Biomedical Engineering Reference
In-Depth Information
that. a. number. of. different. factors,. including. salt. concentration,. pH,. and. temperature. of.
the. vaccine,. may. all. affect. the. eficiency. of. DNA. transfection.
20
. Nevertheless,. this. study.
raised.concerns.and.gave.credence.to.the.argument.that.ineficient.DNA.uptake.is.limit-
ing.vaccine.immunogenicity.in.humans..But.perhaps.the.more.important.observation.that.
Dupuis.et.al..made.was.that.electroporation.of.the.tissue.immediately.following.injection.
could. ameliorate. the. decrease. in. immune.response. observed. with. DNA. injection. alone..
Electroporation,.which.increases.DNA.uptake,.improved.gene.expression.of.the.vaccine.
and.augmented.both.cellular.and.humoral.immune.responses.
14.3 ElectroporationofDNAVaccines
14.3.1 Mechanism of Electroporation
In.1982,.Neumann.et.al..demonstrated.that.pulsing.murine.cells.with.electric.impulses.(8.
kB/cm,.5.microseconds).dramatically.increased.the.uptake.of.linear.or.circular.plasmids.
in.culture.
21
.This.protocol.was.termed.electroporation..Neumann.and.colleagues.proposed.
the.development.of.large.hydrophobic.pores.via.alterations.to.the.transmembrane.voltage.
induced.by.the.electric.pulse.allowed.for.DNA.to.be.taken.up.with.increased.eficiency..
The.enlarged.pores.become.hydrophilic.by.rotation.of.lipids.between.the.aqueous.medium.
and.the.lipid.bilayer,.allowing.for.passage.of.normally.impermeable.macromolecules.such.
as.DNA..A.primary.limitation.with.this.model,.however,.is.that.evidence.for.these.stable.
hydrophilic.pores.is.lacking..To.date,.there.is.not.a.proven.mechanism.for.electroporation,.
although.the.process.likely.involves.the.reversible.change.in.the.impermeability.of.the.cell.
membrane.to.allow.objects,.such.as.drugs,.nucleic.acids,.and.antibodies,.to.enter.the.cell..
A.point.of.highlight,.however,.is.that.electroporation.is.a.reversible.process.inasmuch.as.
electric.pulse-induced.alternations.to.the.cell,.such.as.permeability.of.ions,.are.transient..
This.is.clearly.distinct.from.irreversible.electroporation,.which.leads.to.cell.death.follow-
ing.permanent.changes.to.membrane.physiology.
22
Although.more.details.need.to.be.illed.in,.a.working.model.for.electroporation.has.been.
proposed;.Teissie.et.al..have.described.a.series.of.events.or.steps.that.are.involved.in.elec-
tropermeabilization.
23
.The.irst.is.the.trigger.step,.whereby.the.external.ield.induces.an.
increase.in.the.transmembrane.potential.up.to.the.critical.permeabilizing.threshold..This.
is. followed. by. the. expansion. step,. where. a. time-dependent. membrane. transition. takes.
place.as.long.as.the.ield.is.maintained.at.an.overcritical.value..In.the.stabilization.phase,.
the.membrane.organization.is.recovered.as.soon.as.the.ield.is.subcritical,.leading.to.the.
resealing.and.memory.steps..In.these.steps,.membrane.permeability.recovers.to.its.prior.
state,.commencing.the.reversing.process..The.recovery.and.reversion.back.to.a.prior.state.
are.critical.to.induce.homeostasis.and.prevent.cell.death..(See.Teissie.et.al.
23
.for.more.thor-
ough.and.mechanistic.insight.into.this.process.).
14.3.2 Increased Immunogenicity with Electroporation
In.1990,.Wolff.et.al..made.the.seminal.observation.that.injection.of.naked.DNA.was.suficient.
to.induce.gene.expression.in.vivo.
24
As.mentioned.previously,.this.observation.then.paved.
the. way. for. DNA. plasmids. that. encode. various. antigens. to. induce. immune. responses..
