METHODS FOR OPTIMIZING ELECTROPORATION

Abstract

Embodiments of the invention are directed to a technique for electroporation that allows for a delivery of long electrical pulses of high magnitude in highly conductive buffers and minimizes damage to cells undergoing electroporation.

Claims

1. An electroporation method comprising: (a) determining electroporation parameters such that during an electrical pulse a first time constant representative of electrical conductivity increase in electroporation medium (t.sub.1) during the pulse is not less than a second time constant representative of capacitor discharge (t.sub.2), wherein the pulse duration is less than either t.sub.1 or t.sub.2; and (b) applying one or more electrical pulses under the electroporation parameters to a sample to be electroporated.

2. The method of claim 1, wherein the electroporation parameters comprise buffer conductivity, power supply capacitance, electroporation chamber geometry, and electric field strength.

3. The method of claim 2, wherein buffer conductivity is between 0 to 3 Ohm/m.

4. The method of claim 2, wherein the power supply capacitance is between 1 and 10.sup.6 F.

5. The method claim 2, wherein the electroporation chamber has dimension of length between 0.1 to 100 cm, width between 0.1 to 10 cm, and a gap between 0.001 and 10 cm.

6. The method of claim 2, wherein the electric field is between 0.01 and 10 kV/cm.

7. The method of claim 1, wherein the electrical pulse is at least 1 sec.

8. The method of claim 1, wherein the electrical pulse is of a magnitude of 0.001 to 10,000 volts.

9. The method of claim 1, wherein the sample comprises a living cell, a cell particle, or a lipid vesicle.

10. The method of claim 9, wherein the cell is a blood cell.

11. The method of claim 9, further comprising loading the cell with a chemical or biological agent.

12. The method of claim 11, wherein the biologically active substance is a nucleic acid or small molecule.

13. An electroporated cell, cell particle, or lipid vesicle produced using the method of claim 1.

14. The electroporated cell, cell particle, or lipid vesicle of claim 13, wherein the cell particle is a platelet.

15. A population of electroporated cells, cell particles, or lipid vesicles having a loading efficiency of at least 50, 60, 70, 80, or 90%.

16. An electroporation apparatus configured to perform the method of claim 1.

17. An electroporation method comprising: (a) determining electroporation parameters such that during a decaying electrical pulse the rate of conductivity increase in an electroporation medium is lower that the rate of voltage decay; and (b) applying one or more electrical pulses under the electroporation parameters to a sample to be electroporated.

18. The method of claim 17, wherein the electroporation parameters comprise buffer conductivity, power supply capacitance, electroporation chamber geometry, and electric field strength.

19. The method of claim 17, wherein the rate of voltage decay is 10 s to 10 s.

20. The method of claim 17, wherein the sample comprises a living cell, a cell particle, or a lipid vesicle.

21. The method of claim 20, wherein the cell is a blood cell.

22. The method of claim 20, further comprising loading the cell with a chemical or biological agent.

23. The method of claim 22, wherein the biologically active substance is a nucleic acid or small molecule.

24. An electroporated cell produced using the method of claim 17.

25. A population of electroporated cells, cell particles, or lipid vesicles having a loading efficiency of at least 50, 60, 70, 80, or 90% after electroporation.

26. An electroporation apparatus configured to perform the method of claim 17.

27. A method of treating a subject having or suspected of having a disease or condition by administering an effective amount of a drug, a biologic or other bioactive molecule comprised in a particle of claim 13, claim 15, claim 24, or claim 25.

28. The method of claim 27 wherein the disease is an infectious disease.

29. The method of claim 28, wherein the infectious disease is a bacterial, fungal, parasite, or virus infection.

30. The method of claim 29, wherein the bacterial infection is a mycobacterial infection.

31. The method of claim 27, wherein the viral infection is a retroviral infection.

32. The method of claim 31, wherein the retroviral infection is a HIV infection.

33. The method of claim 27 wherein the disease is an inflammatory disease.

34. The method of claim 27 wherein the disease is cancer.

35. The method of claim 27 wherein the disease is a vascular occlusive disease.

36. The method of claim 27, wherein the treatment's efficacy is limited because of drug delivery impediments and/or the treatment's toxicity is dose-limiting.

Description

DESCRIPTION OF THE DRAWINGS

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] FIG. 1 Results of a simulation of a current increase in the same chamber at different field strengths and short (left column) and long (right column) time scales.

[0032] FIG. 2 Reduction of capacitance to 100 F allows application of a long pulse at 4 kV/cm.

[0033] FIG. 3 Reduction to 10 F enables use of a shorter pulse at 8 kV/cm.

[0034] FIG. 4 Electrical field of 0.5 kV/cm was applied in two consecutive pulses 1 ms duration and 1 second apart to a conductive medium. There is a mild short-term conductivity increase in the beginning of each pulse.

[0035] FIG. 5 Electrical field of 1 kV/cm was applied in four consecutive pulses 1 ms duration and 1 second apart to a conductive medium. There is a noticeable conductivity increase during each pulse as well as with each subsequent pulse.

[0036] FIG. 6 Electrical field of 1.5 kV/cm was applied in four consecutive pulses 1 ms duration 1 second apart to a conductive medium. There is a noticeable conductivity increase during each pulse as well as with each subsequent pulse. The conductivity values at the beginning of pulses 2-4 are approximately the same as the values at the end of the pulses before them. This illustrates slow rates of buffer cooling during the intervals between the pulses.

[0037] FIG. 7 Electrical field of 2 kV/cm was applied in four consecutive pulses 1 ms duration 1 second apart to a conductive medium. At this field strength the sample conductivity rapidly increased during each pulse, exceeding 200 percent of its initial value. Arcing occurred during the pulses 3 and 4.

[0038] FIG. 8 Platelets were loaded with AllStars Negative Control siRNA (Qiagen, Inc.). The stock solution of siRNA was added to platelet suspension to final concentration of 1 M, the sample was divided into Coincubation and Electroporation samples, and the latter was processed by application of electrical pulses on MaxCyte System. The power supply capacitance was set to 20 F, field strength was 5 kV/cm.

[0039] FIG. 9 Platelets were prepared as described for FIG. 8 and loaded with Alexa-488 (Invitrogen, Inc.) by electroporation.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Certain embodiments of the invention are directed to a technique for electroporation that allows for a delivery of long electrical pulses of high magnitude in highly conductive buffers and minimizes damage by electrical arc or a heat shock.

I. Electroporation

[0041] The process of electroporation generally involves the formation of pores in a cell membrane, or in a vesicle, by the application of electric field pulses across a liquid cell suspension containing cells or vesicles. During the poration process, cells are often suspended in a liquid media and then subjected to an electric field pulse. The medium may be electrolyte, non-electrolyte, or a mixture of electrolytes and non-electrolytes. The strength of the electric field applied to the suspension and the length of the pulse (the time that the electric field is applied to a cell suspension) varies according to the cell type. To create a pore in a cell's outer membrane, the electric field must be applied for such a length of time and at such a voltage as to create a set potential across the cell membrane for a period of time long enough to create a pore.

[0042] Applying a voltage across a plasma membrane that exceeds a certain threshold level forms a pore in the membrane. If the strength of the applied electrical field and/or duration of exposure to it are properly chosen, the pores formed by the electrical pulse reseal after a short period of time, during which extracellular compounds have a chance to enter into the cell. However, excessive exposure of live cells to electrical fields can cause apoptosis and/or necrosisprocesses that result in cell death.

[0043] Generally, the process of electroporation is often used for the transformation of bacteria, yeast, plant protoplasts, cultured cells and other cells or vesicles as a way of introducing some substance into a cell or a vesicle, such as loading it with a molecular probe, a drug that can change the cell's function, or pieces of DNA or forms of RNA, such as mRNA, siRNA or microRNA. This procedure is also highly efficient for the introduction of chemical or biological agents that specifically intervene in molecular pathways in tissue culture cells or primary cells, especially mammalian cells. For example, electroporation is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy.

[0044] During an electroporation process the electrical current flowing through a conductive media causes its heating and subsequent increase in its conductivity. If not properly controlled, such conductivity increase leads to drawing even more current from the power source and may lead to arching and loss of sample. This effect is typically observed at relatively high field strengths (>2 kV/cm). However, electroporation of platelets, for example, requires relatively strong electrical fields and therefore either the buffer conductivity or pulse width can be limited. Many electroporation instruments deliver voltage pulses to a sample from capacitors precharged to a desired voltage. During a pulse, the voltage on a capacitor decreases, and this decrease can be viewed as compensation to the sample conductivity increase.

[0045] Many electroporation instruments deliver voltage pulses to a sample from capacitors, which are pre-charged to a desired voltage. A capacitor is an electrical/electronic device that can store energy in the electric field between a pair of conductors (called plates). The process of storing energy in the capacitor is known as charging, and involves electric charges of equal magnitude, but opposite polarity, building up on each plate. A capacitor consists of two conductive electrodes, or plates, separated by a dielectric. Capacitors are used in electrical circuit and electronic circuits as energy-storage devices.

[0046] The capacitor's capacitance (C) is a measure of the amount of charge (Q) stored on each plate for a given potential difference or voltage (V) which appears between the plates: C=Q/V. In SI units, a capacitor has a capacitance of one farad when one coulomb of charge is stored due to one volt of applied potential difference between the plates. Since the farad is a very large unit, values of capacitors are usually expressed in microfarads (F), nanofarads (nF), or picofarads (pF).

[0047] Conditions for compensating for capacitor voltage decrease relative to a sample conductivity increase are analyzed as follows.

[0048] The current through the buffer is represented by

[00001]$\begin{array}{cc}I\left(t\right)=\frac{U\left(t\right)}{R\left(t\right)}=U\left(\mathrm{Tt}\right).Math..Math.\left(t\right)& \mathrm{Equation}.Math..Math.1\end{array}$

[0049] where I(t) is current [A], U(t) is voltage [V], R(t) is resistance [Ohm] and (t) is conductance [S].

[0050] The conductance change is represented by

[00002]$\begin{array}{cc}.Math.\left(t\right)=\frac{A}{}.Math.\left(t\right)& \mathrm{Equation}.Math..Math.2\end{array}$

[0051] where A is electrode surface (cm.sup.2), is distance between electrodes [cm], and (t) is conductivity [mS/cm].

[0052] The conductivity change is represented by

(t)=.sub.0(1+0.02(T(t)T.sub.0))Equation 3:

[0053] where .sub.0 is the conductivity at time zero (T.sub.0) [S], and T(t) is the buffer temperature [K].

[0054] The temperature increase can be represented by

[00003]$\begin{array}{cc}\frac{\mathrm{dT}\left(t\right)}{\mathrm{dt}}=\frac{1}{}.Math.\frac{\mathrm{dQ}\left(t\right)}{\mathrm{dt}}& \mathrm{Equation}.Math..Math.4\end{array}$

[0055] where is buffer volume (cm.sup.3), is the heat capacity of the buffer [J/(cm.sup.3K), and Q(t) is heat energy [J].

[0056] Heat generation can be represented by

[00004]$\begin{array}{cc}\frac{\mathrm{dQ}\left(t\right)}{\mathrm{dt}}=U\left(t\right).Math.I\left(t\right)& \mathrm{Equation}.Math..Math.5\end{array}$

[0057] Combining Eq. 1 through Eq. 3 results in

[00005]$\begin{array}{cc}I\left(t\right)=U\left(t\right).Math.\frac{A}{}.Math.{}_0\left(1+0.02.Math.\left(T\left(t\right)-T_0\right)\right)& \mathrm{Equation}.Math..Math.6\end{array}$

[0058] Substituting Eq. 5 into Eq. 4 results in

[00006]$\begin{array}{cc}\frac{\mathrm{dT}\left(t\right)}{\mathrm{dt}}=\frac{1}{}.Math.U\left(t\right).Math.I\left(t\right)& \mathrm{Equation}.Math..Math.7\end{array}$

[0059] The system of Eq. 6 and Eq. 9 may not be solved analytically, only a numerical solution can be computed (FIG. 1). The equations also can be analyzed as follows.

[0060] Case 1: Constant Voltage.

[0061] If the power supply has a very large capacitance or a DC battery, the voltage applied to sample is almost constant during a pulse:

[0062] Then differentiating Eq. 6 over time results in:

[00007]$\begin{array}{cc}\frac{\mathrm{dI}\left(t\right)}{\mathrm{dt}}=\frac{A}{d}.Math.{}_0\left(0.02.Math.U\left(t\right).Math.\frac{\mathrm{dT}\left(t\right)}{\mathrm{dt}}+\frac{\mathrm{dU}\left(t\right)}{\mathrm{dt}}.Math.\left(1+0.02.Math.\left(T\left(t\right)-T_0\right)\right)\right)& \mathrm{Equation}.Math..Math.8\end{array}$

[0063] Substituting Eq. 7 into Eq. 8 results in:

[00008]$\begin{array}{cc}\frac{\mathrm{dI}\left(t\right)}{\mathrm{dt}}=\frac{A}{d}.Math.{}_0\left(0.02.Math.\frac{1}{}.Math.{U\left(t\right)}^2.Math.I\left(t\right)+\frac{\mathrm{dU}\left(t\right)}{\mathrm{dt}}.Math.\left(1+0.02.Math.\left(T\left(t\right)-T_0\right)\right)\right)& \mathrm{Equation}.Math..Math.9\end{array}$

[0064] Under constant voltage conditions dU(t)/dt=0, U(t)=U.sub.0. Substituting these expressions into Equation 9 results in:

[00009]$\begin{array}{cc}\frac{\mathrm{dI}\left(t\right)}{\mathrm{dt}}=0.02.Math.\frac{A}{}.Math.\frac{{}_0}{}.Math.U_0^2.Math.I\left(t\right)=0.02.Math.\frac{{}_0}{}.Math.E^2.Math.I\left(t\right)& \mathrm{Equation}.Math..Math.10\end{array}$

[0065] Solving the equation result in

[00010]$\begin{array}{cc}I\left(t\right)=I_0.Math.e^{\left(\frac{t}{1}\right)}& \mathrm{Equation}.Math..Math.11\\ I_0=U_0.Math.\frac{A}{}.Math.{}_0& \mathrm{Equation}.Math..Math.12\\ \frac{1}{1}=0.02.Math.\frac{{}_0}{}.Math.E^2& \mathrm{Equation}.Math..Math.13\end{array}$

[0066] There is an exponential current increase due to a conductivity increase that is dependent mostly on the applied field strength.

[0067] Case 2: Compensation.

[0068] If the 1, the time constant of conductivity increase, is matching 2, the time constant of capacitor discharge, both processes are likely to compensate for each other during a short pulse (when duration of the pulse is less than either time constant). The discharge of a capacitor can be described by

[00011]$\begin{array}{cc}I\left(t\right)=-C.Math.\frac{\mathrm{dU}\left(t\right)}{\mathrm{dt}}& \mathrm{Equation}.Math..Math.14\end{array}$

[0069] where C is the electrical capacitance [F]

[0070] The time constant of this exponential process can be expressed in terms of initial conductivity and capacitance

[00012]$\begin{array}{cc}\frac{1}{2}=\frac{A.Math..Math.{}_0}{.Math..Math.C}& \mathrm{Equation}.Math..Math.15\end{array}$

[0071] The compensation condition .sub.1=.sub.2 becomes

[00013]$\begin{array}{cc}\frac{0.02}{}.Math.E^2=\frac{A}{.Math..Math.C}& \mathrm{Equation}.Math..Math.16\end{array}$

[0072] The five variables in the Eq. 16 can be expressed through other parameters (e.g. the ratio of chamber volume to the electrode gap V/ can be used instead of the electrode surface area A) without changing mechanism of the processes described above and ways to avoid arcing.

[0073] As an example, the buffers developed for platelet handling and electroporation having relatively high conductivity and very short electrical pulses are used; one probably has to use long electrical pulses to load platelets with RNA and any other large charged molecules. To achieve the best outcome of such process, a user would have to use very high voltages applied to highly conductive media for relatively long intervals of time. These three conditions are not easily met concurrently. A solution to this problem is the disclosed methods for electroporation that allow for a delivery of long electrical pulses of high magnitude in highly conductive buffers and minimizes the chance of damaging the cell or vesicle by electrical arc or a heat shock. In the present invention, during a pulse in electroporation, the voltage on a capacitor decreases, and this decrease can be viewed as compensation to the sample conductivity increase.

[0074] Typically five variables can be aligned to achieve either constant or decreasing current in a processing chamber. These variables together define how fast the current through the buffer will decrease due to the capacitor discharge and/or increase due to a change in the buffer conductivity. Typically, the dimensions of the processing chamber (A and ) are predefined and the field strength (E) is dictated by the electroporation protocol. This leaves conductivity and capacitance as the most convenient variables to be manipulated with capacitance being the most flexible variable due to the existence of upper limits on conductivity as a result of maximal current rating on hardware. Typically, variables are optimized to achieve constant current in a processing chamber. In the present invention, any of the variables could be adjusted to achieve the desired compensation effect.

[0075] The data for FIG. 1 were generated using Mathematica software (Wolfram Research, CA) as a numerical solution of a system of two differential equations.

[0076] Code Example.

[0077] Specify heat capacity of water k [J/(K mL), cuvette width w [cm], cuvette height h [cm], cuvette gap g [cm], capacitance c [F], pulse voltage u0 [V], initial buffer

[00014]$.Math.k=4.18;w=0.5;h=0.5;g=0.2;$$.Math.c=0.00001;u.Math..Math.0=1600;S.Math..Math.0=0.18;\mathrm{tmax}=0.0002;$$\mathrm{solution}=\mathrm{NDSolve}\left[\left\{u^{}\left[t\right]=-\frac{u\left[t\right]}{c}.Math.\frac{\mathrm{wh}}{g}.Math.S.Math..Math.0.Math.\left(1+0.02.Math.\left(y\left[t\right]-25\right)\right),y^{}\left[t\right]=-\frac{c}{\mathrm{kwhg}}.Math.u\left[t\right].Math.u^{}\left[t\right],u\left[0\right]=u.Math..Math.0,y\left[0\right]=25\right\},\left\{u,y\right\},\left\{t,0,\mathrm{tmax}\right\}\right];$$\mathrm{Plot}\left[\mathrm{Evaluate}\left[-c.Math.{}_t.Math..Math.u\left[t\right].Math.\text{/}.\mathrm{solution}\right],\left\{t,0,\mathrm{tmax}\right\},\mathrm{PlotRange}.fwdarw.\left(0,150\right),\mathrm{AxesLabel}.fwdarw.\left\{\mathrm{Time},s,\mathrm{Current},A\right),\mathrm{PlotLabel}.fwdarw.8.Math..Math.\mathrm{kV}.Math..Math.{\mathrm{cm}}^{-1}\right];$

conductivity S0 [S/cm], pulse width tmax [s], y is the temperature [K]:

[0078] FIG. 1 shows results of a simulation of a current increase in the same chamber at different field strengths and short (left column) and long (right column) time scales. Capacitance is reduced to 100 F and allows application of a long pulse at 4 kV/cm (FIG. 2) and a reduction to 10 F enables use of a shorter pulse at 8 kV/cm (FIG. 3).

II. Electroporation Targets

[0079] Targets for electroporation include a number of cell types or particles derived from a number of organisms and sources. In some embodiments, the target can be nucleated or anucleated cells or particles. Cells or particles of the invention can be primary cells or a cell line or a particle derived therefrom. For example, a target may be prokaryotic, yeast, insect, mammalian, rodent, hamster, primate, human, bird, plant cells, or portions/fragments thereof. In certain aspects, the present invention relates to compositions, methods, and apparatus for the introduction of chemical or biological agents into various types of living cells or cell particles or synthetic vesicles or liposomes. More particularly, the present invention relates to a method and apparatus for the introduction of chemical or biological agents into cells or cell particles. These electroporation targets can be utilized as a drug delivery system (an intelligent liposome) to target a site of infection, metastasis, or other pathologic lesionparticularly if drug is otherwise toxic if not in a delivery vehicle.

[0080] If the patient population is infected with a virus and may exhibit viremia, the electroporation target can be allogeneic rather than autologous or synthetic in order to not exacerbate the patient's condition. Allogeneic cells or material can be obtained from standard sources (hospital services). Donors can be screened using histories and blood tests for infectious diseases.

[0081] A. Electroporation Target Manufacture/Collection

[0082] Compositions described herein can be used in therapeutic applications. They are either isolated from collected samples to make a therapeutic dose. One example of the therapeutic use of the compositions described herein is formulating a therapeutic agent in appropriate buffer at a required concentration and processing the formulation using systems such as the MaxCyte system. If the formulated drug and electroporation target are sterile filled into a container with appropriate port(s), this can be run through a closed, sterile system in routine laboratory environment (filling room-like controlled environment not needed). The process can be completed within 2 to 3 hours. Performance variables of the system are generated in real time and will assist QC operations.

[0083] Typically, manufacturing of the drug-loaded target can be done at a central facility or at the point(s)-of care. If there is to be a central facility (or several regional ones), the stability of the drug-loaded target is an important factor. Stability for at least several days could support a custom order operation. In a point-of-care system, formulated drug would be supplied to the sites where therapy is required. Targets or delivery vehicles can be obtained at those sites and the final manufacturing step carried out processing facility by technical personnel using detailed standard operating procedures (SOPs). This would be analogous to final preparation of transfusion products on site. In this case, the final product stability is not critical.

[0084] B. Therapeutic Applications

[0085] The invention further encompasses methods for delivering therapeutic agents using an electroporated entity or target, (e.g., cell, liposome, platelet, or derivative thereof) as a delivery vehicle. The active agent formulations produced using the methods described herein typically have a sustained effect and lower toxicity allowing less frequent administration and an enhanced therapeutic index. Therapeutic agents are delivered by first preparing platelets, cells, liposomes, or segments thereof loaded with at least one therapeutic agent, as obtained according to the methods described herein.

[0086] In certain embodiments of the present invention, substances or drugs can be introduced into a delivery vehicle (i.e., an electroporation target). The delivery vehicles can also be loaded with bioactive compounds. Examples of suitable bioactive compounds include, but are not limited to, stabilizing agents, tracers, fluorescent tags and other imaging substances such as radiolabels, cryoprotectants, nucleic acids, and bioactive materials. Bioactive materials particularly suited to incorporation into platelets include, but are not limited to, hemostatic effectors, wound healing factors, anti-cancer agents, cytokines and drugs. Suitable bioactive materials also include therapeutic and prophylactic agents. Examples of bioactive materials include, but are not limited to, proteins and peptides (synthetic, natural, and mimetics), oligonucleotides (anti-sense, ribozymes, etc.), nucleic acids (e.g., inhibitory RNA, siRNA, miRNA, expression vectors, etc.), viral vectors, hemotherapeutic agents, anti-cancer drugs, antiinflammatory drugs, anti-fungal drugs, anti-viral drugs, anti-microbial drugs, thrombomodulating agents, immunomodulating agents and the like.

[0087] Thrombus dissolving substances can be introduced by delivery vehicles, such as tissue plasminogen activator, urokinase or streptokinase and the like can be introduced into a population of delivery vehicles. Tissue plasminogen activator is sold under the trademark ACTIVASE by Genentech Corporation, South San Francisco, Calif. These delivery vehicles loaded with the thrombus dissolving substances can then be introduced into a patient who is suffering from a thrombus blocking a blood vessel. Because the delivery vehicles contain active thrombus dissolving enzymes, the thrombus is then dissolved. The thrombus dissolving substances can be introduced into the delivery vehicles by a variety of methods with the most preferable method according to the present invention.

[0088] It is to be understood that other drugs can be introduced into the delivery vehicle or other cells for delivery to damaged tissue. These drugs include, but are not limited to, smooth muscle inhibitors, antiinfective agents (e.g., antibiotics, antifungal agents, antibacterial agents, antiviral agents), chemotherapeutic/antineoplastic agents and the like.

[0089] In one embodiment, the drug is an antiinfective. Antiinfectives are agents that act against infections, such as bacterial, mycobacterial, fungal, viral or protozoal infections. Antiinfectives covered by the invention include but are not limited to aminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin, and the like), tetracyclines (e.g., chlortetracycline, oxytetracycline, methacycline, doxycycline, minocycline and the like), sulfonamides (e.g., sulfanilamide, sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and the like), paraaminobenzoic acid, diaminopyrimidines (e.g., trimethoprim, often used in conjunction with sulfamethoxazole, pyrazinamide, and the like), quinolones (e.g., nalidixic acid, cinoxacin, ciprofloxacin and norfloxacin and the like), penicillins (e.g., penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillin indanyl, ticarcillin, azlocillin, mezlocillin, piperacillin, and the like), penicillinase resistant penicillin (e.g., methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin and the like), first generation cephalosporins (e.g., cefadroxil, cephalexin, cephradine, cephalothin, cephapirin, cefazolin, and the like), second generation cephalosporins (e.g., cefaclor, cefamandole, cefonicid, cefoxitin, cefotetan, cefuroxime, cefuroxime axetil; cefmetazole, cefprozil, loracarbef, ceforanide, and the like), third generation cephalosporins (e.g., cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime, cefixime, cefpodoxime, ceftibuten, and the like), other beta-lactams (e.g., imipenem, meropenem, aztreonam, clavulanic acid, sulbactam, tazobactam, and the like), betalactamase inhibitors (e.g., clavulanic acid), chlorampheriicol, macrolides (e.g., erythromycin, azithromycin, clarithromycin, and the like), lincomycin, clindamycin, spectinomycin, polymyxin B, polymixins (e.g., polymyxin A, B, C, D, E1 (colistin A), or E2, colistin B or C, and the like) colistin, vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide, aminosalicylic acid, cycloserine, capreomycin, sulfones (e.g., dapsone, sulfoxone sodium, and the like), clofazimine, thalidomide, or any other antibacterial agent that can be lipid encapsulated.

[0090] In certain aspects, anti-microbials include anti-mycobacterials, including, but not limited to isoniazid, rifampin, streptomycin, rifabutin, ethambutol, pyrazinamide, ethionamide, aminosalicylic and cycloserine.

[0091] Antiinfectives can include antifungal agents, including polyene antifungals (e.g., amphotericin B, nystatin, natamycin, and the like), flucytosine, imidazoles (e.g., n-ticonazole, clotrimazole, econazole, ketoconazole, and the like), triazoles (e.g., itraconazole, fluconazole, and the like), griseofulvin, terconazole, butoconazole ciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine, terbinafine, or any other antifungal that can be lipid encapsulated or complexed. Discussion and the examples are directed primarily toward amikacin but the scope of the application is not intended to be limited to this antiinfective. Combinations of drugs can be used.

[0092] In certain aspects, antiinfecives include anti-virals including but not limited to anti-herpes agents such as acyclovir, famciclovir, foscamet, ganciclovir, acyclovir, idoxuridine, sorivudine, trifluridine, valacyclovir and vidarabine; anti-retroviral agents such as ritonavir, didanosine, stavudine, zalcitabine, tenovovir and zidovudine; and other antiviral agents such as, but not limited to, amantadine, interferon-alpha, ribavirin and rimantadine.

[0093] Also included as suitable antiinfectives used in the drug formulations of the present invention are pharmaceutically acceptable addition salts and complexes of drugs. In cases wherein the compounds may have one or more chiral centers, unless specified, the present invention comprises each unique racemic compound, as well as each unique nonracemic compound.

[0094] In aspect, an active agent is an antineoplastic drug. Currently, there are approximately twenty recognized classes of approved antineoplastic drugs. The classifications are generalizations based on either a common structure shared by particular drugs, or are based on a common mechanism of action by the drugs. A partial listing of some of the commonly known commercially approved (or in active development) antineoplastic agents by classification is as follows:

[0095] Structure-Based Classes include Fluoropyrimidines5-FU, Fluorodeoxyuridine, Ftorafur, 5-deoxyfluorouridine, UFT, S-1 Capecitabine; Pyrimidine NucleosidesDeoxycytidine, Cytosine Arabinoside, 5-Azacytosine, Gemcitabine, 5-Azacytosine-Arabinoside; Purines6-Mercaptopurine, Thioguanine, Azathioprine, Allopurinol, Cladribine, Fludarabine, Pentostatin, 2-Chloro Adenosine; Platinum AnaloguesCisplatin, Carboplatin, Oxaliplatin, Tetraplatin, Platinum-DACH, Ormaplatin, CI-973, JM-216; Anthracyclines/AnthracenedionesDoxorubicin, Daunorubicin, Epirubicin, Idarubicin, Mitoxantrone; EpipodophyllotoxinsEtoposide, Teniposide; CamptothecinsIrinotecan, Topotecan, 9-Amino Camptothecin, 10,11-Methylenedioxy Camptothecin, 9-Nitro Camptothecin, TAS 103, 7-(4-methyl-piperazino-methylene)-10,11-ethylenedioxy-20(S)-camptothecin, 7-(2-N-isopropylamino)ethyl)-20(S)-camptothecin; Hormones and Hormonal AnaloguesDiethylstilbestrol, Tamoxifen, Toremefine, Tolmudex, Thymitaq, Flutamide, Bicalutamide, Finasteride, Estradiol, Trioxifene, Droloxifene, Medroxyprogesterone Acetate, Megesterol Acetate, Aminoglutethimide, Testolactone and others; Enzymes, Proteins and AntibodiesAsparaginase, Interleukins, Interferons, Leuprolide, Pegaspargase, and others; Vinca AlkaloidsVincristine, Vinblastine, Vinorelbine, Vindesine; TaxanesPaclitaxel, Docetaxel.

[0096] Mechanism-Based Classes include AntihormonalsAnastrozole; AntifolatesMethotrexate, Aminopterin, Trimetrexate, Trimethoprim, Pyritrexim, Pyrimethamine, Edatrexate, MDAM; Antimicrotubule AgentsTaxanes and Vinca Alkaloids; Alkylating Agents (Classical and Non-Classical)-Nitrogen Mustards (Mechlorethamine, Chlorambucil, Melphalan, Uracil Mustard), Oxazaphosphorines (Ifosfamide, Cyclophosphamide, Perfosfamide, Trophosphamide), Alkylsulfonates (Busulfan), Nitrosoureas (Carmustine, Lomustine, Streptozocin), Thiotepa, Dacarbazine and others; AntimetabolitesPurines, pyrimidines and nucleosides, listed above; AntibioticsAnthracyclines/Anthracenediones, Bleomycin, Dactinomycin, Mitomycin, Plicamycin, Pentostatin, Streptozocin; Topoisomerase InhibitorsCamptothecins (Topo I), Epipodophyllotoxins, m-AMSA, Ellipticines (Topo II); AntiviralsAZT, Zalcitabine, Gemcitabine, Didanosine, and others; Miscellaneous Cytotoxic AgentssiRNA, miRNA, Hydroxyurea, Mitotane, Fusion Toxins, PZA, Bryostatin, Retinoids, Butyric Acid and derivatives, Pentosan, Fumagillin, and others.

[0097] Antiangiogenic drugs can be incorporated into the platelets. Antiangiogenic drugs include, but are not limited to, AGM-1470 (TNP-470) or antagonists to one of its receptors, MetAP-2; growth factor antagonists, or antibodies to growth factors (including VEGF or bFGF); growth factor receptor antagonists or antibodies to growth factor receptors; inhibitors of metalloproteinases including TIMP, batimastat (BB-94), and marimastat; tyrosine kinase inhibitors including genistein and SU5416; integrin antagonists including antagonists alphaVbeta3/5 or antibodies to integrins; retinoids including retinoic acid or the synthetic retinoid fenretinide; steroids 11-epihydrocortisol, corteloxone, tetrahydrocortisone and 17-hydroxyprogesterone; protein kinase inhibitors including staurosporine and MDL 27032; vitamin D derivatives including 22-oxa-1 alpha, and 25-dihydroxyvitamin D3; arachidonic acid inhibitors including indomethacin and sulindac; tetracycline derivatives including minocycline; thalidomide and thalidomide analogs and derivatives; 2-methoxyestradiol; tumor necrosis factor-alpha; interferon-gamma-inducible protein 10 (IP-10); interleukin 1 and interleukin 12; interferon alpha, beta or gamma; angiostatin protein or plasminogen fragments; endostatin protein or collagen 18 fragments; proliferin-related protein; group B streptococcus toxin; CM101; CAI; troponin I; squalamine; nitric oxide synthase inhibitors including L-NAME; thrombospondin; wortmannin; amiloride; spironolactone; ursodeoxycholic acid; bufalin; suramin; tecogalan sodium; linoleic acid; captopril; irsogladine; FR-118487; triterpene acids; castanospermine; leukemia inhibitory factor; lavendustin A; platelet factor-4; herbimycin A; diaminoantraquinone; taxol; aurintricarboxylic acid; DS-4152; pentosan polysulphite; radicicol; fragments of human prolactin; erbstatin; eponemycin; shark cartilage; protamine; Louisianin A, C and D; PAF antagonist WEB 2086; auranofin; ascorbic ethers; and sulfated polysaccharide D 4152.

[0098] Genes to be targeted using nucleic acid agents using the methods and formulation of the invention include, without limitation, those whose expression is correlated with an undesired phenotypic trait. Thus, genes relating to cancer, rheumatoid arthritis and viruses can be targeted. Cancer-related genes include oncogenes (e.g., K-ras, c-myc, bcr/abl, c-myb, c-fms, c-fos and cerb-B), growth factor genes (e.g., genes encoding epidermal growth factor and its receptor, fibroblast growth factor-binding protein), matrix metalloproteinase genes (e.g., the gene encoding MMP-9), adhesion-molecule genes (e.g., the gene encoding VLA-6 integrin), tumor suppressor genes (e.g., bcl-2 and bcl-X1), angiogenesis genes, and metastatic genes. Rheumatoid arthritis-related genes include, for example, genes encoding stromelysin and tumor necrosis factor. Viral genes include human papilloma virus genes (related, for example, to cervical cancer), hepatitis B and C genes, and cytomegalovirus (CMV) genes (related, for example, to retinitis). Numerous other genes relating to these diseases or others might also be targeted. In certain embodiments nucleic acids can target mRNA encoding c-myc, VEGF, CD4, CCRS, gag, MDM2, Apex, Ku70, or ErbB2.

[0099] Methods of gene regulation include administering siRNA, miRNA, and other inhibitory nucleic acids; administration of nucleic acid encoding a therapeutic nucleic acid, protein, or peptide. In another aspect, the invention provides a method of preparing and/or administering to the subject a dosage of a therapeutic inhibitory oligonucleotide or nucleic acid (antisense, ribozyme, siRNA, dsRNA) molecule wherein the administered nucleic acid inhibits a biological process such as transcription or translation. The present invention provides methods of administering one or more therapeutic nucleic acid molecules to a subject, using a nucleic acid delivery vehicle prepared using the methods described, to bring about a therapeutic benefit to a subject. As used herein, a therapeutic nucleic acid molecule or therapeutic nucleic acid is any nucleic acid (e.g., DNA, RNA, non-naturally occurring nucleic acids and their analogues such as peptide nucleic acids, and their chemical conjugates) that, as a nucleic acid or as an expressed nucleic acid or polypeptide, confers a therapeutic benefit to a subject. The subject preferably is mammalian such as a mouse, and more preferably is a human being.

[0100] The agents, including thrombus dissolving substances, can be introduced into the delivery vehicle by a variety of methods with the most preferable method being according to the apparatus and/or methods of the present invention.

[0101] The dosage of any compositions of the present invention will vary depending on the symptoms, age and body weight of the patient, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the subject composition. Any of the subject formulations may be administered in a single dose or in divided doses. Dosages for the compositions of the present invention may be readily determined by techniques known to those of skill in the art or as taught herein.

[0102] In certain embodiments, the dosage of the subject compounds will generally be in the range of about 0.001, 0.01, 1, 5, 10 pg/ng/mg to about 0.1, 1, 5, 10 pg/ng/mg/g per kg body weight, including all values and ranges there between.

[0103] The present invention also includes a method of treating a patient in need of a therapeutic agent comprising administering to the patient an effective amount of platelets containing the therapeutic agent.

[0104] A number of disease states or conditions are related to abnormal platelet function or levels. These diseases or conditions include, but are not limited to thrombocytopenia (e.g., Idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura, drug-induced thrombocytopenia, heparin-induced thrombocytopenia (HIT)), Gaucher's disease, aplastic anemia, fetomaternal alloimmune thrombocytopenia, HELLP syndrome, hemolytic-uremic syndrome, chemotherapy, Dengue, alpha-delta platelet storage pool deficiency (aSPD), thrombocytosis (e.g., benign essential thrombocytosis), Bernard-Soulier syndrome, Glanzmann's thrombasthenia, Scott's syndrome, von Willebrand disease, Hermansky-Pudlak Syndrome, decreased cyclooxygenase activity (induced or congenital), storage pool defects (acquired or congenital), haemophilia, atherosclerosis, coronary artery disease, myocardial infarction, cerebrovascular disease, Stroke, CVA (cerebrovascular accident), peripheral artery occlusive disease (PAOD), angiogenic disorders, or cancer (e.g., delivery of anti-angiogenic substances).

III. Description of a Representative Electroporation Device

[0105] Electroporation is done with electroporators, appliances that create the electric current and send it through the cell solution. The solution is pipetted into a glass or plastic cuvette that usually has two aluminum electrodes on its sides. For instance, for bacterial electroporation, a suspension of around 50 microliters is usually used. Prior to electroporation it is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage is set on the electroporator (several hundred volts is often used) and the cuvette is inserted into the electroporator. Immediately after electroporation 1 milliliter of liquid medium is added to the bacteria (in the cuvette or in an eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more and then it is spread on an agar plate.

[0106] The flow electroporation devices that can be used in conjunction with the present invention is, in one embodiment, comprised of the following: instrument (electronics module (power box and tower)); computer (communicates with electronics module and actually runs the process in real time and manages process-associated data); monitor (displays graphical user interface [GUI] and enables user interaction); and disposable set or processing chamber (component in which flow electroporation of cell suspension occurs).

[0107] In certain embodiments, the present invention uses flow electroporation to overcome the practical limitations in respect to the number of cells that can be electroporated, the time in which they can be electroporated and the volume of solution in which they are suspended that attend to static or batch electroporation methods. With this method, a cell suspension is passed across parallel bar electrodes that are contained in a flow cell that is preferably disposable. It is to be understood that different configurations of flow cells can be used in the present invention. During this passage, the cells are subjected to electrical pulses with predetermined characteristics. The molecule of interest then diffuses into the cell following concentration and/or electrical gradients. The present invention is optionally capable of subjecting the cells to a range of electric field strengths. Generally speaking, field strengths useful in the present invention are greater than approximately 5 kV/cm; preferably, approximately 5 kV/cm to 7 kV/cm.

[0108] The process is initiated by attaching the flow cell with solutions and cell suspensions in the containers with the necessary fluids and samples. The flow cell snaps into place and is secured by closing a hinged panel. Priming solution (saline) and cell suspension are introduced by providing the required commands to the electroporation system, which controls operation of the pump and pinch valves. As the cells transit the flow path between electrodes, electric pulses of the chosen voltage, duration, and frequency are applied. Product and waste fluids are collected in the designated containers.

[0109] The user inputs the desired voltage and other parameters into the flow electroporation system of the present invention. As noted above, a range of settings is optionally available. The computer communicates to the electronics in the tower to charge the capacitor bank to the desired voltage. Appropriate switches then manipulate the voltage before it is delivered to the flow path to create the electric field (the switches provide alternating pulses or bursts to minimize electrode wear brought on by prolonged exposure to the electric field). The voltage is delivered according to the duration and frequency parameters set into the flow electroporation system of the present invention by the operator or as determined in accordance with the inventive method. Details of an example of a flow electroporation system is described in U.S. Pat. No. 7,186,559 incorporated by reference in its entirety.

[0110] Electroporation parameters will be determined using the procedures and methods described herein. Aspects of the invention can be used in context with static and flow electroporation systems. The dimension of the electroporation chambers will be considered in determining the parameter for optimal electroporation.

[0111] The electroporation arts are replete with ex vivo means of transfecting biological cells and vesicles. For example, U.S. Pat. No. 5,720,921 to Meserol discloses an electroporation chamber that is designed as a continuous flow chamber wherein vesicles are transferred to the chamber, electroporated and flushed out after electroporation pulses are applied. Other flow chambers include U.S. Pat. Nos. 5,612,207 and 6,623,964, and U.S. Patent publication 2001/0001064, each of which is incorporated herein by reference in its entirety.

[0112] Other electroporation chambers have also been disclosed wherein continuous flow of the medium carrying the vesicles is not used but the electroporation chamber device includes various elements. For example, U.S. Pat. No. 4,906,576 discloses a chamber having among other elements a magnetic core. U.S. Pat. No. 6,897,069 discloses an electroporation sample chamber with removable electrodes. Other chambers are cuvette style for handling small samples. Still other chambers such as disclosed in WO04/083,379 provide for larger volumes.

[0113] Electrodes of electroporation chambers can comprise two or more plate electrodes. The electrode plates can comprise any useful biocompatible and conductive material including titanium and gold. The plates can comprise a width dimension that is generally greater than the distance, or gap, between opposing electrodes, and even more preferably greater than twice the gap distance. The electrode plate can be addressable with an electric pulse as determined by the present invention. The electrodes can comprise an array of between 1 and 100 cathodes and 1 and 100 anodes, there always being an even number of cathodes and anodes so as to form pairs of positive and negative electrodes.

[0114] The cathode and anode electrodes can be space on opposing interior sides of the reservoir at a distance of between 0.4 and 1 cm apart.

[0115] Each pair of said anodes and cathodes can be energized at a load resistance (in Ohms) depending upon the chamber size.

IV. Examples

[0116] The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

Conductivity Changes Associated with High Voltage Pulses

[0117] An electrical field of 0.5 kV/cm was applied in two consecutive pulses 1 ms duration and 1 second apart to a conductive medium (FIG. 4). There is a mild short-term conductivity increase in the beginning of each pulse.

[0118] An electrical field of 1 kV/cm was applied in four consecutive pulses 1 ms duration and 1 second apart to a conductive medium (FIG. 5). There is a noticeable conductivity increase during each pulse as well as with each subsequent pulse.

[0119] An electrical field of 1.5 kV/cm was applied in four consecutive pulses 1 ms duration 1 second apart to a conductive medium (FIG. 6). There is a noticeable conductivity increase during each pulse as well as with each subsequent pulse.

[0120] The conductivity values at the beginning of pulses 2-4 are approximately the same as the values at the end of the pulses before them. This illustrates slow rates of buffer cooling during the intervals between the pulses.

[0121] An electrical field of 2 kV/cm was applied in four consecutive pulses 1 ms duration 1 second apart to a conductive medium (FIG. 7). At this field strength the sample conductivity rapidly increased during each pulse, exceeding 200 percent of its initial value. Arcing occurred during the pulses 3 and 4.

Example 2

Platelet Preparation and Processing

[0122] Platelet Isolation:

[0123] Platelets are isolated from PRP by first adjusting pH to 6.5 using 0.3 M citric acid40 mL PRP+400 l Citric Acid and then centrifuging at 500 g for 15 min.

[0124] Platelet Washing:

[0125] 2 wash cycles in Wash Buffer: 36 mM citric acid, 90 mM NaCl, 10 mM EDTA and 5 mM glucose, pH 6.5. Either PGE (at 1 M final) or PGI (at 0.05 g/mL final) is added prior to each wash.

[0126] Platelet Preparation for EP:

[0127] After washing platelets are resuspended in 1 volume of MaxCyte EP Buffer (+2 mM EGTA)+1 vol of 300 mM taurine (+2 mM EGTA). Platelet count is adjusted at 600K/L.

[0128] Platelet Resealing

[0129] EP and No-EP platelet suspensions are transferred into a water bath at 37 C. for 30 min.

[0130] Platelet Washing to Remove the Loaded Compound:

[0131] 1 mL of Tyrode Buffer (135 mM NaCl, 4 mM KCl, 6 mM NaHCO.sub.3, 0.5 mM NaH.sub.2PO.sub.4, 1 mM glucose, 10 mM HEPES, pH 6.5) is added to each sample tube containing the 150 L of platelet suspension followed by centrifugation at 500 g for 10 min. 2 wash cycles with 1 mL of Tyrode Buffer (free of Ca++ and Mg++) at pH 6.5 are performed.

[0132] Post-EP Analysis

[0133] After washing each aliquote of platelets is suspended in 300 L PBS and analyzed by FACS.

Nucleic Acid Loading into Platelets

[0134] Platelets.

[0135] Platelets were obtained from Lifeblood Biological Services (Memphis, Tenn.). Platelets were isolated, washed and prepared for electroporation as described above.

[0136] Loading Platelets.

[0137] Platelets were loaded with AllStars Negative Control siRNA (Qiagen, Inc.). The stock solution of siRNA was added to platelet suspension to final concentration of 1 M, the sample was divided into Coincubation and Electroporation samples, and the latter was processed by application of electrical pulses on MaxCyte System. The power supply capacitance was set to 20 F, field strength was 5 kV/cm.

[0138] After electroporation the platelet suspensions were re-sealed and washed as described above. Results are illustrated in FIG. 8.

Example 3

Small Molecule Loading into Platelets

[0139] Platelets were prepared as previously described and loaded with Alexa-488 (Invitrogen, Inc.) by electroporation as described above. Results are illustrated in FIG. 9.

[0140] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

REFERENCES

[0141] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference: [0142] U.S. Pat. No. 5,676,646 [0143] U.S. Pat. No. 7,186,559 [0144] Authi et al., FEBS Lett 254(1-2): 52-8, 1989. [0145] Crawford and Chronos, Semin Interv Cardiol 1(1): 91-102, 1996 [0146] Hughes and Crawford, Biochim Biophys Acta 981(2): 277-87, 1989 [0147] Pfliegler et al., Thromb Haemost 72(4): 604-10, 1994 [0148] Rols and Teissie, Biophys J 75(3): 1415-23, 1998 [0149] Valeri et al., Transfusion, 45:1890-98, 2005. [0150] Wolf et al., Biophys J 66(2 Pt 1): 524-31, 1994 [0151] WO1998/034478