Transcript PHYSICAL ENHANCEMENT techniques

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
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ELECTROPORATION
DEFINITION
BACKGROUND
PROCEDURE
PHENAMENON OF ELECTROPORATION
ADVANTAGES AND DISADVANTAGES
APPLICATIONS
 MICRONEEDLES
• DEFINITION
• ADVANTAGES
• APPLICATIONS
• CURRENT REASEARCH IN MICRONEEDLE
TECHNOLOGY
 CONCLUSION
 REFERENCES
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
ELECTROPORATION
MICRONEEDLES
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.
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
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
 Diagram of the major components of an
electroporator with cuvette loaded
Laboratory Practice
Electroporation Cuvettes
Electroporators
Benchtop 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
They can also be set to different
operating parameters depending on
whether or not the cell has a cell
wall
Handheld electroporators
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.
Pipectrode
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).
INOVIO’S ELECTROPORATION TECHNOLOGY
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
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)
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
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 corneumthe 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 (Praustnitz
et. al., 1993).
 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).
Fig. Diagram of the method of gene therapy using electroporation.
 MICRONEEDLES : A Novel Approach to
Transdermal Drug Delivery
• A microstructured transdermal
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
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.
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)
In general terms, make needles that:
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•
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Go into skin easily
Deliver drugs effectively
Don’t hurt
Are biocompatible
The needles need to:
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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
Figure : (a) A microformed microneedle 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
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.
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
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
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.
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.
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.
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
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.
DISSOLVING MICRONEEDLE PATCH
CONCLUSION
Molecular Tool: Electroporation
In molecular biology, the process of electroporation is often used for the
transformation of bacteria, yeast, and plant protoplasts.
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.
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.
REFERENCES
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