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SEMINAR. ON. ELECTROPORATION AND MICRONEEDLES . PRESENTED BY. P.RENUKA. M.PHARMACY- II SEM. DEPARTMENT OF INDUSTRIAL PHARMACY. UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES. KAKATIYA UNIVERSITY,WARANGAL. CONTENTS. INTRODUCTION ELECTROPORATION DEFINITION BACKGROUND

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slide1

SEMINAR

ON

ELECTROPORATION AND MICRONEEDLES

PRESENTED BY

P.RENUKA

M.PHARMACY- II SEM

DEPARTMENT OF INDUSTRIAL PHARMACY

UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES

KAKATIYA UNIVERSITY,WARANGAL

slide2

CONTENTS

  • INTRODUCTION
  • ELECTROPORATION
  • DEFINITION
  • BACKGROUND
  • PROCEDURE
  • PHENAMENON OF ELECTROPORATION
  • ADVANTAGES AND DISADVANTAGES
  • APPLICATIONS
  • MICRONEEDLES
  • DEFINITION
  • ADVANTAGES
  • APPLICATIONS
  • CURRENT REASEARCH IN MICRONEEDLE TECHNOLOGY
  • CONCLUSION
  • REFERENCES
slide3

INTRODUCTION

  • Until very recently, the only drugs that could permeate transdermally were those possessing a very narrow and specific combination of physicochemical properties.
  • However, rapid advances in bioengineering have led to the emergence of various new "active" enhancement technologies designed to transiently circumvent the barrier function of the stratum corneum.
  • These novel systems, using iontophoresis, sonophoresis, electroporation, or microneedles arrays, will greatly expand the range of drugs that can be delivered transdermally.
physical enhancement techniques
PHYSICAL ENHANCEMENT techniques

ELECTROPORATION

MICRONEEDLES

slide5

ELECTROPORATION

  • DEFINITION
  • Electroporation or Electropermeabilization is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electric field
  • Basic principle of electroporation; Short pulses of high voltage current are applied to the skin producing hydrophilic pores in the intercellular bilayers via momentary realignment of lipids.
slide6

BACKGROUND;

  • Many research techniques in molecular biology require a foreign gene or protein material to be inserted into a host cell. Since the phospholipids bilayer of the plasma membrane has a hydrophilic exterior and a hydrophobic interior (fig 1), any polar molecules, including DNA and protein, are unable to freely pass through the membrane
  • FIG 1; Diagram of the Phospholipids Bilayer.
  • Many methods have been developed to surpass this barrier and allow the insertion of DNA and other molecules into the cells to be studied. One such method is electroporation
slide7

PROCEDURE

Electroporation System

Figure 2. Diagram of the basic circuit setup of the electroporation apparatus. This diagram shows the basic electric circuit that provides the voltage for electroporation.

Electroporation Cuvettes

slide8

Diagram of the major components of an electroporator with cuvette loaded

Laboratory Practice

Electroporation Cuvettes

slide9

Electroporators

  • Benchtop electroporators
  • Handheld electroporators
  • Generally used as common lab equipment, residing atop a central bench or hood.
  • They offer the advantage of electroporating multiple samples at the same time
  • Cordless, rechargeable and use disposable pipectrodes, which combine elements of both cuvettes and pipettes.
  • Their operating parameters are preset to the optimal parameters for transforming either bacteria or mammalian cells.
  • They can also be set to different operating parameters depending on whether or not the cell has a cell wall

Pipectrode

slide10

How Electroporation Works

  • The phenomenon of electroporation

Physical Mechanism

  • Schematic diagram showing the theoretical arrangement of lipids in a hydrophobic core (top) and a hydrophilic pore (bottom).
slide12

Advantages:

  • . Versatility:Electroporation is effective with nearly all cell and species types
  • .Efficiency: A large majority of cells take in the target DNA or molecule.
  • In a study on electro transformation of E. coli, for example, 80% of the cells received the foreign DNA
  • Small Scale: The amount of DNA required is smaller than for other methods
  • In vivo: The procedure may be performed with intact tissue .
  • A paper published in Developmental Biology showed the successful transfer of a DNA construct with a fluorescent reporter gene into intact mouse brain tissue
slide13

Disadvantages:

  • . Cell Damage: If the pulses are of the wrong length or intensity, some pores may become too large or fail to close after membrane discharge causing cell damage or rupture (Weaver, 1995).
  • · Nonspecific Transport: The transport of material into and out of the cell during the time of electro permeability is relatively nonspecific. This may result in an ion imbalance that could later lead to improper cell function and cell death (Weaver, 1995)
slide14

APPLICATIONS

  • Electroporation is widely used in many areas of molecular biology research and in the medical field. Some applications of electroporation include:
  • DNA Transfection or Transformation: This is likely the most widespread use of electroporation. Specific genes can be cloned into a plasmid and then this plasmid introduced into host cells (bacterial or otherwise) in order to investigate gene and protein structure and function. (Nickoloff, 1995)

Fig. Microscope images of the results of transfection by electroporation. In this experiment, a gene construct was inserted by electroporation into the cells shown on the right. The fluorescence of the protein produced by the reporter gene included in this construct shows that the DNA was properly uptake in the majority of cells. These cells could now be used in further experimentation

slide15

Direct Transfer of Plasmids Between Cells:

  • Bacterial cells already containing a plasmid may be incubated with another strain that does not contain plasmids but that has some other desirable feature. The voltage of electroporation will create pores, allowing some plasmids to exit one cell and enter another.
  • The desired cells may then be selected by antibiotic resistance or another similar method . This type of transfer may also be performed between species. Thus, large numbers of plasmids may be grown in rapidly multiplying bacterial colonies and then transferred to yeast cells by electroporation for study (Gunn et. al., 1995).
  • Trans-dermal Drug Delivery:
  • Just as electroporation causes temporary pores to form in plasma membranes, studies suggest that similar pores form in lipid bilayers of the stratum corneum- the outermost dead layer of skin. These pores could allow drugs to pass through to the skin to a target tissue.
  • This method of drug delivery would be more pleasant than injection for the patient (not requiring a needle) and could avoid the problems of improper absorption or degradation of oral medication in the digestive system (Praustnitzet. al., 1993).
slide16

Cancer Tumor Electro chemotherapy:

  • Scientists are investigating the potential of electroporation to increase the effectiveness of chemotherapy. As in electroporation for DNA transfection, the applied electrical pulse would disrupt the membrane of the tumor cell and increase the amount of drug delivered to the site.
  • Some studies have suggested that increased tumor reduction is seen when this method is applied to cancerous cells in animal model systems (Maeda et. al., 1998).
  • Gene Therapy:
  • Much like drug delivery, electroporation techniques can allow vectors containing important genes to be transported across the skin and into the target tissue.
  • Once incorporated into the cells of the body, the protein produced from this gene could replace a defective one and thus treat a genetic disorder (Fig.) (Inovio, 2002).
slide18

MICRONEEDLES: A Novel Approach to Transdermal Drug Delivery

  • A microstructuredtransdermal system also called microneedle consists of an array of microstructured projections coated with a drug or vaccine that is applied to the skin to provide intradermal delivery of active agents, which otherwise would not cross the stratum corneum.
  • The mechanism for delivery, however, is not based on diffusion.Instead, it is based on the temporary mechanical disruption of the skin and the placement of the drug or vaccine within the epidermis, where it can more readily reach its site of action
slide19

Microneedle devices are tiny arrays of needles, typically about the size of a dime, that can be fashioned from silicon, glass, or biodegradable polymers using techniques

  • such as microlithography and etching, which are widely employed in the manufacture of electronics products.
  • needles themselves can be rendered hollow or solid.
  • Devices with hollow needles can be attached to a syringe, enabling a solution of drug to be injected through the microneedles. 
  • In contrast, solid needles are coated with drug, so that once the device is pressed into the skin, the drug simply dissolves off, being deposited in the dermis. 
slide20

However, since the holes that are made in the skin by solid needles are so small, allowing only a tiny amount of drug to enter the body, the number of needles is the primary factor determining the how much drug actually can be administered by a single device. 

  • Collectively, hundreds of coated needles on one device can deliver up to one milligram of drug, which far exceeds the amount necessary for most vaccines to be effective.
  • But perhaps the two most important factors, the elements that determine successful drug delivery while ensuring a pain-free experience, are the sharpness and the length of the needles. 

FIG; Microscope image shows an array of hollow microneedles next to a hypodermic needle typical of those now used to inject drugs and vaccines. (Georgia Tech Image: Shawn Davis)

slide21

In general terms, make needles that:

  •   Go into skin easily
  •   Deliver drugs effectively
  •   Don’t hurt
  •   Are biocompatible
  • The needles need to:
  •   Withstand typical handling
  •    Deliver controlled amount of drug at specific rate
  •    Deliver to precise depth in body
  •   Withstand insertion without buckling, fracture, or delamination
  • Needles have been made from:
  •   Glass
  •   Silicon
  •   Metal—stainless steel, solid or coat of gold over Ni, Pd or Pd-Co, and Pt
  •   Biodegradable polymers, if a tip snaps off while inserted, it will easily biodegrade
slide22

Figure : (a) A microformedmicroneedle with opening at the apex; (b) 4 pyramid microneedles microformed on a thin aluminium sheet; (c) 9 microneedles with high aspect ratio microformed on thin metal sheet; (d) 4 solid metal microneedles via electroplating; (e) Beveled tip microneedles via electroplating; (f) microneedles in 3x3 array; (g) silicon microneedles with 250 micron height

slide23

Microneedle fabrication

  • The needle fabrication process involved four steps.
  • First, arrays of microneedles made of SU-8 epoxy photo resist were fabricated by patterning SU-8 onto glass substrates and defining needle shape by lithography.
  • Then, the tips of the needles were sharpened using reactive ion etching. The next step involved laser drilling holes through the microneedles and base substrate oriented off-center, but parallel to the Microneedle axis.
  • This created holes that serve as the micro fluidic needle bores for injection or infusion, which terminate in side-opening holes along the needle shaft below the needle tip.
  • Finally, the needle arrays were coated with nickel by electroplating to increase their mechanical strength.
slide24

Advantages of Microneedles

  • 1. As for microneedles they can be fabricated to be long enough to penetrate the stratum corneum, but short enough not to puncture nerve endings. Thus reduces the chances of pain, infection, or injury.

Instead of one big injection, why not lots of tiny ones? Microneedle devices look and feel like a patch, but they actually consist of hundreds of microneedles that can be programmed to deliver drugs steadily and painlessly

slide25

2. By fabricating these needles on a silicon substrate because of their small size, thousands of needles can be fabricated on a single wafer. This leads to high accuracy, good reproducibility, and a moderate fabrication cost.

  • 3. Hollow like hypodermic needle; solid—increase permeability by poking holes in skin, rub drug over area, or coat needles with drug .
  • 4. Arrays of hollow needles could be used to continuously carry drugs into the body using simple diffusion or a pump system.
  • 5. Hollow microneedles could be used to remove fluid from the body for analysis – such as blood glucose measurements – and to then supply microliter volumes of insulin or other drug as required .
  • 6. Very small microneedles could provide highly targeted drug administration to individual cells.
  • 7. These are capable of very accurate dosing, complex release patterns, local delivery and biological drug stability enhancement by storing in a micro volume that can be precisely controlled
slide26

APPLICATIONS OF MICRONEEDLES

  • Blood glucose measurements
  • As stated previously microneedles can be fabricated to only penetrate the 10-15 μm of the skin.
  • This means there is no pain when taking blood samples for glucose measuring devices. There is a huge market in glucose testers due to diabetic patients and hospitals.
  • Kumetrixs is an example of a company that fabricates such a device. The micro-needle is penetrating to the skin and draws a very small volume of blood (less than 100 nanoliters) into the disposable.
  • Chemical reagents in the disposable react with the glucose in the blood to produce a color.
  • The blood-glucose concentration will be measured either electrochemically or optically, and the resultant value displayed on the monitor.
slide27

TRANSDERMAL DRUG DELIVERY

  • Since microneedles that are long enough and robust enough to penetrate across this layer, but short enough to not stimulate the nerves in the deeper tissue, have the potential to make transdermal delivery a painless and much more viable option .
  • With the use of hollow microneedles it allows the delivery of medicines, insulin, proteins, or nanoparticles that would encapsulate a drug or demonstrate the ability to deliver a virus for vaccinations .
  • An array of needles ranging from 300-400 needles can be designed to puncture the skin and deliver the drug.
slide28

Molecular and cell biology

  • Microneedles have been applied for the delivery of membrane impermeable molecules into cells.
  • For application in molecular cell biology, methods for the delivery of peptides, proteins, oligonucleotides,
  • Arrays of microneedles were fabricated and utilized to deliver DNA into plant and mammalian cells, as a method for transforming cells
  • Target drug delivery
  • Additionally, microneedles have been utilized to target drug delivery to a specific region or tissue in the body, thus avoiding detrimental effects that can result from administering certain drugs systemically.
  • This targeting can reduce side effects, minimize the dose of an expensive drug, and/or provide a means of delivery to a location that is difficult to treat.
  • For instance, a multichannel silicon microneedle has been microfabricated to deliver bioactive compounds into neural tissue while simultaneously monitoring and stimulating the neurons in vivo.
slide29

Future applications; microneedle skin therapy

  • Skin Microneedling is a collagen induction / transdermal drug delivery treatment for skin rejuvenation, scar repairing, depigmentation and other skin condition improvement.
  • Microneedle roller (or meso-roller) is a roller with numerous microneedles that penetrate the skin layer to 1) stimulate the wound healing process so that it induces new collagen and elastin and to 2) create clear routes to increase absorption of cosmetics or medical ingredients.
  • These channels should close up within an hour. Many clinical studies have proven that skin microneedling is more effective or as powerful as others including laser resurfacing, chemical peels or dermabrasion
slide30

CURRENT REASERCH IN MICRONEEDLE TECHNOLOGY

  • Several new and interesting microneedle concepts have been recently proposed which may find great utility in the future. For example, biodegradable polymer microneedles have recently been fabricated and characterized.
  • Gill et al (2007) have been studied on coating of Microneedle. A novel micron-scale dip-coating process was designed to reliably produce uniform coatings on both individual and arrays of microneedles. This process was used to coat compounds including calcein, vitamin B, bovine serum albumin and plasmid DNA.
  • Recently Lee et al (2008) has studied on dissolving microneedles for transdermal drug delivery. This study presents a design that encapsulates molecules within microneedles that dissolve within the skin for bolus or sustained delivery and leave behind no biohazardous sharp medical waste.
slide32

CONCLUSION

  • Molecular Tool: Electroporation
  • In molecular biology, the process of electroporation is often used for the transformation of bacteria, yeast, and plantprotoplasts.
  • This procedure is also highly efficient for the introduction of foreign genes in tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy.
slide33

Microneedles : The option for painless delivery

  • Many people, particularly children, are ‘needle-phobes’. In addition, there are several patients, such as diabetics who are dependant on multiple injections on a daily basis.
  • A solution to the problems posed by needle-based injections is the development of microneedles. This technology will help realise the development of new and improved devices, which will be smaller, cheaper, pain-free and more convenient with a wide range of biomedical and other applications.
  • The future of drug delivery is assured to be significantly influenced by microfabrication technologies.
  • These microfabricated drug delivery devices can enable efficient drug delivery that was unattainable with conventional drug delivery techniques, resulting in the enhancement of the therapeutic activity of a drug.
  • It is not difficult to imagine that microneedle system can be easily combined with micro electronic elements which can fully control the delivery rate . It can be envisioned that such a “pharmacy on a chip” may be the future of drug delivery.
slide34

REFERENCES

  • J. C. Weaver and Y. A. Chizmadzhev."Theory of electroporation: A review " Biochemistry and Bioenergetics. 41. (1996) 135-160.
  • K. C. Melikov, V. A. Frolov, A. Shcherbakov, A. V. Samsonov, Y. A. Chizmadzhev and L. V. Chernomordik."Voltage-Induced Nonconductive Pre-Pores and Metastable Single Pores in Unmodified Planar Lipid Bilayer " Biophysical Journal. 80. (2001) 1829-1836.
  • R. P. Joshi and K. H. Schoenbach."Electroporation dynamics in biological cells subjected to ultrafast electrical pulses: A numerical simulation study." Physical Review E. 62. (2000) 1025-1033.

4.Prausnitz MR et al. 1993. Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery. Proc NatlAcadSci USA 90: 10504-8.

5. Saito, Tetsuichiro. 2001. In Vivo Electroporation. <http://www.frontier.kyoto-u.ac.jp/rc01/in_vivo_electroporation.html> Accessed 2003 15 Feb.

6. Weaver JC. 1995. Electroporation Theory: Concepts and Mechanisms. In: Nickoloff JA, editor. Electroporation Protocols for Microorganisms. Totowa, New Jersey: Humana Press. p 1-26.

7.Inovio. 2002. Technology Platform: Electroporation. <http://www.inovio.com/technology_electroporation.shtml> Accessed 2003 15 Feb

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8. S Henry, D V McAllister, M G Allen and M R Prausnitz, MicrofabricatedMicroneedles: A Novel Approach to Transdermal Drug Delivery, Journal of Pharmaceutical Sciences, 1998, 87: 922-925.

9. Microneedles: Report Describes Progress in Developing New Technology for Painless Drug and Vaccine Delivery, Georgia Research Tech News. (2003

  • 10. McAllister, D V Allen, M G Prausnitz, M R. Microfabricatedmicroneedles for gene and drug delivery. Annu. Rev. Biomed. Eng.2000, 2: 289–313.
  • 11. Trimmer W, Ling P, Chin C K, Orten P, Gaugler R, Hashmi S, Hashmi G, Brunett B, Reed M. Injection of DNA into plant and animal tissues with micromechanical piercing structures. In: Proceedings of the IEEE Microelectromechanical Systems Workshop 8th, Amsterdam, 1995: 111–115.
  • 12. J Zachary Hilt, Nicholas A. PeppasMicrofabricated drug delivery devices International Journal of Pharmaceutics, 2005,306: 15–23
  • 13. Meidan Victor M, MichniakBozena B. Emerging Technologies in Transdermal Therapeutics. American Journal of Therapeutics, 2004, 11(4): 312-316.
  • 14. Microneedles Give Painless Shots, Technology Research News.http://www.technologyreview.com/articles/rnb_112503.asp. (2003)