Biomedical Engineering Reference
In-Depth Information
through.a.diazonium.coupling.reaction.(Hooker.et.al..2004)..This.reaction.can.take.place.
in. empty. capsids. after. RNA. hydrolysis. and. results. in. >95%. coupling. eficiency.. Kovacs.
et.al..showed.the.feasibility.of.this.approach.in.2007.by.modiication.of.the.interior.empty.
capsids. with.∼60. luorescent. dye. molecules. conjugated. to. Tyr85.. Lysine. residues. on. the.
capsid. exterior. were. then. modiied. with. PEG. chains. through. N-hydroxysuccinimide.
(NHS).linkages.(Kovacs.et.al..2007)..As.a.proof-of-concept,.the.dual-modiied.MS2.capsids.
were.tested.in.an.ELISA.assay.against.an.anti-MS2.polyclonal.antibody,.and.the.pegylated.
capsids.exhibited.a.90%.reduction.in.binding.
Genetic.modiications.of.MS2.have.also.been.used.for.the.purpose.of.generating.drug.
delivery.constructs..In.2009,.Wei.et.al..developed.an.antisense.RNA.delivery.system.using.
MS2. capsids. by. a. two. plasmid. coexpression. system.. One. plasmid. encoded. for. the. MS2.
maturase.and.coat.protein,.and.the.other.encoded.for.the.operator.sequence.alongside.the.
antisense. RNA. for. 5′-untranslated. region. (UTR). and. internal. ribosome. entry. site. (IRES).
of.the.hepatitis.C.virus.(HCV).(Wei.et.al..2009)..These.antisense-carrying.MS2.“virus-like.
particles”.(VLPs).were.delivered.to.cells.containing.an.HCV-luciferase.reporting.system.by.
conjugating.the.human.immunodeiciency.virus.(HIV-1).TAT.peptide.to.lysine.residues.on.
the.capsid.exterior.via.a.succinimide-based.heterobifunctional.cross-linker..Their.results.
showed.a.decrease.in.luciferase.activity.in.Huh-7.cells.with.increasing.concentrations.of.
TAT.decorated.MS2.VLPs.loaded.with.HCV.antisense.RNAs.
In. addition. to. peptides,. DNA. aptamers. can. also. be. conjugated. to. the. exterior. of. MS2.
capsids. to. act. as. a. targeting. ligand.. A. nucleotide. sequence,. 41. base. pairs. in. length. that.
binds. to. a. tyrosine. kinase. receptor. on. Jurkat. T. cells,. has. been. chemically. conjugated. to.
lysine.residues.on.the.MS2.capsid.exterior.(Tong.et.al..2009)..A.luorescent.dye.(AlexaFluor.
488). could. be. attached. to. genetically. engineered. cysteine. residues. (N87C). on. the. capsid.
interior..With.this.dual-modiication.approach,.speciic.uptake.of.dye-labeled.MS2-aptamer.
conjugates.in.cells.was.demonstrated..In.2010,.Stephanopoulos.et.al..used.the.same.aptamer.
sequence;.however,.in.this.case,.the.interior.of.the.capsid.was.modiied.with.sulfonated.
porphyrins.(Figure.7.8)..These.porphyrin-loaded.MS2.capsids.were.used.to.show.targeted.
photodynamic.therapy.(PDT),.by.illuminating.the.Jurkat.cells.after.MS2-porphyrin.uptake.
with.415.nm.light..The.encapsidated.porphyrins.generated.reactive.oxygen.species.(ROS).
upon.illumination,.and.resulted.in.generating.necrosis.and.death.in.almost.80%.of.the.cell.
population.(Stephanopoulos.et.al..2010b).
As.an.alternative.to.chemical.and.genetic.modiications.for.the.placement.of.cargo.inside.
viral.capsids,.it.is.also.possible.to.load.capsids.by.a.charge-interaction.approach..Recent.
results. reveal. that. native. MS2. capsids. eficiently. can. be. loaded. with. approximately. 250.
positively. charged. porphyrins.. In. this. case,. the. porphyrins. are. able. to. enter. the. capsid.
pores. and. interact. with. the. negatively. charged. nucleic. acid. backbone. coiled. inside. the.
capsid.(Cohen.et.al..2012).
7.4.3 Diagnostics
The.spherical.structure.and.multivalency.of.MS2.capsids.make.them.suitable.candidates.
for.delivery.of.contrast.agents.for.use.in.diagnostics..VLPs.can.be.modiied.on.the.interior.
or.exterior.with.luorophores,.quantum.dots,.or.magnetic.resonance.imaging.agents..As.
with. the. delivery. of. therapeutic. molecules,. capsid. alteration. is. advantageous. to. attach.
multiple.contrast-generating.compounds.with.a.single.modiication.step.
Preliminary. research. using. MS2. as. a. carrier. of. contrast. agent. involved. chemical.
conjugation.of.the.gadolinium-based.MRI.contrast.agent.Magnevist
®
.to.each.of.the.three.
lysine. residues. on. the. exterior,. leading. to. approximately. 360. gadolinium. ions. per. viral.
