Biomedical Engineering Reference
In-Depth Information
speciic.transactivator.binding.sequence.(TABS).on.RNA-1.that.causes.a.structural.change.
in.the.RNA.complex,.recognized.by.the.capsid.protein.(Basnayake.et.al..2009).
The.RCNMV.capsid.structure.is.affected.by.both.pH.and.the.concentration.of.Mg
2+
/Ca
2+
.
ions..At.low.pH.(below.pH.6.5).the.diameter.is.∼36.nm.whereas.in.neutral.pH.(pH.∼6.5-8).
the.capsid.swells.to.∼45.nm..In.basic.solutions.above.pH.8,.the.virus.capsid.starts.to.disas-
semble..In.its.native.form,.the.capsid.can.bind.several.hundred.atoms.of.Ca
2+
.and.Mg
2+
,.
respectively..Removing.the.divalent.ions.causes.a.major.structural.change.in.the.capsid,.
which. leads. to. the. formation. of.∼1.3.nm. pores. at. each. trimer. axis. (Sherman. et. al.. 2006)..
This.ion-dependent.shape.change.is.thought.to.be.part.of.the.natural.function.of.the.virus..
It.is.transmitted.through.soil,.where.the.concentration.of.divalent.cations.is.in.the.milli-
molar.range..Upon.infection.the.virus.enter.the.cell.cytoplasm.that.has.a.low.concentration.
(micromolar),.causing.Mg
2+
/Ca
2+
.to.leach.from.the.capsid..The.loss.of.ions.causes.a.shape.
change.that.aids.the.release.of.RNA..The.structural.change.is.reversible.where.the.pores.
close.when.increasing.the.concentration.of.divalent.ions.
7.7.2 Drug Delivery
The. structural. dependency. of. RCNMV. on. divalent. cations. and. pH. can. be. leveraged. as.
a.strategy.for.triggered.loading/release.of.small.molecules..Franzen.et.al..demonstrated.
successful.loading.of.small.luorescent.molecules,.including.the.cancer.drug.doxorubicin.
(DR).(Loo.et.al..2008)..Initial.treatment.of.the.virus.with.200.mM.EDTA.at.pH.8.were.used.
to.form.pores.in.the.capsid,.followed.by.incubating.with.dyes.at.high.dye/capsid.molar.
ratio..Once.loaded,.the.solution.was.dialyzed.against.a.solution.with.200.mM.Ca
2+
.at.pH.
6.to.close.the.pores.and.entrap.the.molecular.cargo..They.noted.that.while.dye.molecules.
could. be. encapsulated. at. ratios. about. 70-90. molecules. per. capsid,. several. thousand. DR.
molecules.could.be.contained..The.DNA.intercalating.properties.of.DR.were.thought.to.be.
the.reason.for.this.higher.loading.eficiency.
RCNMV.can.be.used.for.drug-delivery.applications.if.they.can.be.targeted.to.speciic.cells..
Recently,.Lockney.et.al..used.the.heterobifunctional.chemical.linker.sulfosuccinimidyl-4-
(N-maleimidomethyl)cyclohexane-1-carboxylate.(Sulfo-SMCC).to.couple.100-220.copies.of.
a.CD46.receptor-targeting.peptide.to.the.capsid.exterior..Combining.the.peptide-targeting.
approach.with.cation-dependent.loading.of.DR.allowed.successful.delivery.of.DR-loaded.
RCNMV. particles. to. HeLa. cancer. cells. (Lockney. et. al.. 2011).. The. authors. noted. that. the.
SMCC. approach. caused. some. dimerization/aggregation. and. suggested. other. coupling.
strategies,.such.as.“click”.chemistry,.which.might.provide.more.homogenous.nanoparticle.
distribution.
7.7.3 Material Templating
The.RNA-dependent.assembly.of.the.RCNMV.capsid.can.be.leveraged.for.triggered.encap-
sulation. of. nanoparticles. (NPs).. Loo. et. al.. (2006). demonstrated. that. gold. NPs. of. various.
sizes.can.be.“packaged”.inside.the.capsid.using.this.approach..They.irst.immobilized.a.
thiolated.20-nucleotide.DNA.to.gold.NPs.that.was.based.on.the.RNA-2.stem.loop.sequence.
and.added.RNA-1.to.obtain.an.“origin.of.assembly.”.Subsequently,.they.introduced.puri-
ied.capsid.protein.(prepared.at.pH.9).and.dialyzed.against.pH.5.5.buffer,.which.triggered.
assembly.around.the.particle.(Figure.7.14)..The.reassembly.was.performed.at.pH.5.5,.which.
resulted.in.the.most.“native-like”.capsids..They.noted.that.5,.10,.and.15.nm.gold.particles.
were.encapsulated.while.20.nm.particles.were.not..The.interior.capsid.core.is.∼17.nm.and.
it. was. hypothesized. that. assembly. around. larger. nanoparticles. prevented. formation. of.
