1. Introduction

        In recent years, biological and biomedical applications of microstructures have become one of the promising research areas of microelectromechanical systems (MEMS). These micro-scale devices for biological/biomedical applications are often referred to as “BioMEMS” [1]. Electroporation has been used for electrogenetherapy and electrochemotherapy [2]. For application of electroporation on skin, conventional macro-needle electrodes have been used [3], but they cause pain during insertion, can lead to tissue burning during electroporation, and require a high-voltage pulse generator.

        The objective of this research is to develop a microdevice to deliver biomolecules such as DNA into skin by means of electroporation. This is particularly interesting for delivery of DNA vaccines into the skin's dendritic cells [4]. The basic requirements of the device are the need to penetrate across the stratum corneum layer and provide an electrical pathway to generate an electric field. In addition to that, the device should provide methodologies to reduce pain during injection and tissue burning, two issues that plague traditional therapies.

        One promising approach to satisfy these requirements is to implement electrical functionality in a microneedle array (i.e., an array of electrically conductive needle s measuring hundreds of microns in length that are electrically isolated from each other to enable generation of strong field strengths across short electrode-electrode spacings). The microneedle array can provide minimally invasive, painless insertion into skin and can act as an electrode after proper metallization process. Due to the shallow insertion depth of the microneedles, pain during an application of an electric field by disturbing nerves can also be minimized. In addition, the voltage required for electroporation can be reduced due to the small size scale, avoiding the use of a high voltage source, which is not suitable for portable applications.

2. Experimental methods and results

      The device contains a 10x10 array of microneedles, with adjacent microneedles electrically isolated, which gives a “checkerboard” pattern of electrical polarity (Figure 1). The height of the microneedles is 500µm, bottom is 200µm square, and spacing between microneedles is 200µm.

(a)                                                     (b)

(c)                                                     (d)

(e)                                                     (f)

Figure 1. (a)-(d) SEM picture of the fabricated device; (e),(f) Photomicrograph of the device after metal transfer micromolding

To verify mechanical strength, an insertion test was performed with human cadaver skin. The fabricated microneedle array was inserted and removed from the skin, and the skin was dyed with trypan blue. Microscopic imaging showed blue stains in the microneedle array pattern, indicating that the microneedles pierced into the skin (Figure 2).

Figure 2.  Photomicrograph of human cadaver skin after piercing with microneedle array and staining with blue dye

The capability of delivering molecules into cells using this electroporation microneedle array was examined in vitro. DU-145 prostate cancer cells were used for the electroporation experiment, and calcein was used as the model compound for delivery into the cell. A cell suspension was mixed with calcein and applied as a droplet over the microneedle array. After applying voltages of 12-91V for 2.5ms, the cells were collected, followed by adding propidium iodide (PI) to stain dead cells. When 12 V was applied for 2.5 ms, the average uptake of calcein was 7 % of the cells (Figure 3). The uptake of calcein increased until 69 V was applied and then decreased as the applied voltage increased. At 69 V, which corresponds to the nominal field strength (applied voltage divided by the distance between electrodes at the bottom of the device) of 3.45 kV/cm, a maximum uptake of 48 % of cells was achieved. However, cell viability suddenly dropped at that point, indicating that the field strength induced by the applied voltage started to cause cell death rather than uptake of calcein.

3. Conclusion

The ultimate goal of this research is to develop a micro-scale electroporation device that can deliver molecules into skin. We designed and fabricated electrically active and appropriately patterned microneedle arrays and showed that they were strong enough to insert into skin. We then observed that uptake of calcein occurred at conditions which correspond to macro-scale electroporation experiments, indicating that the fabricated device has the capability to deliver molecules into cells by electroporation. Overall, electroporation with a microneedle array can be a promising alternative method to deliver molecules into skin.

Figure 3.  Uptake of calcein and cell viability at each applied voltage

References

1. R. S. Shawgo, A. C. R. Grayson, Y. W. Li, and M. J. Cima, BioMEMS for drug delivery, Current Opinion in Solid State & Materials Science (2002) 6, 329-334.

2. M. J. Jaroszeski et al., Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery, Humana Press, Inc., Totowa, 2000.

3. G.A. Hofmann et al., Electroporation Therapy: A New Approach for the Treatment of Head and Neck Cancer, IEEE Transactions on Biomedical Engineering (1999) 46, 752-759.

4. Roos AK, Moreno S, Leder C, Pavlenko M, King A, Pisa P, Enhancement of cellular immune response to a prostate cancer DNA vaccine by intradermal electroporation, Molecular Therapy (2006)13, 320-327