Biomedical Engineering Reference
In-Depth Information
group.and.hydrocarbon.cross-sectional.areas,.i.e.,.of.cylindrical.shape,.prefer.to.form.bilayers.
(Figure.24.4b)..Meanwhile,.lipids.with.a.small.head.group.and.bulky.cis-unsaturated.hydro-
carbon.groups.favor.the.H
II
.phase.(Figure.24.4c;.Hafez.and.Cullis.2001,.Hafez.et.al..2001)..
Poly-cis-unsaturated.lipids.are.the.most.effective.in.this.regard..For.instance,.cationic.lipids.
with.C18:2.alkyl.chains.have.been.shown.to.have.higher.transfection.eficiency.than.compa-
rable.cationic.lipids.with.C18:1.chains.(Heyes.et.al..2005,.Huang.and.Liu.2011).
As.shown.by.cryo-TEM.and.small-angle.x-ray.scattering.(SAXS),.the.siRNA.LNPs.exhibit.
a.lamellar.liquid.crystalline.phase.L
α
.(Pozzi.et.al..2009)..The.L
α
.structure.is.required.for.sta-
bility.during.in.vitro.culture.and.in.vivo.systemic.administration,.while.the.H
II
.structure.is.
required.for.escaping.the.endosome..Therefore,.the.key.for.designing.new.cationic.lipids.is.to.
enable.eficient.transition.from.L
α
.to.H
II
.inside.the.endosome.in.response.to.the.change.in.pH.
Parameters. that. determine. the. structure. and. stability. of. siRNA-LNPs. include. the.
selection.of.the.cationic.and.helper.lipids.as.well.as.the.lipid-to-siRNA.ratio.(Niculescu-
Duvaz.et.al..2003,.Pozzi.et.al..2009)..For.example,.formulations.containing.the.helper.lipid.
DOPE. usually. give. high. transfection. eficiency. because. DOPE. is. able. to. promote. the.
transformation.to.the.inverted.hexagonal.structure,.H
II
.(Ma.et.al..2007,.Pozzi.et.al..2009)..
The.transfection.eficiency.of.LNPs.usually.increases.with.the.increase.in.weight.fraction.
of.DOPE.in.the.formulation..However,.high.DOPE.content.reduces.particle.stability.
Furthermore,.controlling.the.number.of.layers.of.multilamellar.structure.is.an.alternative.
strategy.to.improve.transfection.eficiency..For.endosome.escape.or.membrane.fusion.of.
the.LNP.with.multilamellar.layers,.it.may.be.necessary.to.“peel.off”.each.layer.to.release.
the. entire. payload. of. siRNAs,. which. is. an. ineficient. process.. A. great. deal. of. siRNA.
payload.may.remain.trapped.within.the.interior.lamellar.layers.of.the.lipoplex,.leading.to.
lysosomal.degradation..To.reduce.lamellarity,.LNPs.with.a.“core-shell”.structure.may.be.
used.instead.of.LNPs.with.an.“onion-like”.structure.
24.8.3 Proton Sponge Model
After.internalization.by.endocytosis,.the.endosome.acidiies.and.amine.groups.on.LNPs.
that.have.a.pKa.in.this.range.(typically.between.5.and.7).become.protonated..The.inlux.
of. additional. protons. as. well. as. chloride. ions. follows.. The. uptake. of. ions. results. in.
osmotic. swelling. and. rupture. of. the. endosome. membrane.and. the. release. of. its. content.
in.the.cytoplasm.(Dominska.and.Dykxhoorn.2010,.Huang.and.Liu.2011)..There.are.many.
materials.that.have.amines.with.pKa.values.in.the.range.of.5-7,.such.as.polyethylenimine.
(PEI).and.β-amino.esters..The.tertiary.amines.found.on.ionizable.cationic.lipids.also.have.
a.pKa.in.this.range.(Heyes.et.al..2005,.Semple.et.al..2010).
In.fact,.the.cationic.siRNA-LNPs.may.utilize.two.or.three.endosomal.escape.mechanisms.
to. facilitate. release. of. siRNA.. The. concept. of. incorporating. an. ionizable. cationic. lipid.
with.a.tertiary.amine.head.group.is.highlighted.in.SNALP.technology.(Heyes.et.al..2005,.
Zimmermann.et.al..2006)..In.this.case,.the.positive.charge.density.of.the.LNP.is.minimal.at.
the.pH.of.the.culture.medium.or.blood.circulation,.but.increases.substantially.in.the.acidic.
environment. once. the. LNP. enters. the. endosome.. The. membrane-destabilizing. property.
of. the. LNP. is. signiicantly. increased. via. protonation. of. tertiary. amines. and. associated.
increases.in.charge.intensity.
24.8.4 Structure-Function relationship Study
Signiicant.efforts.are.being.made.to.better.understand.the.structure-activity.relationship.
(SAR). of. the. cationic. lipids.. Surface. charge. can. be. characterized. via. zeta. (ζ). potential.
