Biomedical Engineering Reference
In-Depth Information
resulted.in.improved.LNP.size.control.and.enhanced.biological.activity..Moreover,.we.have.
reported.a.novel.single-step.method.for.synthesis.of.ODN.LNPs.by.rapid.evaporation.of.
ethanol.using.a.coaxial.electrospray.device.(Wu.et.al..2009).
The. resulting. LNP. particles. generally. have. onion-like. multilamellar. structures. based.
on.results.from.cryo-transmission.electron.microscope.(cryo-TEM).(Weisman.et.al..2004,.
Koh.et.al..2010,.Yang.et.al..2009).images..The.mechanism.of.formation.of.these.“onion”-like.
nanostructures.is.illustrated.by.a.“zipper”.effect.model.(Weisman.et.al..2004)..According.to.
this.model,.the.negatively.charged.oligonucleotides.act.as.bridges.between.adjacent.lipid.
bilayers.containing.a.cationic.lipid..Although.our.newly.developed.MHF.method.has.been.
shown.to.produce.more.uniform.and.smaller.nanoparticles,.the.onion-like.nanostructures.
still.remained.(Koh.et.al..2010).
24.6 Rational Design of Cationic Lipids
siRNA-based. therapeutics. require. delivery. of. siRNAs. into. the. cytoplasm.. siRNA-LNPs.
need.to.be.capable.of.cellular.binding,.transport.to.the.endosome,.escape.of.siRNAs.from.
the.endosome,.and.loading.into.RISC..To.achieve.the.highest.cellular.uptake,.slightly.posi-
tively.charged.LNPs.are.desirable..Endocytosis.is.the.most.common.pathway.for.the.cel-
lular.entry.of.LNPs.(Hillaireau.and.Couvreur.2009,.Sahay.et.al..2010)..siRNA-LNPs,.upon.
internalization,. become. entrapped. in. early. endosomes. (pH. 6.5),. followed. by. late. endo-
somes.(pH.5.5).and.lysosomes.(pH.4.5).where.they.can.be.degraded..In.order.to.avoid.deg-
radation,.it.is.important.for.the.siRNA.molecules.to.escape.from.the.early/late.endosomes.
24.7 Endocytosis Pathway for LNPs
The. endocytosis. route. is. considered. the. predominant. pathway. for. the. uptake. of. siRNA-
LNPs.into.cells..Endocytosis.is.further.sub-classiied.into.clathrin-dependent.endocytosis.
(also.known.as.clathrin-mediated.endocytosis.[CME]),.caveolae-mediated.endocytosis,.and.
macropinosome-mediated.endocytosis.(i.e.,.macropinocytosis).(Sahay.et.al..2010)..Generally,.
most. siRNA-LNPs. are. internalized. through. clathrin-mediated. endocytosis.. In. addition,.
cell.type,.particle.size,.shape,.composition,.and.surface.chemistry/charge.may.determine.
the.speciic.route.of.endocytosis..It.should.be.noted,.however,.that.some.LNPs.do.not.enter.
cells. via. CME.. For. example,. Anderson. et. al.. reported. that. the. best-performing. lipidoid,.
C12-200,.did.not.enter.cells.through.the.classical.endocytic.pathway—CME—but.rather.by.
macropinocytosis.(Love.et.al..2010)..It.is.believed.that.in.certain.cell.types,.the.luid.content.
of.macropinosomes.is.not.transported.to.lysosomes,.thereby.avoiding.the.lysosomal.degra-
dation.characteristic.of.the.regular.CME.pathway.(Love.et.al..2010,.Sahay.et.al..2010).
24.8 Mechanism of Endosomal Escape
Upon. endocytosis,. the. cationic. lipids. interact. with. the. anionic. phospholipids. in. the.
endosomal.membrane..The.process.of.endosomal.membrane.destabilization.is.associated.
