High speed, high power apparatus for vesicle prealignment, poration, loading and fusion in uniform electric fields and method therefor

Claim 1

An apparatus for treating vesicles, wherein the treatment is of the type capable of producing stable pores through membrane walls of said vesicles contained in a liquid suspension so that materials can be loaded into said vesicles or unloaded therefrom, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous and uniform electric field across said chamber containing said liquid suspension so that all of said vesicles throughout said suspension in said chamber are subjected to the same electric field potential, means connected to said providing means for generating a high voltage, high current rectangular wave treatment having a power of at least one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than 50 nanoseconds to said providing means so that immediate application of said charge to said providing means is obtained in order to subject said vesicles throughout said suspension in said chamber to said charge at the same time, means connected to said delivering means for adjusting said rise time of said charge, and means connected to said delivering means for producing a variable pulse length to said charge, said producing means being capable of high speed termination of said charge to said providing means, said pulse length being sufficient to permit said charge to treat said vesicles in said suspension.

Claim 2

An apparatus for treating vesicles, wherein the treatment is of the type capable of producing stable pores through membrane walls of said vesicles contained in a liquid suspension so that materials can be loaded into said vesicles or unloaded therefrom, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a homogeneous and uniform electric field across said chamber containing said liquid suspension so that all of said vesicles throughout said suspension in said chamber are subjected to the same electric field potential, means connected to said providing means for generating a high voltage, high current rectangular wave treatment having a power of at least one megawatt, means connected to said generating means for selectively delivering said charge at a high speed rise time of less than 50 nanoseconds to said providing means so that immediate application of said charge to said providing means is obtained in order to subject said vesicles throughout said suspension in said chamber to said charge at the same time, means connected to said delivering means for adjusting said rise time of said charge, and means connected to said delivering means for producing a variable pulse length to said charge, said producing means being capable of high speed termination of said charge to said providing means, said pulse length being sufficient to permit said charge to treat said vesicles in said suspension.

Claim 12

An apparatus for the high speed application of high voltage, high current electric field pulses in a homogeneous electric field across a liquid suspension containing vesicles having membrane walls, said electric field pulses being capable of producing stable pores through said membrane walls of said vesicles, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber comprising: (a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing said homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, (b) dielectric material separating said electrodes for forming the sides of said chamber, and (c) means in said dielectric material for the insertion and removal of said liquid suspension, means connected to said chamber for matching the impedance of said chamber when said electric field pulses are applied and, means connected to said matching means and to said electrodes for applying said electric field pulses, said applying means comprising: (a) means for generating a high voltage, high current rectangular wave treatment charge having a power of at least one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, (b) means connected to said generating means for selectively delivering said charge at a high speed rise time of less than 50 nanoseconds to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained, (c) means connected to said delivering means for producing a pulse signal of variable length, said producing means being capable of high speed shorting of said charge at the end of said variable length thereby terminating the application of said high voltage, high current charge between said electrodes by providing a high speed fall time to said charge, said variable length being sufficient to permit said charge between said electrodes to produce said stable pores through said membrane walls of said vesicles in said suspension.

Claim 13

An apparatus for the high speed application of high voltage, high current electric field pulses in a homogeneous electric field across a liquid suspension containing vesicles having membrane walls, said electric field pulses being capable of producing stable pores through said membrane walls of said vesicles, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber comprising: (a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing said homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, (b) dielectric material separating said electrodes for forming the sides of said chamber, and (c) means in said dielectric material for the insertion and removal of said liquid suspension, means connected to said chamber for matching the impedance of said chamber when said electric field pulses are applied and, means connected to said matching means and to said electrodes for applying said electric field pulses, said applying means comprising: (a) means for generating a high voltage, high current rectangular wave treatment charge having a power of at least one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, (b) means connected to said generating means for selectively delivering said charge at a high speed rise time of less than 50 nanoseconds to said electrodes in said chamber so that immediate application of said charge to said electrodes is obtained, (c) means connected to said delivering means for producing a pulse signal of variable length, said producing means being capable of high speed shorting of said charge at the end of said variable length thereby terminating the application of said high voltage, high current charge between said electrodes by providing a high speed fall time to said charge, said variable length being sufficient to permit said charge between said electrodes to produce said stable pores through said membrane walls of said vesicles in said suspension.

Claim 19

An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber comprising: (a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing a homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, (b) dielectric material separating said electrodes for forming the sides of said chamber, and (c) means in said dielectric material for the insertion and removal of said liquid suspension, means for generating a high current, high voltage rectangular wave poration charge having a power grater than one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to one of said parallel electrodes, means connected to said high speed switch for moving said switch electrodes together to cause said charge to be delivered from said one switch electrode to said opposing switch electrode in less than 50 nanoseconds, and means connected to said opposing electrode of said high speed switch for producing a variable pulse length to said charge commencing with said delivery of said charge, said producing means being capable of high speed shorting of said charge at the end of said pulse length thereby terminating said charge to said parallel electrodes, said pulse length being sufficient to permit said charge between said parallel electrodes to produce stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles.

Claim 20

An apparatus for producing stable pores in and through membrane walls of vesicles contained in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, said chamber comprising: (a) a pair of parallel electrodes spaced a predetermined distance apart on opposing ends of said chamber for providing a homogeneous and uniform electric field throughout said chamber so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, (b) dielectric material separating said electrodes for forming the sides of said chamber, and (c) means in said dielectric material for the insertion and removal of said liquid suspension, means for generating a high current, high voltage rectangular wave poration charge having a power grater than one megawatt so that all of said vesicles throughout said suspension in said chamber are subjected to said charge at the same time, a high speed switch having two opposing electrodes, one of said switch electrodes being connected to said generating means, the opposing switch electrode being connected to one of said parallel electrodes, means connected to said high speed switch for moving said switch electrodes together to cause said charge to be delivered from said one switch electrode to said opposing switch electrode in less than 50 nanoseconds, and means connected to said opposing electrode of said high speed switch for producing a variable pulse length to said charge commencing with said delivery of said charge, said producing means being capable of high speed shorting of said charge at the end of said pulse length thereby terminating said charge to said parallel electrodes, said pulse length being sufficient to permit said charge between said parallel electrodes to produce stable pores in and through said membrane walls of said vesicles in said suspension while maintaining the viability of said vesicles.

Claim 21

A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotationally aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current having a power greater than one megawatt across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second field having an adjustable rise time of less than 50 nanoseconds, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles.

Claim 22

A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotationally aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current having a power greater than one megawatt across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second field having an adjustable rise time of less than 50 nanoseconds, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles.

Claim 23

An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotationally aligning said vesicles along either their major or minor axis with the direction of said electric field, and second means connected to said providing means for generating a second uniform, homogeneous electric field at a high voltage, high current rectangular wave portion charge having a power greater than one megawatt for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said second field having an adjustabIe rise time of less than 50 nanoseconds, said power and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles while maintaining the viability of said vesicles.

Claim 24

An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding more than one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotationally aligning said vesicles along either their major or minor axis with the direction of said electric field, and second means connected to said providing means for generating a second uniform, homogeneous electric field at a high voltage, high current rectangular wave portion charge having a power greater than one megawatt for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said second field having an adjustabIe rise time of less than 50 nanoseconds, said power and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles while maintaining the viability of said vesicles.

Claim 25

A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second pulse having an adjustable rise time of less than 50 nanoseconds and a power greater than one megawatt, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.

Claim 26

A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis with the direction of said electric field, and applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second pulse having an adjustable rise time of less than 50 nanoseconds and a power greater than one megawatt, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.

Claim 27

A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together at locations throughout the chamber, applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, said second pulse having an adjustable rise time of less than 50 nanoseconds and a power greater than one megawatt, said second uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said vesicle pairs while maintaining the viability of said vesicle pairs, and waiting a predetermined period of time to permit the paired vesicles to fuse into a single vesicle in response to the step of applying said second uniform, homogeneous electric field pulse.

Claim 28

A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together at locations throughout the chamber, applying a second uniform, homogeneous electric field rectangular wave poration pulse at a preselected high voltage and high current across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency so that all of said vesicles throughout said suspension in said chamber are simultaneously subjected to the same electric field potential, said second pulse having an adjustable rise time of less than 50 nanoseconds and a power greater than one megawatt, said second uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said vesicle pairs while maintaining the viability of said vesicle pairs, and waiting a predetermined period of time to permit the paired vesicles to fuse into a single vesicle in response to the step of applying said second uniform, homogeneous electric field pulse.

Claim 29

A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field from a random function generator at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis with the direction of said first electric field, and applying a second uniform, homogeneous electric field rectangular poration pulse having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles.

Claim 30

A method for producing stable pores in and through the membrane walls of ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field from a random function generator at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically aligning said vesicles along either their major or minor axis with the direction of said first electric field, and applying a second uniform, homogeneous electric field rectangular poration pulse having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles.

Claim 31

An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of aligning said vesicles along either their major or minor axis, and second means connected to said providing means for generating a second uniform, homogeneous rectangular wave electric field having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said predetermined value and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles.

Claim 32

An apparatus for producing stable pores in and through membrane walls of aspherical, ellipsoidal, electrically or dielectrically aspherical vesicles placed in a liquid suspension, said apparatus comprising: a chamber for holding at least one milliliter of said liquid suspension containing said vesicles, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, means in said chamber for the insertion and removal of said liquid suspension, means operative with said chamber for providing a uniform, homogeneous electric field across said chamber containing said liquid suspension, first means connected to said providing means for generating a first uniform, homogeneous electric field at at least one preselected frequency across said chamber for a first predetermined time interval, said at least one preselected frequency being capable of aligning said vesicles along either their major or minor axis, and second means connected to said providing means for generating a second uniform, homogeneous rectangular wave electric field having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less for a second predetermined time interval across said chamber to provide substantially the same transmembrane electric field potential simultaneously across each aligned vesicle, said predetermined value and said predetermined time interval being based upon the time constant of said membrane to produce said stable pores in said vesicles.

Claim 33

A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis, applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of leading said substances so that said vesicles contain said substances.

Claim 34

A method for loading substances into ellipsoidal, electrically or dielectrically aspherical vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at at least one preselected frequency across in said chamber for a first predetermined time interval, said at least one preselected frequency being capable of electrically rotational aligning said vesicles along either their major or minor axis, applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rise time of fifty nanoseconds or less across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said second uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said second uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of leading said substances so that said vesicles contain said substances.

Claim 35

A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together, applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rise time less than 50 nanoseconds across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said vehicle pairs while maintaining the viability of said vesicle pairs, and waiting a predetermined period of time to permit the paired vesicles to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.

Claim 36

A method for fusing vesicles together, said vesicles being placed in a liquid suspension, said method comprising the steps in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a first uniform, homogeneous electric field at a preselected frequency across said chamber for a first predetermined time interval, said preselected frequency being capable of pairing said vesicles together, applying a second uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rise time less than 50 nanoseconds across said chamber for a second predetermined time interval in response to the step of applying said preselected frequency, said uniform, homogeneous electric field pulse being capable of creating pores at contiguous adjacent points in and through the membranes of said vehicle pairs while maintaining the viability of said vesicle pairs, and waiting a predetermined period of time to permit the paired vesicles to fuse into a single cell in response to the step of applying said second uniform, homogeneous electric field pulse.

Claim 37

A method for loading substances into vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rapid rise time of fifty nanoseconds or less across said chamber for a predetermined time interval, said uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.

Claim 38

A method for loading substances into vesicles, said vesicles being placed in a liquid suspension, said method comprising the steps of in the order of: inserting more than one milliliter of the liquid suspension in a chamber, said liquid suspension being capable of being selected from the group consisting of electrolytic and non-electrolytic fluids, applying a uniform, homogeneous electric field rectangular wave poration pulse having a power greater than one megawatt with an adjustable rapid rise time of fifty nanoseconds or less across said chamber for a predetermined time interval, said uniform, electric field pulse being capable of providing substantially the same transmembrane poration electric field simultaneously across each aligned vesicle to produce said stable pores in said vesicles while maintaining the viability of said vesicles, removing said porated vesicles from said suspension in response to the step of applying said uniform, homogeneous electric field pulse, loading said substances through said stable pores formed in said membrane walls of said vesicles in response to the step of removing said vesicles from said suspension by suspending said porated vesicles in a medium carrying said substances, and annealing said pores in said vesicles in response to the step of loading said substances so that said vesicles contain said substances.