WO2018210345A1

WO2018210345A1 - Apparatus for delivery of agent

Info

Publication number: WO2018210345A1
Application number: PCT/CN2018/087547
Authority: WO
Inventors: Wen-Shiang Chen; Yi Kung
Original assignee: Chen Wen Shiang
Priority date: 2017-05-18
Filing date: 2018-05-18
Publication date: 2018-11-22

Abstract

An apparatus for delivery of an agent into a predetermined tissue of a subject and a method of using the apparatus to deliver an agent into a predetermined tissue are disclosed. The apparatus has a concentric-type needle electrode (1) to enable the cellular uptake of exogenous molecules. The apparatus and method of the invention are easy to use and they overcome the drawbacks of the conventional techniques used in electroporation process, such as electrical safety, tissue damage, and more numbers of required wounds. The apparatus and method of the invention effectively reduce tissue damage, wound numbers, processing steps and working areas.

Description

[Title established by the ISA under Rule 37.2] APPARATUS FOR DELIVERY OF AGENT

Field of the Invention

The invention relates to an apparatus for delivery of an agent. Particularly, the invention pertains to an apparatus with concentric-type needle electrodes for agent delivery to a predetermined tissue.

Background of the Invention

Macromolecular drugs such as peptides, proteins, nucleotides or polynucleotides are receiving great attentions from the most pharmaceutical companies due to their valuable druggable potentials. However, the large molecular size and hydrophilic nature cause poor intrinsic permeation across biological membranes and low efficiency of cellular uptake. Many different physical approaches for delivery of macromolecular drugs have been proposed. Physical delivery systems are one of the efficient non-viral methods including electroporation, micro-injection, gene gun, tattooing, laser and ultrasound. Among them, electroporation has developed into a universal methodology to introduce macromolecules, such as DNA and RNA, (including siRNA) , proteins, carbohydrates, dyes, and virus particles into target cells.

Electroporation is a technique to apply an electrical current across a cell membrane, change the membrane structure and enable the cellular uptake of exogenous molecules. It is the most frequently used and efficient method for gene transfection, and shows a high potential for clinical use. Electroporation is often used for treating primary isolated cells (L. Lambricht, A. Lopes, S. Kos, G. Sersa, V. Préat, G. Vandermeulen, 2016, Clinical potential of electroporation for gene therapy and DNA vaccine delivery, Expert Opinion on Drug Delivery, 13 (2) : 295–310) , but it requires specific parameter adaptation and optimization for particular cell types, and incurs extensive tissue damage from ohmic heating and puncture, while also raising serious concerns about electrical safety (C. Schulze, M. Peters, W.

P. Wohlsein, 2016, Electrical Injuries in Animals: Causes, Pathogenesis, and Morphological Findings, Veterinary Pathology, 53 (5) : 1018-1029) . In electroporation, the gap between the gene injection site and the electrodes being triggered can raise precision issues that may be addressed by using a needles-array to increase the area, and to apply a higher intensity of the electric field. Nonetheless, the approaches create additional wounds and produce additional tissue damage (K.E. Broderick, X. Shen, J Soderholm, F. Lin, J. McCoy, A.S. Khan, J. Yan, M.P. Morrow, A. Patel, G.P. Kobinger, S. Kemmerrer, D.B. Weiner., N.Y. Sardesai, 2011, Prototype development and preclinical immunogenicity analysis of a novel minimally invasive electroporation device, Gene Therapy, 18: 258-265) . Many devices have been proposed for in vivo application. US5273525 provides an apparatus for implanting macromolecules into a preselected tissue region of the patient such as a muscle or an organ of a patient; for example, a modified syringe is provided for injecting a predetermined quantity of a fluid medium carrying the preselected macromolecules. US7328064 provides an apparatus for injecting a fluid into body tissue. US9526836 discloses means for the controlled administration of the therapeutic agent through an orifice to the patient, a plurality of penetrating electrodes arranged with a predetermined spatial relationship relative to the orifice, and means for generating an electrical signal operatively connected to the electrodes.

However, those devices are hard to use for surgeon and cannot solve the problem of tissue damage resulting from the treatment process. There is still a need to develop an apparatus for delivering an agent into a tissue in a low voltage and low damage to the tissue.

Brief Description of the Drawings

Figure 1 shows an embodiment of the apparatus of the invention.

Figure 2 shows the concentric-type needle electrode and the fluid channel of the apparatus of the present invention.

Figure 3 shows an embodiment of the array of the invention.

Figure 4 shows comparison of electroporation performance between NTUH 1 and BTX 533. Exposure time is 1 s. Plasmid DNA is 40 μg of pCI-neo-Luc+. The substrate is 4 mg of D-Luciferin Firefly. *and #are respectively NTUH 1 at 10 V vs. 0 V of bias, and NTUH 1 vs. BTX 533 at 50 V of bias.

Figure 5 shows comparison of tissue damage by electroporation with NTUH 1, at 10 V and 100 V, and with BTX 533 at 50 V and 100 V.

Figure 6 shows an equivalent circuit for the proposed needle-electrodes. R _T is the tissue resistance; C _D is the interfacial capacitance of the electrode; R _CT is the charge transfer resistance; W _Diff is the impedance (a Warburg element) originating from the diffusion constraint.

Figure 7 shows the timeline of electroporation bias dose with the proposed NTUH 1 needle-electrode. Exposure time is 1 s. Plasmid DNA is 40 μg of pCI-neo-Luc+. The substrate is 4 mg of D-Luciferin Firefly. *is p < 0.05 vs. un-bias at the same time point in the t-test.

Figure 8 shows luminescent image of electro-transfected mice with needle-electrode array. Mouse (L) is processed with a 2 needle-electrode array; Mouse (R) is processed with a 3 needle-electrode array. Both are treated under 50 V of bias with an insertion depth of 5 mm. Exposure time is 1 s. Plasmid DNA is 20 μg of pCI-neo-Luc+. The substrate is 2 mg of D-Luciferin Firefly.

Figure 9 shows pCI-neo-Luc+ plasmid.

Figure 10 shows (a) luminescent image of electro-transfected zebrafishes, where A and B are respectively the electro-transfected locations and the non-electro-transfected points. (b) Comparison of luminescent flux between A and B in (a) . All are treated under 50 V of bias with an insertion depth of 5mm. Exposure time is 1 min. Plasmid DNA is 20 μg of pCI-neo-Luc+. The substrate is 2 mg of D-Luciferin Firefly. **is p <0.01 in the t-test.

Figure 11 shows (a) fluorescent image of electro-transfected zebrafishes. A and B are respectively the electro-transfected brain and non-electro-transfected body. (b) Comparison of fluorescent flux between A and B in (a) . All are treated under 50 V of bias with an insertion depth of around 1.5 mm. Plasmid DNA is 20 μg of pEGFP-C1. *is p <0.05 in the t-test.

Figure 12 shows pEGFP-C1 vector.

Detailed Description of the Invention

The invention creates an apparatus for delivery of an agent into a predetermined tissue of a subject. The apparatus has a concentric-type needle electrode to enable the cellular uptake of exogenous molecules. The apparatus of the invention is easy to use and it overcomes the drawbacks of the conventional devices used in electroporation process, such as electrical safety, tissue damage, and more numbers of required wounds. The apparatus of the invention effectively reduces tissue damage, wound numbers, processing steps and working areas.

In one aspect, the invention provides an apparatus for delivery of an agent into a predetermined tissue of a subject, comprising:

a concentric-type needle electrode comprising an inner needle and an outer needle;

a fluid channel for delivery of the agent, which is in fluidic communication with the lumen of the inner needle; and

an electrical field generator electrically connected to the electrode, which applies an electric signal to the predetermined tissue to increase intracellular uptake of the agent.

In one embodiment, the electrical field generator provides a bias sufficient to enable the cellular uptake of exogenous molecules but limits damage of cells or tissues of the treated subject. The bias applying to the subject of treatment is adjustable according to the conductivity of the treated cells or tissues. Preferably, it is greater than about 0 V to about 150 V. In some embodiments, the bias is in a range of greater than about 0 V to about 120 V, about 0 V to about 100 V, about 0 V to about 90 V, about 0 V to about 80 V, about 0 V to about 70 V, about 0 V to about 60 V, about 0 V to about 50 V, about 0 V to about 40 V, about 0 V to about 30 V, about 0 V to about 20 V, about 0 V to about 10 V, about5 V to about 120 V, about5 V to about 100 V, about5 V to about 80 V, about5 V to about 60 V, about 0 V to about 40 V or about5 V to about 20 V. Preferably, the bias is 10 V.

In one embodiment, the distance (d) between the electrodes to the area (a) of the electrodes is in a ratio ρ (L/A; cell constant) , wherein ρ = R, if V =IR, where V is the bias applied across the tissue; I is the current through the tissue; R is the resistance of the tissue; ρ is the resistivity; L is the distance between electrodes; A is the cross-sectional area.

The length of the outer needle and the inner needle are adjustable according to the location of the treated cell or tissue in the subject. In one embodiment, the length of the outer needle is shorter than the inner needle.

The concentric-type needle electrode comprises an inner needle and an outer needle as two electrodes with opposite electricity. The inner needle has an outer diameter smaller than the inner diameter of the outer needle and inserts into the shaft of the outer needle to form a concentric arrangement. The diameters of the outer needle and the inner needle are adjustable according to the treated cell or tissue in the subject. In one embodiment, the outer needle and the inner needle are a 20 G needle and a 30 G needle, respectively.

In one embodiment, a printed circuit connects to the inner needle and the outer needle, respectively, to extend the concentric-type needle electrode. In a further embodiment, the printed circuit connects to the inner needle and the outer needle, respectively, and locates along the fluid channel.

The fluid channel is in fluidic communication with the lumen of the inner needle. In one embodiment, the fluid channel is an infusion tube or a syringe.

In one embodiment, the agent to be delivered to the predetermined tissue includes any agent capable of providing a desired or beneficial effect on living tissue. The agents include, but are not limited to, pharmaceutical drugs and vaccines, and nucleic acid sequences (such as supercoiled, relaxed, and linear plasmid DNA, antisense constructs, artificial chromosomes, or any other nucleic acid-based therapeutic) , and any fluidic formulations thereof. Preferably, the agent comprises macromolecules selected from the group consisting of peptides, proteins, nucleotides and polynucleotides.

In another aspect, the invention provides an array comprising two or more modules, wherein a module comprises a concentric-type needle electrode, a fluid channel and a fixing device for fixing and connecting the modules. The array can be used to deliver agents to a target site of the tissue with larger area or deliver various agents to the target tissue. The concentric-type needle electrodes of the array can connect to one electrical field generator, or two or more concentric-type needle electrodes each can connect to one electrical field generator.

Referring to Figure 1, an embodiment of the apparatus of the apparatus of the invention is illustrated. The apparatus comprises a concentric-type needle electrode 1, a fluid channel 2 and an electrical field generator 3. The concentric-type needle electrode 1 connects to the electrical field generator 3 through a pair of wires 13, wherein the concentric-type needle electrode 1 has an outer needle 12 and an inner needle 11 wherein the outer needle 12 is insulted from the inner needle 11 by an insulating layer 14. Figure 2 shows an embodiment of the concentric-type needle electrode 1 and the fluid channel 2. In the embodiment, the fluid channel 2 is a syringe 22 with a hole 211 on the needle hub 21 and the concentric-type needle electrode 1 has an outer needle 12 and an inner needle 11. The outer needle 12 and the inner needle 11 do not touch each other. The outer needle 12 is insulted from the inner needle 11 by an insulating layer 14. A pair of wires 13 connects to the outer needle 12 and the inner needle 11, respectively. One of the wires 13 connects to the inner needle through the hole 211. The other ends of the wires connect to the electrical field generator. The outer needle 12 and the inner needle 11 are provided as electrodes with opposite electricity. The inner needle 11 has an outer diameter smaller than the inner diameter of the outer needle 12 and inserts into the outer needle 12 to form a concentric arrangement.

In one embodiment, an insulating layer is placed between the inner needle and the outer needle to prevent short circuit. Any insulating material can be used to produce the insulating layer. Preferably, the insulating layer is epoxy.

Referring to Figure 3, an embodiment of the array of the invention is illustrated. The array comprises two or more modules, wherein a module comprises a concentric-type needle electrode 1, a fluid channel 2 and a fixing device 4 for fixing and connecting the modules.

In one embodiment, the concentric-type needle electrode of the invention provides a lower bias (10 V) and a smaller working area (≈ 10 mm ³) .

The fabrication of the concentric-type needle electrode of the invention is shown as follows. A hole is punched on the insulin needle hub in order to connect a wire to its needle part. A polyethylene tube (PE 10) is placed on the outer rim of the shaft of needle as an insulating layer. A needle (such as 20 G needle) whose length is tuned beforehand by the tube cutter is then inserted into the shaft of the needle to form a concentric arrangement. A wire is next connected on the outside of the 20 G needle. Epoxy adhesive is applied to all the wires to fix them in position and to form a protective layer.

In another aspect, the invention provides a method of using the aforementioned apparatus to deliver an agent into a predetermined tissue of a subject, comprising inserting the concentric-type needle electrode to the predetermined tissue, delivering the agent to the tissue through the fluid channel, and applying an electric signal to the predetermined tissue through the electrical field generator.

While particular embodiments are illustrated in and described with respect to the drawings, it is envisioned that those skilled in the art may devise various modifications without departing from the spirit and scope of the appended claims. It will therefore be appreciated that the scope of the disclosure and the appended claims is not limited to the specific embodiments illustrated in and discussed with respect to the drawings and that modifications and other embodiments are intended to be included within the scope of the disclosure and appended drawings. Moreover, although the foregoing descriptions and the associated drawings describe example embodiments in the context of certain example combinations of elements and/or functions, it should be appreciated that different combinations of elements and/or functions may be provided by alternative embodiments without departing from the scope of the disclosure and the appended claims

Examples

Materials &methods

1. Bio-and chemical materials

The study proposal was approved by the ethics committee of the Laboratory Animal Center at National Taiwan University College of Medicine (approval No. 20100137 for the use of mice and zebrafish) . All mice (BALB/cByJNarl, 5 weeks) were purchased from the National Laboratory Animal Center (Taipei City, Taiwan) , and were electro-transfected between 7 to 8 weeks of age. Expression vectors pCI-Neo-Luc+ (7187 bp) and pEGFP-C1 (4731 bp) , purified by Qiagen Mega endotoxin free, were acquired from the Biomedical Resource Core of the First Core Laboratory, College of Medicine, National Taiwan University (Taipei City, Taiwan) . D-Luciferin Firefly potassium salt was purchased from Biosynth AG (Lake Constance, Switzerland) . 150 mMNaCl, sterile-filtered by 0.22 μm PES membrane (millipore syringe filter) was purchased from Polyplus-transfection (Illkirch, France) . Dulbecco's phosphate-buffered saline (DPBS 10 ×, Gibco) was purchased from Thermo Fisher Scientific Inc. (Waltham, US) . Forane (Isoflurane) was purchased from AesicaQueenborough Ltd. (Queenborough, UK) . Ethyl 3-aminobenzoate methanesulfonate (Tricaine, 98 %) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US) . All materials and their derivatives were biotechnology grade.

2. Instruments &devices

A square wave electroporation system (ECM 830, BTX) and 2 needle array (No. 533, BTX) were purchased from Harvard Apparatus Inc. (Holliston, US) . A matrix isoflurane vaporizer (VIP 3000) was purchased from Midmark Corp. (Ohio, US) . In vivo imaging system spectrum (IVIS) and XGI-8 gas anesthesia system (PerkinElmer, Waltham, US) were equipped in the Laboratory Animal Center at National Taiwan University College of Medicine. A fixed stage microscope (BX51) was purchased from Olympus Co. (Tokyo, Japan) . A digital microscope camera system (RT-KE Color 3-shot, SPOT) and its software (SPOT advanced) were purchased from Diagnostic Instruments, Inc. (Sterling Heights, US) . A steel tube cutter (58692-U, Supelco) was purchased from Sigma-Aldrich Co. LLC. (St. Louis, US) . Intramedic ^TM Polyethylene Tubing (PE10) was purchased from Becton, Dickinson (Franklin Lakes, US) . An Omnican 50 insulin syringe (30 G with 12 mm needle) was purchased from B. Braun Melsungen AG (Melsungen, Germany) . An AGANI needle (20 G) was purchased from Terumo Medical Corporation (Tokyo, Japan) . Scotch-Weld ^TM Epoxy Adhesive was purchased from 3M Company (Maplewood, US) .

3. Fabrication of needle-electrode (NTUH 1) and needle-electrode array

A hole was punched on the insulin needle hub in order to connect wire to its needle part. A polyethylene tube (PE 10) was placed on the outer rim of the shaft of needle as an insulating layer. A 20 G needle whose length was tuned beforehand by the tube cutter and then inserted into the shaft of the needle to form a concentric arrangement. A wire was next connected on the outside of the 20 G needle. Epoxy adhesive was applied to all the wires to fix them in position and to form a protective layer.

For arraying needle-electrodes, concentric-type needle devices were fixed by a fixing device 4 as shown in Figure 3. Needle-electrodes were connected with the wires to form a needle-electrode array.

4. Electroporation on mice and IVIS analysis

Once the mice were anesthetized with isoflurane, their thighs were fixed and the concentric-type needle electrode was inserted into their quadriceps femoris muscle. 40 μg of pCI-neo-Luc+, pre-dissolved in 50 μl of 150 mM NaCl, was then injected into the quadriceps femoris muscle. Electroporation was then performed 8 times with a 10 V pulse (0.2 ms) in 1 s intervals.

After 24 h, 50 μl of the substrate (40 mg/ml D-Luciferin Firefly) was injected into both sides of each mouse’s lower abdomen and allowed to react for 10 min. The anesthetized mice were then placed into the IVIS scanning chamber to measure gene transfection performance. All IVIS data in the proposed research were analyzed using Living image 3.1 (Caliper Life Sciences, Waltham, US) .

5. Histology section

To evaluate the extent of tissue damage due to electroporation at various bias voltages, the experimental procedure described in the above section 4 of Materials and Methods was modified using pork short fillets injected with 150 mM NaCl as a control solution instead of mice with a plasmid DNA solution. The short fillets were then immersed in 10%formaldehyde solution for 24 h. Subsequently, the specimens were embedded in paraffin and H&E was applied to the short fillets. Slides were analyzed using an Olympus BX51 microscope, along with the Spot microscope camera system and its software, SPOT advanced.

6. Electroporation on Zebrafishes

Zebrafishes were restrained after being anesthetized with 0.2 mg/ml Tricaine, dissolved in distilled water. The concentric-type needle electrode was inserted into the fish abdomen and brain as described in the above section 4 of the Materals and methods. After 24 h, the zebrafishes were analyzed using IVIS to obtain luminescent and fluorescent images (excitation at 465 nm, emission at 520 nm) .

7. Statistics

All data obtained were analyzed statistically using Student's t-test, and presented as mean±SD. A probability value of p < 0.05 was considered indicative of a significant difference.

Example 1 Electroporation on mice

To test the reusability and repeatability of the concentric-type needle electrode of the invention, NTUH 1, electroporation was performed reusing a previously used NTUH 1 cleaned by distilled water along with unused NTUH electrodes. Both electroporation results show similar transfection outcomes (p ＞＞ 0.05) . Therefore, all subsequent experiments were conducted with reused NTUH 1 electrodes, cleaned using distilled water between experiments.

After confirming the reliability of the NTUH 1 was compared with a commercial product, BTX 533 (Harvard Apparatus, Holliston, US) . As shown in Figure 4, the minimal detectable transgene expression was found when 10 and 50 V were applied using NTUH 1 and BTX 533 electrodes respectively. Moreover, comparing the luminescent flux at 50 V of bias between NTUH 1 and BTX 533 shows that the electroporation efficiency of NTUH 1 is much higher than that of BTX 533. This efficiency gap may be caused by the cell-constant of the electrode structure, which was around 0.2 cm ^-1 and 7.06 cm ^-1, respectively, for NTUH 1 and BTX 533.

The concentric-type electroporation needle electrode and array can be used with a lower bias, fewer wounds, fewer processing steps, and a smaller working area to provide significantly improved accuracy and precision (Table 1) . Moreover, the solution can be arrayed to simultaneously treat several target regions.

Table 1. Comparison between NTUH 1 and BTX 533

Conductivity is a key factor for tissue damage and cell membrane breakdown during electroporation. Cell-constancy is the key factor in evaluating electrode conductivity, where

V =EL (1)

I =iA (2)

This suggests the proposed needle-electrode pair has an effective surface area (A) , and given a parallel electric field, the electric field strength (E) will be linearly correlated with the voltage (V) by an effective distance (L) between the electrodes. Moreover, the current intensity (I) is the product of the current density (i) and the effective area (A) of the electrodes. So the conductance (G) by the proposed configuration will be:

Where κ is the cell constant, which is determined by the geometry of the electrode set. G0 is conductivity, which is an intrinsic conductance dependent on the properties of the ionic conductor in the subject tissue, including ion concentration (C) , charge (n) and mobility (μ) .

Figure 6 shows the electrophysiological and electrochemical mechanism of the proposed needle-electrodes. CD, RCT, and WDiff are respectively frequency, bias, and electrode material dependent, and RT is cell constant dependent. CD is small, so an electrode of lower cell constant, such as NTUH 1, can reduce tissue damage and operation bias. Furthermore, the ohmic drop is also reduced, along with impedance matching problems.

Figure 5 shows that electroporation with NTUH 1 and BTX 533 at 100 V of bias produces similar tissue damage results (around 10,000 μm ²) . But to complete electroporation, NTUH 1 only caused 1 wound while BTX 5333 produced 3 (1 for injecting the plasmid DNA, and 2 for inserting the electrodes) . Tissue damage from ohmic heating can be clearly seen as dark shapes marked by arrows in the BTX 533 part of Figure 5 and in R _T of Figure 6. This supports the proposed hypothesis that higher conductivity will produce less tissue damage. The results of Figures 4 and 5 suggest that NTUH 1 is not only effective under 10 V of bias, but can also decrease wound size.

Pharmacokinetics is also a key factor in gene therapy. As shown in Figure 7, gene expression achieved maximum performance after 1 week, and deteriorated thereafter because the plasmid DNA was degraded by lysosomes. Lipo-vector and polymeric vector are added to extend DNA lifespan. As shown in Figure 7, at higher bias values (e.g., 100, 50 V) , the expression can last for 3 weeks before a second electroporation round is needed; however, for lower bias values (e.g., 25 and 10 V) , the expression dropped to the baseline level after the first week.

As shown in Figure 8, mouse (L) is processed with a 2 needle-electrode array; Mouse (R) is processed with a 3 needle-electrode array. Both are treated under 50 V of bias with an insertion depth of 5 mm. Exposure time is 1 s. Plasmid DNA is 20 μg of pCI-neo-Luc+ (see Figure 9) . The substrate is 2 mg of D-Luciferin Firefly. Unlike other commercial products, NTUH 1 was successfully used for array-based electroporation. Moreover, this needle-electrode array is suitable for use in conditions where the target shape and location varies.

As shown in the left mouse in Figure 8, the electrode’s electro-field interaction has difficulty penetrating the vertebral column and the region lacks an electrical mediator. On the other hand, according to the right mouse in Figure 8, in addition to the needle bevel, electro-field interaction also exists in between the needles, which indicates good potential for use in gene therapy, especially when dealing with pathological targets with different shapes and locations.

Example 2 Electroporation on zebrafish

Zebrafishes serve as a common animal model in gene-related experiments in which genes are altered during the embryonic period. Despite the relative difficulty of using tissue and organs from mature fish, gene transfection experiments using mature fish are still important. Figure 10 shows (a) luminescent image of electro-transfected zebrafishes, where A and B are respectively the electro-transfected locations and the non-electro-transfected points; (b) comparison of luminescent flux between A and B in (a) . All are treated under 50 V of bias with an insertion depth of 5mm. Exposure time is 1 min. Plasmid DNA is 20 μg of pCI-neo-Luc+ (see Figure 9) . The substrate is 2 mg of D-Luciferin Firefly. Figure 11 shows (a) fluorescent image of electro-transfected zebrafishes. A and B are respectively the electro-transfected brain and non-electro-transfected body. (b) Comparison of fluorescent flux between A and B in (a) . All are treated under 50 V of bias with an insertion depth of around 1.5 mm. Plasmid DNA is 20 μg of pEGFP-C1 (see Figure 12) . As depicted in Figures 10 and 11, NTUH 1 can be used to transfect genes to abdomen muscle and brain.

Claims

An apparatus for delivery of an agent into a predetermined tissue of a subject, comprising:

(a) a concentric-type needle electrode comprising an inner needle and an outer needle;

(b) a fluid channel for delivery of the agent, which is in fluidic communication with the lumen of the inner needle; and

(c) an electrical field generator electrically connected to the electrode, which applies an electric signal to the predetermined tissue to increase intracellular uptake of the agent.
The apparatus of Claim 1, wherein the electrical field generator provides a bias greater than 0 V to 150 V.
The apparatus of Claim 1, wherein the electrical field generator provides a bias of 10 V.
The apparatus of Claim 1, wherein the length of the outer needle is shorter than the inner needle.
The apparatus of Claim 1, wherein the inner needle has an outer diameter smaller than the inner diameter of the outer needle.
The apparatus of Claim 1, wherein the outer needle and the inner needle of the concentric-type needle electrode are a 20 G needle and a 30 G needle, respectively.
The apparatus of Claim 1, wherein the concentric-type needle electrode is electrically connected to the electrical field generator by wires or a printed circuit.
The apparatus of Claim 1, wherein the fluid channel is an infusion tube or a syringe.
The apparatus of Claim 1, wherein the agent is a pharmaceutical composition.
The apparatus of Claim 9, wherein the pharmaceutical composition comprises macromolecules.
The apparatus of Claim 9, wherein the macromolecules are selected from the group consisting of peptides, proteins, nucleotides and polynucleotides.
The apparatus of Claim 1, wherein the outer needle is insulted from the inner needle by an insulating layer.
An array comprising two or more modules and a fixing device for fixing and connecting the modules, wherein the module comprises:

(a) a concentric-type needle electrode comprising an inner needle and an outer needle;

(b) a fluid channel for delivery of the agent, which is in fluidic communication with the lumen of the inner needle; and

(c) an electrical field generator electrically connected to the electrode, which applies an electric signal to the predetermined tissue to increase the intracellular uptake of the agent.
A method of using an apparatus of Claim 1 to deliver an agent into a predetermined tissue of a subject, comprising inserting the concentric-type needle electrode to the predetermined tissue, delivering the agent to the tissue through the fluid channel, and applying an electric signal to the predetermined tissue through the electrical field generator.
The method of Claim 14, wherein the agent is a pharmaceutical composition comprising macromolecules selected from the group consisting of peptides, proteins, nucleotides and polynucleotides.