Biomedical Engineering Reference
In-Depth Information
measurement..Particle.size.is.typically.measured.through.dynamic.light.scattering.(DLS)..
The.morphology.can.be.determined.by.cryo-TEM..Physical.properties.are.also.measured.
by. small-angle. x-ray. scattering. (SAXS),. differential. scanning. calorimetry. (DSC),. and.
isothermal.calorimetry.(ITC).
Cryo-TEM.is.an.electron.microscopy.technique.that.uses.vitriied.sample.ilm.obtained.
by.rapidly.freezing.the.sample.ilm.(−196°C).on.a.carbon-coated.grid..It.is.an.invaluable.tool.
for.the.study.of.nanostructures.formed.by.lipids/siRNAs.in.aqueous.solutions..Lamellar.
complexes.(bi-.or.multi-).account.for.the.majority.of.nanostructures.of.siRNA-based.LNPs.
reported.in.the.literature..The.lamellarity.is.mainly.dependent.on.the.nucleic.acid-to-lipid.
ratio. (Semple. et. al.. 2001).. Furthermore,. there. are. substantial. similarities. between. LNP.
structures.incorporating.ODN,.siRNA,.or.DNA.(Thierry.et.al..2009)..The.process.of.particle.
formation.for.the.“onion”-like.multilamellar.nanostructures.by.the.“zipper”.effect.model.
is.illustrated.by.Dr..Yeshayahu.Talmon's.group.(Weisman.et.al..2004).
The.roles.of.head.group.design.and.pKa,.hydrocarbon.length,.degree.of.unsaturation,.
and.asymmetry.in.bilayer.disruption.activity.have.been.studied.systematically.to.establish.
SARs.. Overall,. biological. data. have. correlated. well. with. predictions. from. the. molecular.
“shape”.analysis.(Hafez.and.Cullis.2001)..Further.studies.are.necessary.to.establish.the.SAR.
between. the. nanostructure. of. siRNA-LNPs. and. transfection.. Several. novel. technologies.
for. characterizing. intracellular. traficking. pathways,. such. as. Q-dot/FRET,. may. provide.
new.insights.to.this.important.problem.(Wu.et.al..2011).
24.8.5 Advances in Novel Cationic Lipids
A.cationic.lipid.may.be.separated.into.three.functional.moieties:.long.hydrocarbon.chains,.
a.linker,.and.an.amine-based.head.group..The.overall.property.of.a.cationic.lipid.arises.
from.the.design.of.these.three.moieties..Due.to.the.complexity.of.the.delivery.pathway,.no.
precise.schemes.can.be.deduced.to.correlate.the.cationic.lipid.chemistry.with.transfection.
eficacy..Therefore,.the.optimization.of.molecular.structures.for.cationic.lipids.is.still.largely.
an.empirical.exercise..A.large.number.of.cationic.lipids.have.been.synthesized.for.nucleic.
acid.delivery.systems.(Bhattacharya.and.Bajaj.2009,.Montier.et.al..2008)..A.typical.in.vivo.
delivery.system.consists.of.cationic.lipids,.neutral.lipids.(e.g.,.cholesterol).and.PEG-lipids..
For.example,.the.“stable.nucleic.acid.lipid.particles”.(SNALP).formulation.is.composed.of.
ionizable.cationic.lipids.such.as.1,2-dioleyloxy-
N
,
N
-dimethyl-3-aminopropane.(DODMA).
and.1,2-dilinoleyloxy-3-dimethyl.aminopropane.(DLinDMA),.a.diffusible.PEG-lipid.and.
cholesterol..Ionizable.cationic.lipids.with.pK
a
.<.7.0.are.able.to.eficiently.encapsulate.nucleic.
acids.at.low.pH.during.LNP.synthesis,.and.yield.LNPs.with.neutral.or.low.zeta.potential.
at.pH.7.4,.i.e.,.the.physiological.pH.(Heyes.et.al..2005)..The.degree.of.saturation.of.cationic.
lipids. affects. lipid. pK
a
,. fusogenicity,. cellular. uptake,. and. gene. silencing. activity.. It. was.
found.that.a.lipid.with.linoleyl.moieties.containing.two.cis.double.bonds.per.hydrocarbon.
chain.(DLinDMA).was.very.active.for.transfection..Cationic.lipids.with.a.high.fusogenic.
activity.generally.lead.to.LNPs.with.a.higher.siRNA.transfection.eficiency.(Heyes.et.al..
2005,.Semple.et.al..2010).
Semple. et. al.. (2010). synthesized. a. series. of. cationic. lipids. based. on. DLinDMA,. with.
the. aim. of. inding. the. optimal. linker. between. head. groups. and. two. hydrophobic. lipid.
chains..Keeping.the.linoleyl.chains.constant,.they.found.that.the.introduction.of.a.ketal.
ring.linker.signiicantly.increased.activity.in.vivo..The.best-performing.lipid,.DLin-KC2-
DMA,.remarkably.reduced.the.half-maximal.effective.dose.(ED
50
).of.siRNA.from.∼1.0.to.
∼0.03.mg/kg. in. rodents. (Semple. et. al.. 2010).. Meanwhile,. Anderson. et. al.. took. a. differ-
ent. approach. to. screen. novel. lipid-like. materials. termed. “lipidoids”. (Akinc. et. al.. 2008,.
