CA2261703A1

CA2261703A1 - Electroporation employing user-configured pulses

Info

Publication number: CA2261703A1
Application number: CA002261703A
Authority: CA
Inventors: Gunter A. Hofmann
Original assignee: Individual
Current assignee: Genetronics Inc
Priority date: 1996-09-09
Filing date: 1997-01-27
Publication date: 1998-03-12
Also published as: JP2001503208A; JP3963955B2; EP0925647A1; KR100420731B1; DE69711893T2; KR20000035966A; US6096020A; US5869326A; ES2175343T3; ATE216155T1; DE69711893D1; EP0925647B1; WO1998010515A1

Abstract

An electroporation method and apparatus generating and applying an electric field according to a user-specified pulsing scheme. Advantageously, one such pulse includes a low voltage pulse of a first duration, immediately followed by a high voltage of a second duration, immediately followed by a low voltage of a third duration. The low voltage electroporation field accumulates molecules at the surface of a cell, the appropriately high voltage field creates an opening in the cell, and the final low voltage field moves the molecule into the cell. The molecules may be DNA, portions of DNA, chemical agents, the receiving cells may be eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, sperm, or other suitable cells. The molecules are placed in close proximity to the cells, either in the interstitial space in tissue surrounding the cells or in a fluid medium containing the cells.

Description

ELECI'ROPORATION El~LOYING USER-CONFIGURED PULSES

TECHNICAL FIELD
The present invention generally relates to electro-cell manipulation. More particularly, the invention concerns an electroporation apparatus and method forgenerating and applying an electric field according to a user-selected pulsing scheme to more efficiently introduce molecules into cells and minimi7~ damage to cellular tissue.

BACKGROUND ART
A cell has a natural resistance to the passage of molecules through its membranes into the cell cytoplasm. Scientists in the 1970s first discovered "electroporation", where electrical ~lelds are used to create pores in cells without causing permanent damage to them. Electroporation was further developed to aid in the insertion of various molecules into cell cytoplasm by temporarily creating pores in the cells through which themolecules pass into the cell.
Electroporation has been used to implant materials into many different types of cells. Such cells, for example, include eg~s, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria. fungi, yeast, and sperm. Furthermore, electroporation has been used to implant a variety of different materials, referred to herein as "implant materials", "implant molecules", "implant agents". Namely, these materials have included DNA; genes, and various chemical agents.
Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. With in vitro applications, a sample of live cells is first mixed with the implant agent and placed between electrodes such as parallel plates.
Then, the electrodes apply an electrical field to the cell/implant mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM
~ 25 600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc.

W O 98/lOSlS PCT~US97/01088 With in vivo applications of electroporation, electrodes are provided in a caliper that grips the epidermis overlying a region of cells to be treated. Alternatively, needle-shaped electrodes may be inserted into the patient, to access more deeply located cells.
In either case, after the implant agent is injected into the treatment region, the electrodes apply an electrical field to the region. Examples of systems that perform in vivo electroporation include the Electro Cell Manipulator ECM 600 product, and the Electro Square Porator T820, both made by the BTX Division of Genetronics, Inc.
One type of in vivo electroporation application under research is electrochemotherapy, which uses electroporation as to deliver chemotherapeutic agents directly into tumor cells. This treatment is carried out by infusing an anticancer drug directly into the tumor and applying an electric field to the tumor between a pair of electrodes. The molecules of the drug are suspended in the interstitial fluid between and in and around the tumor cells. By electroporating the tumor cells, molecules of the drug adjacent many of the cells are f~rced or drawn into the cell, subsequently killing the cancerous tumor cell.
Electroporation in this application is especially beneficial because electroporation can help minimi~ the amount of implant agent used, these chemicals frequently being harmful to normal cells. ln particular, less of the implant agent can be introduced into the tumorous area because the electroporation will enable more of the implant agent to actually enter the cell. Electroporation is also beneficial for chemotherapy because some of the most promising anti-cancer drugs, such as Bleomycin, normally cannot penetrate the membranes of certain cancer cells. However~ recent experiments with electroporation demonstrated that it is possible to insert the Bleomycin directly into the cells.
Known electroporation techniques (both in vitro and in vivo) function by applying a brief high voltage pulse to electrodes positioned around the treatment region. The electric field generated between the electrodes causes the cell membranes to temporarily become porous, whereupon molecules of the implant agent enter the cells. In known electroporation applications, this electric field comprises a single square wave pulse on the order of 1000 V/cm, of about 100 lls duration. Such a pulse may be generated, for W O 98/10515 PCTrUS97/01088 example, in known applications of the Electro Square Porator T820, made by the BTX
Division of Genetronics, Inc.
Although known methods of electroporation may be suitable for certain applications, the electric field may actually damage the electroporated cells in some 5 cases. For example, an excessive electric field may damage the cells by creating permanent pores in the cell walls. In extreme cases, the electric field may completely destroy the cell.
Attempting to ameliorate these undesirable ef'fects, at least one application has proposed the use of multiple pulses. One application, for example, proposed use of an 10 electromechanical relay to provide consecutive first and second pulses. S.I. Sukharev et al., Biophys. J. Vol. 63, November 1992, pp. 1320-1327. More particularly, Sukharev uses an electric field pulse 100 as shown in Figure 1. The pulse 100 includes (1) a first, narrow duration, high voltage pulse 102, (2) a delay 103 of ~t, during which no pulse is generated, then (3) a second, wide duration, low voltage pulse 104. The first pulse 102 was intended to porate the membrane, whereas the second pulse 104 was intended to electrophorese DNA into the cell cytosol. Sukharev tecoL~nized that the delay 1()3 should not be excessive.
Although the Sukharev system may provide satisfactory results in some applications, this system may not be completely adequate f'or certain other applications.
20 Some users may find, for example, that Sukharev's electroporation does not effectively move enough molecules of the implant agent into the target cells. This results from an excessive delay 103 between Sukharev's first 102 and second 104 pulses, as recognized by the present inventor. The pores of a cell, created by electroporation, stay open for a finite time, largely depending upon the cell's temperature. Thus, the effect of the first 2S pulse may start to significantly decay (thereby closing the cell's pores) during the delay between the first and second pulses. In some applications, this may be sufficient to completely nullify the first pulse's effect upon the cell by the time the second pulse occurs. As a result, the efficacy of Sukharev's electroporation may be insufficient in some cases. Moreover, lacking an effective first pulse, the second pulse of Sukharev's 30 system may need to be increased to the point where it permanently destroys cells.

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The dclay d~s~ ed above i6 inherem to the Sukh~rev system due to the use of ele~ll u,"~,I.anical re~ay~. Sukharev uses independcnt pulse generators, whose outputs ~rc selectively coupled lo output clc~Llu~c5 by a rclay. As known in thc art, howcver, the switching of an d~c~l.,"~echuucal rclay typically takes a ~i~nificant amount of time, 5 sorn~tim~s e~en S0-100 m~. Therefore, the efficacy of the implarlt agent achie~cd by Sukharev n~y be too low for somc applic~tions.
U S. patent 4,663,292 cntitled ~'High-Voltage ~iologica~ h~a~ramolecule Fusior.
System " tcaches ~fan elc,.l~opo~ati~n device using ar~ outp-lt high-vol~age tranformer for pulse duration control of ~n output di~charge pulse of spccificd voltage rn~ tn~l e - 1~ PCT application WO 91118103 entitled Method and Device for MaJ~in~ ing Cells Permeable," teaches of an clcckopora~ion dovicc thllt controh dischargc pulsin~ by applyin~ one ~hor~ h electric field pulse to living cells undergoin~ el~ctroporadon to rn~lce thesc ceJI~ p~ .c~blc to molccular ~ent materials to bc in~roduGed to the cell. I~ex~, this devicc ~cnera~cs at Icust onc longer elc~trical pulse dischar~c of a lowcr elcctric field gn~ de Thu~ as recognized by the prcsent inventor~ cxistin~ clcctroporation sy~terns m~y no~ be s~itable for certQin application~ duc to thc ~cneration of an exce~sive electn& ficld or due to the delsy betwoen adjacent plll9CS. Furthcrmorc~ m~ny existing electropor4tion systems laclc s~mci~t control over the par~neters of the electric field pulses such as 20 amplitude duration, number of pul3e~s, etc.
, ;
D~SCLOSL~F. OF ~IVENT~ON
l~roadly~ thc present invention concerns ~n elcctroporation method and apparatusfor 6~ ating and applyir~ an elcctric field ~ccordit~g to a user-specified pulsing schcme.
One eY~ of such a pulsing schane il-rllld~C ~ low voltage pulse of a first duration, 25 ir~me~ roly followed by 8 high voltage of a ~ccont dur~tion, i.,.l"cl;~tely followed by l~w vol~a~e of a third duration. The invention providcs thc low volt~6c electropora~ion field to ~ccum,Jl~te ~nolec~ at tho surf~cc of a ccll, the appropriately high volta~e field t~ croate arl openin~ in the coll, ~nd the final low volta~e fleld tO move the n~leeul~ in~o the eeU.

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The molcatles may be ~cncs or drugs ~uch ~8 DNA~ p~rtions of DN~, chemical a~ent~ or any otha mnlec~ . The r~olecules arc placod in close proxirnity to the cclls, either in the interstitial tissue surrounding thc cells or in a tluid rTleclium containin~ thc cells.
5~ceordingly, onc aspect of thc prcscnt invention concerns a mcthQd of ~cncrating and applyin~ an clectric fielt according to a user-s~'e~ pulsing schcmc to more efficiently introduce rnolecule~ into cell~ and ~hlh~ze damage t~ cellular tiDsue.
A different aspect of the invention conccrns an a~ar~tus comprising an electrical pulse ~norator to ~nerate ~nd apply such a pulsjng scheme. One crnhodim~n~ of sllch an 10apparatUs utilizes the following co.,.pol,e.,~s. First and second power surpliçs provide first and sccond rcJ~ec~ e output voltages A transtormer, with prima~y and secondaly windin~s, ha~ a pair of output te~.~.in&ls cc)uplet to the secon~ y windjngs. A first switch, rcsponsive to ~ first ~atin8 signal, applies thc first output voltage to the primary windin~.
A second switcl; responsivc to a second gatin~ si~nal, applies the ~econd volts~e directly 1~to thc output terminals. ~ controller, ~cc; ~es uscr ~rerifi~ti~ln cf an output pul~c pattcrn, and pro~ides the tlrst and ~econd ~atin~ 6igna~1s to generate the ~l~erified output pulse p~ttcrn at the output terminal~
The prcsent invcntion provides a number of distinct bene~ts. Generally, thc invention is usoful to introduce ~nol~Gllles of an in~plant a~ent into cells with ~ignificantly 20increascd cf~ectiveness~ The treatment ~cnt, for examplc, may inclute drugs for tresting ,. .cancer, karposi's sarconta~ and a num~er of other ~e~seq and condition~.
ln contrast to prior ~ ngcn.enls u~ng a const~nt IcveJ clectr;ic fieJd, the stcpped pul~c of thc inveJ~tion minimizes ccll darn~ge by using a low volta~o dectric ~ield before and aRer cell pcre~ ue createt. The inve~tion thus l ~lilLl~U~s th¢ exposure of cclls tc hi~h 25volt~ge eJectrica~l ficld~, rcducing possible damage to the cclls. Moreover, the 3tepped pulsc of thc invcn~i~n aJso saves cnerW, sincc the ~irst and third pulses usc Icss voltage than prior ~rrangements.
~n attition, by ~sin~ elccl. o~or~L,on to open cell~ for receipt of nlrle lJle~ of an implant agent, the invention i~cre&~s the efficacy of the a~ent. Consequcnt~y, Icss of the 30implant agent i5 n~eded, thcrcby rcducing any ~idc c~rccls of thc implant agent.

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Another benefit of the invcntion i5 that, regardless of how long voltage is applied to ~he trar,;,l~, .,.~,'~ prima~y winding, resultant tran~f~rmer saturation limits the duration of the co. . e~pon~ g O~ltpUt ~ignal c~n thc transformer's secondary windin~. Thi3 prevents dama~ge to the treated cells, whi-;h mi~ht otherwise resull from prolon,ged applic~ion of 5 volta~c to ~he transformer's primary winding. Another atv~ta~,e of the invencion is that the tra~sformcr's output is floatinR, and therefore no ~ubst~nti~l current will flow if the patient i~ c~nnccted to snother carth or ground polentia].
The invention also pro~ides a number of othcr b~ncfi~c, as di scl~sed in greater detail below.

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BR~FF D~.C~ TlON OF DRAWTNG
Thc objects, ad~an~a~es and feature~ of this invcntion uill be rnore readily appreciated ~om thc following detailed de~cription, when rcad in conjunction with the accompanyin6 drawin~, in which:
FIG I is a di~~rarn illustratin~ an electroporation wan~eform in accordancc with the ~s pri~r art;
~G. 2 i~ a dia~ra~n of the hardw~rc components ~nd im~reoJmec~ions of a pul~c generator pursuant to one aspect of the present invention;
FIG. 3 is a di~ram of ~n e~cemplaly ar~iclc of m~ f~c~l~re~ comprising a datA
storage medium, in ~ccord~ncc with one aspect of the present invention;
FlG. 4 is a flowch3r~ illustr~ting an exemplary sequcncc of method steps in -.. ;.
accord~ncc with nne aspect of the prescnt invcntion;
FlGS. S-9 arc dre in~s of ilh~strative elc~troporation pulsing schemc~, pursuant to thc invenlion; asld FlG. 10 is- 8 flowchart illustr~tin~ an .-Yon~r~ equence of method steps in ''5 accordsnco with one cxample of thc prescnt invention.

N~ED SHEET

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BEST MODE FOR CARRYING OUT T~E lNV~~1TrON

~ARDWARE COMPONENTS ~ lNTERCONNECTlONS
As mentioned abo\re, one aspect of thc present invention concerns an improved electrical pu~se ~c~ tor capable ot'generating and ~pplyin~ ~n elcctric field ~ccording to 5 a user-sekrted pulsing scheme ta more efficicntly introduce moiecules into cells and mininnize dama~e to cellular tissue. Fi~ure 2 depicts an excmplary pulse generator 200 The gcnera~or 200 inrl~des a number of diffierent corrlpon~r~t~, described ~ follows.

- Power S~IY
A power supply 202 providcs a rcl~ble source of desired v~llagc !evels ~r use bythe generalor 200. The p~wa supply 202 reccives an input voltayc~ such as 11 OV or 220 VAC from a power source 203. ~ dividcr 204 convert~ the input vcltage into rnultirle refore~cc voltages ln the illu6trated embodl-.~cr.l. r~ voltzgcs of 500 ~t (~. C ~ reside on thc divider ou~put lines 204a-204b.
Thesc voltages are provided to c~ ctors 2û6b-2û7h of first and second re~pective~5 transistors 2~6-207. Thc tr~nsistors 706-207 arc selectively gated to apply thcir input voltages to stop voltage nodes 208.209. The selective ~ting ofthe transistors 206-207 is performed by re~pcctive cornparators ~ 213~ whlch tri~ger gA~es 206a-207a of thetransistors 20~-207 when voltages at ;he s~ep voltagc nodes 708-209 dips below volta~es est~hl;r~ d on 3tep voltage inp.n line~ ~16-21 î For example, when the c~ pQlalor 2l2 20 detcrn~ines th~t the voltage on the step voltage nodc 208 is less than the voltage on the prc-set input line 216, the comp~rator 212 activ~te~ the ~ate 206a of the transistor 206, c~using the transistor 206 to couple tlne input volt~ge of the divider 204 directly to the 5tep voltage node 208. Thus, thc transifi~ors 206 maintain substantially con~t~n~ volta~es at the respecli~e step volta~o nodc~ 208-209 in accordance ~r;ith thc stcp voltagc input lines 21~-A,~~NL~!,-3 K~Y.'~O~ IflX "~j CA 02261703 1999-01-27 - --8- REPL~CII MENT P~GE

Ençr~v Re~eryoir~
Thc ~cncrator 200 also inrl-~d~ cllcr~y reselvoirs 220-2~1 coupled to respectivestep voi~a~e nodes 208-209 E~empl~y cnerW rcscrvoirs 220-221 may comprise cap~cit~r3, ~uch as 3200 ,uF, S00 V electrolytic capacitors These capacitors arc aFpropriate for r~m~lm otcp voltagcs 2~8-20~ of 500 ~/ (D.C,).

Tran~for~er Thc generator 200 also in~ludes a transfonn~r 224, which include~ a primary windin~ ~24a and a second~ry w~nding 224b Thc t,~r~fo.,..e~ 224 p;ef~)y demon~trates - - Iow leal~e induc~nce to advanta~eously provide A fast pulse rise time, on the order of se-eral micro~econds. Prefcrably, thc transformer 224 exhibits low inductancc, on the ord~r of several ,IH Thc~c featurcs rn&y bc provided by winding the ~r~nsr~ n.er ~4 w~th a single cable of twelve separate, twisted conductors of which six are c~nn~ctetl in parallel for the primary, six are connected in series fcr the 9ecol~Ao~y. This provide~ a 1 6 ste~up ratio In addition, A separate low volta~c D C bias winding around the core may be used I S to employ d~e fi~ll flux sw~ng of the tran~o~ ,c. 's core. A3 an example, the transformer may utilize a core ~n4de of laminated iron.
Thc ~ ,.cr 224 may advant~e~)ucly be constructed ~o ~a~urate if the pu1sc Iength exceeds a maximum prcscr~bed value~ thereby protccting a patient fron~ excesiive electrical ener~y Pl cfc~bly, the trar~formcr ~24 is capab~e of carrying ~ 3 V-scc (3000 V x 100 ~L~C) before saturation, Anothcr ~ aMBge ofthe transformer 2~4 is that its output is floatin~, and no sub3t~ntial currcnt will flow if the patient is connected to anothcr earth or ground potential.
The second~y wlnting 2Z4b is coup~ to outpus nodcs 230-231, which ~re emboticd by clcc~rode~ in the illustrated application. The electrodes (not shown) may comprisc para~lel plate electrodcs, necdLe clcetr~des, caliper clc~,lrodes, or ancther arran~ement of electrotes Furthcr discudsion of ca~iper elec~lûdcs ~ppcars in (1) U.S
r'atcnt Application No. Q8f 5 37,265, cntitlcd "Mcthod of Troatment U~in~ Elc.,~rupc~l~tit~n l~lediatcd Delivery of Dru~s ant G~nos", filed on Sc,~ . 29, 1~9S, and (2) ~ev et ~1., "Elec~rochemoshorapy- a novel mcthod of canccr ~r~ nt~ll Cancer T~ ..c.,l Rcview~
(l994) 20, 1 OS- I I S A usefill example of neetlc Iccll~des is di~G~ ed in U.S, Plltent Al~/J~N~ED S~EET

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5,702,359, entitled "Nocdle Eloctrndos For Electroporation Mediated l~elivcry Of Drugs and Gcnes "
The load b~ween the electr~des 230-231 is represented by a resistor 234. In the illustrated c,~bodi.~ent, thc load 234 comprises a number of cells, ~hich may be in v~
or in vivn ~amp!es of eggs, plate~e~s, human cell3, red blcod cells, mammalian cells, plcnt pro~npl~etq, plant polle~l, liposnmes, b~cteri~ .3i, y~st, sperm, or othcr colls.
To protect th~ energy reselvoir ~20 and power ~upply 202, a diode 236 may be placcd between the energy rcscrvoi. 220 and thc electrodc 23~. Likewise, to protect the cncrgy rcser~oir 221 and powcr ~uppiy 220, a diode 237 may be placct betwcen t~he seco~ Rry winding 2~40 and the olcctrodc Z30.

Switches The ~enaator 200 al90 include~ switcbes 22~-227 to selectively enabie current t~flow through thc primuy and second~r~ winding~ 224a-224b, r~Qp~c~ ely. In one exempla~ Gonstruction, each switch 226-~27 may comprise an insulatcd ~ate bipolar ~ransistor ("lGBT"). such as a Fuji Electric brand 1MB1400F-060 model IGBT.
The switch ~6 and ~he ener~y rescrvoir 221 3re cnlJpled in serics, this ~eries combination bein~ att~ched ir. parsIlel wi~h thc primaly winding 224a. When vo!tage is applied 10 a ~,3te 226a of the swi;ch 2~6, thc collectDrs 226b and cmitter 226c are clectncally con d. Thus, Ihc encr~y reservoir 221 is cfl~:ctively placed in parallel with the prim~ry windin~ 224~. This pt,'rTnit3 currcnt from the enerW reservoir 121 to flow through the primary winding 224a.
Simila~ly~ the sw~teh ~Z7 and cnergy reservoir 220 aro coupled in senes, this scries c~mbinatior. bcing auachcd in puallel wilh the sccc~n~ - ry winding 224b. When voltage is ~pplied to ~ Bat~-227a of the swi~ch 227, thc collectors 227b and emittor 227c are electr~cal'y connected. Thus, the cnergy rescrvoir 220 is cfleclively placed in puallcl with ~he secc~ndary windin~ 224b. Tnis pamits currcnt from the cnergy rescrvoir 220 to flow throu~h the loHd 234.
Advant~geousl~, none nf thc ener~y re~e~Qirs 220-221 or ~witches 226-227 ~rounds ~e winding~ 2~ 224b. Th~ windings 22~a-224b arc thacfore clectric~

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floating. As a result, no ~ubqt~nti~l curTcnt will flow throu~h a patient or other load 234 that is coMectcd to at~other earth or ~round pot~

Controllcr ~nothcr componcnt of the 3enerator 200 i~ thc controller 240, which mana~es S operation ~f thc switchcs 22~-227. Broadly, the controller 240 re~ te~ the on-times and off-times ofthc switches 226-227 in accordancc 8spcrifi~d schedule, thercby ~enerating a prcdctcrmined pulsing scheme at the electrodcs 230-231. When the controller 240 trig~er~ tho switch 227, the volta8e of the onergy rcscrvoir 220 is applied ta the eJectrodos 230-231. When the wntroller 240 triggers the switch 226, thc voltage of the energy rcser~oir Z20 is applied t~ the transformor 224, where it i~ multiplied by six and applied to the elcctJodes 23 0-~3 1 . The contrclla 220 ma~ also trigger both svvltchos 226-~27 to apply anadditivevolta~e,comprisingthcsumofthestepvol~agcs208-209,totheelectrodes230-2~1 .
The controller 240 rnRy comprise a computer, di~ital or anal~g proces~in~
apparat~, pro~rammable logic array, hard-wircd logic circuit, application specific intcgratet circuit ("ASIC"), or anothcr suitsble device. ln an excmplary embodiment, the control~cr 240 may comprisc a PIL 16C64 M;crochip microprocessor accomp~nied by appropriatc RAM and ROM modules, as desired.
Prcfcrably, the controllcr 240 is coupled to a u~cr interfacc 242 for ~Ych~ngine d~ta , 20 with a uscr. In the illustratcd examp~e, ihe u~er may nperate the user interface 242 to input a desired pul~ing pattern eO bc applicd t~ the elcctrotcs 2~0-'~31.
As an cxample, thc u~cr interf~ce 242 may includc ~n ~Irl~numenc Iccypad, touch screen, computer mouse, push-buttons ~nt/or togglc switchcs, or another suitablccomponent to rccoivc inpu~ ~om a human user. The user interface ''42 may al90 includc a CRT scrcen, LED s~reen, I CD screen, liquid crystal display, printer, displ~y pancl, audio speaker, or ~nother suitable CO~ JOnCllttO convey data to a hurnan user.

Preferable n~U~n P7~neters Tho cloctricsl requiremcnt~ can be deri~ed from the ficld strength, which wa~
dcte "~ ~1 efflcac~ous flrom in vitro e~c,~",~ w~th tumor cells and dru~, typiullly 1200 fND~D S,lEET

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- 13û0 V/cn~, Qnd a pul~e Icnyth of about 100 ~9CC. The maximun~ VOItBgC ofthc generator derives frons the nlaximum tumor 3i2:C onc w~nt~ to trcat. rn ordcr to treat tumors up to 2 crn diamcter with caliper elcctrodes (parallel plates) at ficld strength of 1~00 ~Jcm, an Qperating voltage of 1300 x 2 - 26~0 V i6 required; thc gcncratDr W~l9 dc~i~ned to ~cncratc 5 3ûD~ V maximum to providc ~omc extrs mar~pn.
Thc tissucJtumor specific resis~ivi~y was aisumed ta be as low as 10û Oh~n x cm.With an clectrodc arca of 3 cm x 3 cm = 9 cma~ the rcsi~tance is ~2 Ohm. thc intcrnal impcd3nce of the generator sbDuld be at least a hctor 10 lowcr than 27 Olun so that no subslantia~ drop in volta~c occurs bctwccn charg~n~ and delivered volta~e. With th~
10~ m voltage of 3000 V and a lo~d impcdance O f 22 Ohm, the switchin~ requi. en~e.,ls ~~ from B partial c~p~ritQr dischar3e to generate a squuc pulsc are a very Ylhs~A~ 4C0 kW.
The dcsired m~y~ m permeation pulse len~h is 100 ,lsec; this re~ults in ~n energy per pulse of 40 J. For the cnllection an~ clc~ phoresis pu!se parameters, a maximum volta~ce of 5(:)d V and m~ximum pulse length ~f 20û mscc may be used.
15The r~s~ m load currcnt is about 136 A. which translatej int~ a primary current of 6 136 - 816 A, which the switch has to carry and turn on and o~ Thc switches 2~6-227 can prefcrably maintain continuous cur.rent 800 A for I msec. The n~Yiml Im volta~e is 600 V. Transicnt spikes are limited to a rn~ m of 5~0 V for a 10% safety mar~in This requirod carcfill low induct~nce l..cc~~ ~ 1 assembly to rc~uce trano;~nt~ and ~ be 20 able to get as c30se as safe~y feasible to the maximum volta~e limit of the lGBT.
The l~ad i.npcd~.cc of 22 Ohm is transformed to the primaly: 2V6x6 - 0.61 Ohm.
- A to~al inten~al impedance of 0.0~5 Olun was achieved on thc primaty side of the transfonncr, which tr~nslates to an equi~alent impe~ rc of 1.98 Ohm ~n the secondary.
Such a low i~ ,cd~.nco ca~ lead to excessi~e curronts in case of ~n arc or short circuit and 2~ these would destroy the expensiv~ ~witchin~ lGBT. The ~GBT can be confi~1red to contain a currem lin~ting feuu~e, which tums the switch offwilhin a few ,u~ec in casc of e~c~s1ive load currcnt~ ~uch a~ might occur in case of an arc ~r a short circuit. By in~hci~g an arc in the secnnd~ NC mcasurcd ~ beniBn shut down ofthe IGBT within S ~lsec, as soon as thc cwrent exceeda ~bout 9ûO A in the primary, co~ .onding to I SO A in the secQr~rl~ry.

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The necess~ry capacitor sizc can bc ecti~ ted from the n ~Yiml~m allowable voltage drop across th~ load of S%. The charge conducted in the prirnary pulse is 100 ,us x 816 A -0 08 Cb. If thj~ :Ihould be 5% ofthe capwitor b~nk, the bcnk needs IO hold 20 ~ 0 08 = 1.6 Cb. ~t 500 V Tnsxirnum, the requircd c~pacity ~s C = Q~V = 1.6/jOO = û.0û32 F or 32~0 5 ,uF. The ener~y ~tored in thesc c~r~citors is 400 Joule.
Fcr the collection and clccllul)horesis pul~e, a second capacitor dischar~c circuit delivers the lon~er puls~ Ico~ths (srvcral 100 mscc~ and low voltage (50~ ~J) without the pulse tran~fonner. The low vnltage circuit and the hi~h voltage circuit are decoupled from esch other by ~t~cks of diodes 237 and 236.
,., I û ~PERAT10N
In addition to the vanous hardware c"lbod;n ,c.~t~ describcd abovo, ~ different aspect of the invention broadly co~c~.,.s ~ method for ~4enerating a user-s~,c~,if~d electric field pulsin~ p~ttern to achieve improv~d elcctroporation.

P~la Stora~e !~edia T~i~ method ~ay be imple~lrntP~ for example, by operating the controller 240 to execute a sequence of machine-readable instructions. Thcse instructions may residc in varioug types of data storaue media. In this re~pcc~, one aspect of the present invention concerns ~n ~rticlc of manufActl~re, con~ .,y a dsta storsge medium tan~ibly cmbodying a program o~ machine-re~d~blc instructions executable ~y a di~ital data proce~sor to 20 pcrf~rm mothod steps to ~eneratc a user-specified electric field pulsing patlern tO achieve i..lpr~vcd elearop~ration This d~ta storage mcdium may co..~rise, for exarnplc, R~M con~ained within thc controller 240. Altema~ively; ~he in~uuctions may be co..~ned in another data storage mcdium, such ~8 a rnagnctic data st~rage diskette 300 (Fi~urc 3). Whether conla;nc~ in thc 2S controller 2~0 or clsewherc, the instmc~ion3 may instud be stored on another type of data stora~e medium ~uch as ~)ASD st~rage (e.g. a convension~l "bard drive" or a RAID a~ray)~
magnetic t~pc, clectronic read-~nlsr ".e.no,~ (c.~. ROM), optical ~tora~e dcvicc (c.~.
WORM), papcr "punch" cnsdc~ or othcr data storage media. In an i~lustrative r mhod~

N~D S~EET

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LoeN~ p~ pc~

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of thc invcntion~ thc ms~l~inP-re~dable instruction~ m~y comprisc line~ of compiled PIL
16C~4 Microchip m~chinc code Opcrational Steps ~s merltioned above, one aspect of thc ~vention broAdly concerns ~ nlethod for 5 generatin~ a user~spccified electric field p~llsing pattcrn to achicve improved electroporatio~ Figure 4 ~how~ a sequonce of mcthod s~eps 400 to ilJustrate one examplc of this a~pect of lhe prescnt inventi~n. F~lr ea~e of cxp!~tion, but without ~ny limitation intended thaeby, thc sequcnre of Figure 4 is de~ cd in the specific context of the pL;lse - generator 200 descli~cd abovc.
Af'~cr the steps 400 are initisted in t~sk 40Z, the controller 240 in tas~ 404 rcccives user input spec;fi~ing an nutput pul~c pattan of one or more o~ltput pulses. As an cY~mrle!
thi~ uscr input may be recei~ed fiom the uscr intcrface 242. As an alternative, the user input may bc reccived from another electronic dcvice, or even a pre-stored record.
P~fL.~bly~ the uw input specifie~ a duration for each pulse and also specifying either a "high" ~utput volt~gc or a "low" output ~olt~ge. Next, f~r each pulsc of low predeterrnincd voltage, ~he pulsc ~encrator in task 404 geT~erate~ thc "low" predetermined voltagc at the outpu~ terminals 2~ and 231 for the s~ec;~c~ duration. More particularly, the comroller 24~ may gene~ate a l~w voltage pulse by ~ating the s~4itch 227, thercby permitting thc cnergy rese~oir 220 to discl~rgc through the load 234.
,_. 70 ~Igo in task 404, high voltage pul~es ~re gencratcd a~ the second~y winding te~rLinals ~y concu~t~ applyin~ another voltage to thc pnmary winding ternuTlals of thc transt-ormer for the specified dlJration. Mor~ p~rticularly, ~he high voltago pulsc involves gencr3tin~ thc volt~e a~ di~cu~c~ above, while conclJrrently tng~cring thc switch 226 to penT it the oncrgy rescrvoir 221 to discharRc thrau~h thc prima~y wind~ng 224. As tho voltagc of the re~ervoir 221 is ml~3~ ied by the transformcr 224, a high volt~c is crcated at the electrodes 23~-231. This voltage i5 the additive sum of the voltagcs 3tored in tbe energy reservoirs 220-221. .~J~c~ cly, a lesser "high~ voltage OUtpUI ma~ be createt sololy by tri~8ering thc swi~ch 226, without involving thc switch 22~.

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14 REPLACEMENT PA&E

Onc or morc of the above-rnentiDned pulses are therefore gcnera~ed in task 404 to produce ~he us~r-~pecified pulsc pattern~ er thc u6er-crocified pulsin~ pattern i9 creatcd complcted in task 4041 ;he routine 400 ends in taYI~ 106.

~p:ration ~,Yith rrcf~ ~ PU~ Pattern As ~nc~ti~n~d abc~e, the p~e ~enerator Z00 prnvides ~ uscr-specified pulsc pa~:tern compnsing ~ne nr m~re pulses nf "hi~h" andlor "low~ output voltage. Fi~urcs S-~ illustrat~
v~rious exe~nplary pulse shapcs, which ~y be ~sed alone or in comb~nation to constitute the user-spccificd pul3in~ 3chemc.
-. Although cach of the pulsin~ pattcms of ~i~gures 5-9 may provide distinct adv~ 6es for diffcrent S-rpli~ ons, the folJowing description highlightC the f~atures md opcration ~f a pattern ~00 (Figure 7) to illustratc thc opcra~ion of the invention, both electrically in the pulsc gencrator 200 as well as physiolc)gically in a ceJI sample.
Thc p~ttcm 100 compsiscs ~ "steppcd pattemU, in th~t it provides first, ~econd, and third vol~age ievcls 702-?û4. One, tw~, or all of these volta~cs m~y bc the sarne, if de~ired.
I S The pulses have first, second, and third durations 7û6-?08 ~n thc prc~cnt eY~n rle, the firs~
and third voltagas 706, 708 providc a 500 V (D C.), v.hereas the ~ccor.d volta~e 707 provltes 3000 V ~D.C.) Figure 1~ describcs ar~ illustrative sequcncc 1000 involved in gener~ting and apply~ng the stepp-d pa~tam 700, and thc physiological effiects caused by app' cati~n ofthe pat~ern 700. A.~r th~ sc~uen.e be~n6 in task 1002, the user intertace 242 recc~es user input in taok 1004. In thc illustraled embodimcnt, thc user input includcs thc user's specificuion of a desircd clcctrnporation pul~inR patt~rn, including a duration ~d volta~e ~evel f~r each portion of the pattern.
In an ~Iternative rn~odiment thc user m~y speci~ a desired ~rq~ih~e of electric field to bc appiicd by thc tra~sformer 224, and a me~surement of tho 8ap between the electrodes 230-2~1. In thi~ case, the controller 240 rnay compute the appropnate voltagc for the transformer 2~4 to gen~ratc in order to ~?Piy the desired electric fidd, for example by nrJXiplying thc elcctrie ficld by the ~ap. In ~ne e~nb~diment~ the ~ap measurcm~nt may be input by the uscr manually. Alternatively, thc 8~P may be mechanically measured and A~JD~D SHEEt ........ . . . . .

~ CA 02261703 1999-01-27 F '''~ g EPLACEI~ENr PAGE

electronically fed to the controller 24~ by automatcd means such a~ ~hown in U.S. Patent No. S~439~440.
Concurrently with task 1~04, the power supply ZO~ ~cn~- ~lcs the reference voltages at the output n~des ~8-209. In thc prc~ent cxamp~c, the rcfer~nce volta~e~ 208-20~ of S00 S V (D.C.) uc used. Gcncration of thc re~èrence voltages in task 1008 char~es ~hc energy reservoirs in task 100~
A~or task 1008, ~n oper~tor in ~ask 1010 ~pplies molcculcs of an implant agcM toa treatment sitc. Thc implant a~ent m~y comprisc one or more types of DNA, genes, andlor vanous chemical ~Cnt9. ln the case of elcctroch~ th~raphy, one b~fi-~l implant ~ent i9 Bleomycin.
With a livc patient, the trea~ncnt site coll.pl;9e9 a region of livc CC~3, and the operator is pr~faably a nurse or physician who applies a liquid implant ~gent by il~iec~ g i~ with a hypodcnnic noodle.
In the case of in vitro ap~licP~ion of the implant a~ent, however, the trea~n~ s~te 1~ may c~ ti~lte a cell samplo placcd in an approp..ale containe~. In this cxample, the opaation may be ~ laboratory tce~nici~n ths~ applies a liquld implimt agerlt by pourin~, eye-dropping, or other~vise introdurin~ thc a~ent into Ihe cell sample.
Step 1010, whether performcd in v~w or in vitro, places thc implant a~cnt b~,lwecn the in~c~ticcs ofthc cc118 at thc tre~tn~t site. ~ext, in task 1012 the operator ~lpplies ~0 electrodcs to thc ~rwt~.,e..~ site. In the case of a live patient, this m~y involve ~ripping region of cells through thc dcrmi~ with a c~ljper, inscrtin~ 9 nccdlc ~rray i~to the patient'3 ~~ tissue, or anothcr ,croccd~re. With an in vitro t~e~ lwl~ sitc, task 1012 may i~vol~e placing the cell samplc bdwccn a pur of plate-shaped electrode~ provided for that purpose~. A~
an ~. d...jl~, p~ue shaped clc~.~rodcs may be used, such a~ thc BTX ~rand cuvettes, part 25 numbcr ~40. ~ -A~ter t~ lû 12, thc co.,tr~ 24û in task 1014 8ates the switch 227, dischsrgingthc ener~y rêscrvoir 220 ant thercby applying the rlow" volta~e to the electrodes 230-231.
Tbis step ~c~m~llat~ r ~ 9 of the impl~nt s~ent r~ulr the n)c..ll)rancs of thc cells i~
the cdl ~~nple. ~ di~cG.~.cd by the pre~cnt mvcntors, thi9 StCp m~y bc ~dequately 30 pe.~,n.cd with a rcduced voltage. Acccrtin~ly, the "low" vol~age ofthe cnergy re~crvoir A~DED SHET

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220 acl,ie~ this purposc, whilc still avoidin~ dam~c to the cells in the sample and saving elc_~lical encrgy.
Figuro 7 illustratcs tssk 1~14 as thc volta~,c pulse tO2. A~ shown, this puJse prefcrably ccmpn~cs a squ~rc wa~e having a dur~tion ~6 of about 1~ 20~ nuec Md ~5 vol~age of abaut S00 V (D C ). D~pPn~ing upon the ap~lic~ n~ however, Jill~,.,.n paramelcr~ may be sub~ti~u~ed to define the pulse 702.
A~er task ]û14, thc controller ~40 in ta~k ~016 gatcs the ~witch 2~6 ~while cont~nuing to gatc the switch 227). This create~ a "hi~h" voltagc upon thc clcctrode~ 230-231, cG.~ponding to the sum of the ~cf~ uce voltages 208-209. This hi~h volt~e is 10 suffit,ienr to s~fely create small pores in the cells af thc tissue sample. This inventors '~ believe this d~ect ~o result ~rom electroph~fw;s, the actiDn of Coloumb fDrces ~n the chargcd ~olecules of the implant agcnt.
Fig~Iro 7 illustratcs this ~tep as the volta~rc pulse 703. As illwtrated, this pulse p~c~. ably comprises a sg~are w~ve h~vi ng a duration 7~1 of ~bout 100 ~9ccand an clectric 15 fidd n~ dc of ~bau~ 1"00 Vlcm. ~!epen~ing upon the ~ppl ration~ howevcr, ~ c.ent parameter~ may be substituted to definc thc pulsc ?02.
Advant~o!~oly, thc pulsc generator ~00 au~mntica1ly limits damage to c~lls ofthetissue sample d~lring this stcp. In particular, when thc volta~c ~om th~ pr~nary winding ~24a ~ tcsîhesecon~rywindin~224b, thevalsa~eprcscntedbythc~econtarywinding ~0 224b ~cg~ns to decay, in ~ccord~nce with known principles of tl a,.sro, ..,~ operaticn. Thu~, _ eYen if the volta~c applicd to thc primary winding 224a i~ ~pplied fDr an cxces~ve length of time, the 3ac~,)Jn,y wlntin~ 224b 8'JtomatiC8J]ylimit9 ~he ~issue samplc's exposure to t~S ~ ~ v21tage pulse.
Ncxt, in taslc 1018 the c~ntrollor 240 ce~scs ~ting of thc swi~ch 226 w~le 2~ cor~tinuing to ~atc tho switch 227. This step pern~ts thc mol~cul~ o~the impJant agent tc transit thc ccll~' pc. ~ le ~uc~ rdnc~l and entcr the cells' cytoplasm.
Fi~urc 7 illustr~tes this step a~ thc volta~c pulse J04. As illu~tr~ted, thi~ pulse pref~rably compAses a squ~re wElve hav~ng a duration 708 of about 1-200 mscc snd a voJtagc of abaut 500 V (D.C.). Dopentin~ upon thc ~pplirqtion, howcver. difEerent 30 parameler~ may be ~ubstinlt~d to define the pulse 704.

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REPl.ACEMENT PA~E

Afhr task 1018, the controller 240 relea~es gatin~ of the switch 227, ending thepulsc 700. The4 thc operalor removes the elec~rodcs from the cell sarnple in ta~ 2~, and the sequ~ce 10~0 ends in task 102~.

TTIE EMBODl~ENTS
While there havc bcen shown what are presently conc;~lered to be preferred embodi.~ents of the invention, it will be a~ t to those skilled in thc ut that various ch~nge~ uld modifications carl be m~de herein without deputing from the 9eOpe of thc inventioQ as dcfirled by the ap~...dcd cl~ims , ,. -- - .

Allh~ L{~

Claims

1. In an electroporation apparatus for generating a specified electroporation pulse shape that includes a power source, a transformer, a controller and output terminals configured for connecting to electrodes, an improvement comprising:
a first power supply (209, 221) to provide a first output voltage;
a second power supply (208, 220) to provide a second output voltage, wherein thefirst (209, 221) and second (208, 220) power supplies are separate sources of electrical energy;
a transformer (224) having electromagnetically coupled primary (224a) and secondary (224b) windings, the secondary winding (224b) being interposed between a pair of output terminals (230, 231);
a first switch (227) coupled to the first power supply (209, 221) and the primary winding (224a) and being responsive to a first gating signal to apply the first output voltage from the first power supply (209, 221) to the primary winding (224a);
a second switch (226) coupled to the second power supply (208, 220) and the secondary winding (224b) and responsive to a second gating signal to apply the second output voltage from the second power supply to the secondary winding (224b); andthe controller (240) to receive user specification of an output pulse shape and provide the first and second gating signals to generate the specified output pulse shape at the output terminals (230, 231).

2. The apparatus of claim 1, wherein:
the first power supply provides the first output voltage having a magnitude proportional to a first supply signal;
the second power supply provides the second output voltage having a magnitude proportional to a second supply signal; and the controller provides the first and second supply signals to generate the specified output pulse shape at the output terminals.

3. The apparatus of claim 1, each of the first and second power supplies are electrical power sources that are selectively coupled to and energy storage reservoir and to the first and second switches.

4. The apparatus of claim 3, each energy storage reservoir comprising a capacitor.

5. The apparatus of claim 3, the first and second switch comprising a transistor.

6. The apparatus of claim 3, the first and second switch comprising an insulated gate bipolar junction transistor.

7. The apparatus of claim 1, the transformer having a leakage inductance of less than 10 µHenry.

8. The apparatus of claim 1, the controller including a microprocessor.

9. The apparatus of claim 1, further comprising a user interface coupled to the controller.

10. The apparatus of claim 1, further comprising a pair of electrodes that are each electrically connected to a different one of the output terminal.

11. The apparatus of claim 1, further comprising a diode electrically interposed between one of the output terminals and the secondary winding.

12. The apparatus of claim 1, the second switch and second power supply being coupled in series, the series coupling being electrically attached in parallel with the secondary winding.

13. The apparatus of claim 12, further comprising a diode coupled in series with the second switch and second power supply.

14. The apparatus of claim 1, the first switch and first power supply being coupled in series, the series coupling being electrically attached in parallel with the primary winding.

15. The apparatus of claim 10, wherein the electrodes comprising plate electrodes.

16. The apparatus of claim 10, wherein the electrodes comprising needle electrodes.

17. The apparatus of claim 10, further comprising:
a gap sensor to sense a distance between the electrodes and provide a representative electronic gap distance output signal.

18. The apparatus of claim 1, wherein the controller includes programmed instructions to generate an electroporation pulse pattern by performing steps comprising:
receiving the user specification of an output pulse shape of one or more output pulses, the user specification includes a duration for each pulse and also specifying either a first predetermined output voltage level or a second predetermined output voltage level;
for each pulse of the second predetermined output voltage level, applying a gating signal to one of the switches for the specified duration; and for each pulse of first predetermined output voltage level, applying a gating signal to the first switch while concurrently applying a gating signal to the second switch for the specified duration.

19. The apparatus of claim 1, wherein the controller includes means for electrically coupling the first and second power supplies, and programmed to perform method steps comprising :
(a) to generate a first voltage at the secondary winding terminals by applyinga first voltage to the secondary winding terminals of the transformer;

(b) a predetermined delay after initiating step (a), to maintain application of the first voltage at the secondary winding terminals while concurrently applying a second voltage to the primary winding terminals; and (c) a predetermined delay after initiating step (b), to cease application of the second voltage to the primary winding terminals while maintaining application of the first voltage at the secondary winding terminals for a predetermined duration.

20. The apparatus of claim 19, the controller further being programmed to perform, prior to step (a) from the user specification, method steps comprising:receiving an input specifying a desired electric field;
receiving an input specifying an electrode gap distance; and calculating the first voltage to provide the specified electric field across thespecified electrode gap distance.

21. The apparatus of claim 19, further comprising:
a pair of electrodes electrically connected to the output terminals of the transformer;
a gap sensor to sense an electrode gap distance between the electrodes and provide a representative electrode gap distance output signal;
wherein the controller is further programmed to perform steps comprising:
receiving an input of a desired electric field;
receiving the electrode gap distance output signal; and computing the first voltage to provide the input desired electric field across the electrodes.

22. The apparatus of claim 18, wherein the programmed instructions to generate an electroporation pulse pattern to performing the steps is contained in a portable tangible data storage medium executable by the controller.

23. In an electroporation method for generating an electroporation pulse pattern using an electroporation apparatus that includes a power source, a controller, a transformer having a primary winding that electromagnetically couples to a secondary winding, and electrodes, an improvement comprising:
receiving user input (1004) specifying an output pulse pattern of one or more output pulses, the user input (1004) specifying a duration for each pulse and also specifying either a first predetermined output voltage level (702) or a second predetermined output voltage level (703);
for each pulse of the second predetermined output voltage level (703), applying a first predetermined voltage (702) to the secondary winding terminals (224b) of the transformer (224) for the specified duration; and for each pulse of the second predetermined output voltage level (703), applying the first predetermined voltage level (702) to the secondary winding terminals (224b) of the transformer (224) while concurrently applying a second predetermined voltage level (703) to the primary winding terminals (224a) of the transformer (224) for the specified duration (707).

24. The method of claim 23, further comprising steps before the applying steps of:
arranging the electrodes about a treatment site, the electrodes being coupled across the secondary winding terminals;
delivering molecules of a predetermined implant agent to the treatment site, andafter the applying steps, removing the electrodes from the treatment site.

25. The method of claim 23, the treatment site comprising a region of tissue within a living being.

26. The method of claim 23, the treatment site comprising cells removed from a living being.

27. The method of claim 23, the user input further including at least one of the first and second predetermined voltage levels.

28. The method of 23 wherein the method includes:
(a) applying an electric field at the first predetermined voltage level to a region of cells for a first predetermined duration;
(b) increasing the electric field to the second predetermined voltage level greater than the first predetermined voltage level;
(c) reducing the electric field to a third predetermined voltage level less than the second predetermined voltage level.

29. The method of claim 23 wherein the method further comprising the steps of:
(a) positioning the electrodes relative to a region of cells and delivering a predetermined implant agent to the region of cells;
(b) moving molecules of the implant agent toward the cells by applying a voltage of the first predetermined voltage level to the electrodes for the specified duration;
(c) creating pores in a plurality of the cells by applying the second predetermined voltage level greater than the first predetermined voltage level to the electrodes for a second predetermined time; and (d) moving molecules of the implant agent into a plurality of the pores by applying a third predetermined voltage level less than the second predetermined voltage level.

30. The method of claim 29, the second predetermined voltage level is applied to provide a resultant electric field at the electrodes in the range of 300-3000 V/cm.

31. The method of claim 30, further comprising the steps of computing the second predetermined voltage level by multiplying a desired electric field by measurement of a gap existing between the electrodes.

32. The method of claim 28, the first and third predetermined voltage levels being minimized to minimize damage to the cells due to thermal beating.

33. The method of claim 23, wherein the user input specification to generate anelectroporation pulse pattern to performing the steps is read from a portable tangible data storage medium executable by the controller.