Baer. Generally, such process involves generation of electrical energy forming an electrical field between electrodes within which electroporation occurs. The generation of an ultra-sound acoustic field within which different chemical compounds are administered by delivery to the tissue cells of aquatic animals or their eggs for fish farm treatment purposes, is disclosed in U.S. Pat. No. 5,076,208 to Zohar et al. However, infusion of animal tissue cells with biological agents such as tagging compounds was heretofore performed by electroporation subject to various disadvantages such as tissue cell disruption by high voltage and reduced processing efficiency because of limits imposed on the number of tissue cells capable of being simultaneously processed. It is therefore an important object of the present invention to provide an improved process for infusion of biological material into living tissue cells of relatively large aquatic animal populations within underwater environments with improved efficiency. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, large numbers of aquatic animals are serviced by poration and fusion of their tissue cells with certain biological compounds transferred from solution within an underwater treatment zone. The poration and infusion process is performed within the treatment zone under an acoustic energy field of limited duration produced by amplification of a steady state electrical power signal converted into ultrasound waves within a cavitation frequency range establishing the acoustic field within the treatment zone. The tissue cells after being so treated are extracted from the treatment zone. 
    
    
     
       BRIEF DESCRIPTION OF DRAWING FIGURES 
       A more complete appreciation of the invention and many of its attendant advantages will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawing wherein: 
         FIG. 1  is a block diagram schematically illustrating the process or method of the present invention; 
         FIG. 2  is a partial section view and block diagram illustrating transducer conversion apparatus associated with the method depicted in  FIG. 1 ; and 
         FIG. 3  is a graphical illustration of processing parameters from which maximized treatment duration is obtained. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Referring now to the drawing in detail,  FIG. 1  diagrams the aquatic animal treatment method of the present invention, generally referred to by reference numeral  10 . Pursuant to such method, the tissue cells  12  of aquatic animals to be treated such as migratory fish including rainbow trout as denoted in  FIG. 1 , are to be fused with a biological agent  14  by insonification which includes perforation and fusion performed within an underwater zone  16  during periods of limited duration so as to accommodate treatment of large populations of the aquatic animals. The agent  14  according to one embodiment is of a commonly used type from a group of tagging compounds including calcein (Fluorexine) and oxytetracyline hydrochloride (Terramycin) forming a solution with the water in the treatment zone  16  from which it is transferred to tissue cells such as the larvae and/or eggs of the fish being tagged. After completion of such treatment the tissue cells are removed from the treatment zone  16  and may undergo further processing such as washing so as to extract unfused agents from the treated tissues  20 . 
     The foregoing referred to treatment within zone  16  is performed in response to establishment of an acoustic field therein by generation  22  of acoustic waves as denoted in FIG.  1  and under certain conditions established by water flow rate and temperature by controls  24  for inflow and temperature and controlled outflow  26  from the treatment zone  16 . Timing of treatments within zone  16  are regulated by treatment duration control  28  applied to acoustic field energy generation  22  and controls  24  as also denoted in FIG.  1 . As hereinafter indicated, treatment efficiency pursuant to the present invention is maximized by so limiting the treatment duration. 
     The parameters of method  10  associated with the generation  22  of acoustic field waves for performance of the treatment, were obtained in connection with the use of apparatus as depicted in  FIG. 2  showing treatment zone  16  enclosed by a cylindrical shaped transducer  30  within which water is confined. Such transducer  30  is of a piezoelectric type made of materials such as lead zirconium titanate ceramic (PZT), covered by a water protective coating  32 . Such transducer  30  converts electrical energy supplied thereto into ultrasound energy in the form of acoustic waves  34  as symbolically denoted in  FIG. 2 , establishing the acoustic field within the water treatment zone  16 . The electrical energy was converted by the transducer  30  into a steady state AC signal of approximately 40 kHz within a cavitation frequency range derived from a signal generator  36  and amplified to a voltage level of approximately 200 volts by a power amplifier  38  as denoted in FIG.  2 . Such AC signal may be either continuous or pulsed having a selected duty cycle. 
     Fish eggs and/or larvae obtained from rainbow trout were exposed during experimental treatment to the foregoing described acoustic field within zone  16  by placement into a test tube  40 , onto which the acoustic waves  34  are focussed as shown in  FIG. 2 , mixed with a body of seawater  39  having agent  14  dissolved therein. Such treatment was maximized by passage of a continuous flow of water from a coolant supply  42  to maintain the body of seawater  39  chilled to a predetermined constant temperature of 10° C., while air was injected into the seawater within the test tube by flow from air supply  44  forming bubbles therein during a limited treatment duration between 5 and 30 minutes. The test tube  40  was made of an acoustically transparent plastic material so as to accommodate exposure of its contents to the acoustic field generated during experimental treatment within zone  16  as hereinbefore described. 
     From the foregoing referred to experimentation, various parameters of the method  10  were determined as graphically diagrammed in FIG.  3 . Curved  52  in  FIG. 3  depicts the variation in absorption by infusion of a tagging compound type of agent  14  into fish tissue cells as denoted on the vertical scale  46  for different concentrations of such tagging compound (calcein) as denoted on horizontal scale  48  during a 5 minute treatment duration. Such graphically depicted treatment reached maximized absorption at a peak point  50  on curve  52 , for the same concentration of the tagging compound corresponding to a maximized absorption peak  54  on curve  56  for a 30 minute treatment duration. Accordingly, treatment is maximized by limiting its duration between 5 and 30 minutes under the conditions of flow, temperature and acoustic field energy within the treatment zone  16  as hereinbefore specified. 
     It should be understood that the transducer  30  may be replaced by a flat or curved plate type of transducer with which an unconfined body of water has the acoustic field disposed therein in contact with the transducer. Also, frequencies other than 40 kHz for the electrical energy converted to ultrasound energy by the transducer may be utilized, as long as it is within the cavitation frequency range. The fusion treatment was performed under the conditions described herein at a relatively high delivery rate causing detachment of the mucous membrane of the fish larvae. Such membrane is expected to grow back on the fish being tagged if maintained in a clean environment by washing for an adequate period of time following fusion treatment. Treatment for purposes other than tagging, such as DNA infusion, may be achieved by the present invention because of its capability for penetration of single living cells under the conditions described herein. The method of the present invention is therefore applicable to single cell organisms such as bacteria as well as plant cells. 
     Obviously, other modifications and variations of the present invention may be possible in light of the foregoing teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.