Biomedical Engineering Reference
In-Depth Information
8.4 Trastuzumab-ConjugatedNanoparticles
ofPLA-TPGS/TPGS-COOHCopolymerBlend
In. this. section,. we. report. a. strategy. developed. in. our. earlier. research. on. how. to. pre-
pare. nanoparticles. of. biodegradable. novel. copolymers. for. targeted. chemotherapy,. by.
which.the.targeting.effect.can.be.controlled..The.NP.formulation.consisted.of.a.blend.of.
two-component.copolymers..One.is.poly(lactide)-D-α-tocopheryl.polyethylene.glycol.suc-
cinate. (PLA-TPGS),. which. is. of. a. desired. hydrophobic-lipophilic. balance. (HLB). and. can.
thus.result.in.high.drug.encapsulation.eficiency.and.high.cellular.adhesion/adsorption..
Another.is.carboxyl.group-terminated.TPGS.(TPGS-COOH),.which.plays.the.role.of.linker.
molecules.when.appearing.on.the.NP.surface..This.combination.provides.a.simple.tech-
nique.for.conjugation.of.the.molecular.probes.on.the.NP.surface..Moreover,.the.targeting.
effect.can.be.quantitatively.controlled.by.adjusting.the.copolymer.blend.ratio..Docetaxel.
was.used.as.a.prototype.drug;.it.is.a.hydrophobic.anticancer.drug.and.has.excellent.thera-
peutic. effects. against. a. wide. spectrum. of. cancers,. such. as. breast. cancer,. non-small-cell.
lung.cancer,.ovarian.cancer,.and.head.and.neck.cancer.
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.Docetaxel.is.semisynthetic.analog.
of.paclitaxel,.but.more.effective.as.an.inhibitor.of.microtubule.depolymerization.due.to.its.
ability.to.alter.tubulin.processing.within.the.cells..Moreover,.there.is.a.synergistic.antitu-
mor.effect.when.trastuzumab.is.combined.with.docetaxel.
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Docetaxel-loaded.nanoparticles.of.a.PLA-TPGS.and.TPGS-COOH.blend.were.prepared.
by.a.modiied.solvent.extraction/evaporation.method..Four.levels.of.the.mass.ratio.between.
PLA-TPGS.and.TPGS-COOH.were.used.in.this.research.(1:0,.9:1,.4:1,.and.2:1),.for.which.the.
weight.amounts.of.TPGS-COOH.in.the.blend.were.0,.10,.20,.and.33.3%..In.brief,.10.mg.of.
docetaxel.and.100.mg.of.the.PLA-TPGS/TPGS-COOH.blend.at.various.weight.ratios.were.
dissolved.in.8.ml.of.dichloromethane.(DCM)..The.solution.formed.was.poured.into.120 ml.
of. aqueous. phase. solution. containing. 0.03%. (w/v). TPGS. as. the. emulsiier. under. gentle.
stirring,.and.sonicated.at.25 W.output.for.120 s..The.emulsion.was.evaporated.overnight.
and.then.centrifuged.at.10,500.rpm.for.20.min..The.pellet.was.resuspended.in.water.and.
freeze-dried.for.2.days..Hereinafter,.the.docetaxel-loaded.PLA-TPGS/TPGS-COOH.NPs.of.
0,.10,.20,.and.33.3%.TPGS-COOH.are.termed.NP0,.NP10,.NP20,.and.NP33,.respectively,.and.
the.NPs.with.no.trastuzumab.conjugated.are.called.nude.NPs.
Before. trastuzumab. conjugation,. the. docetaxel-loaded. PLA-TPGS/TPGS-COOH. NPs.
were.irst.activated.according.to.a.procedure.similar.to.that.described.by.Wuang.et.al.,
91
.
where.polypyrrole.nanoparticles.were.synthesized.to.formulate.iron.oxides.(IOs).for.tar-
geted.MRI..In.brief,.10.mg.of.the.PLA-TPGS/TPGS-COOH.NPs.were.dispersed.in.4.ml.of.
deionized. (DI). water,. followed. by. adding. 20. mg. of. 1-ethyl-3-(3-dimethylamino)-propyl.
carbodiimide.(EDC).and.8.mg.of.N-hydroxysuccinimide.(NHS)..One.milligram.of.N-Boc.
ethylenediamine.was.added.to.introduce.amine.groups.to.the.NPs..The.pH.of.the.reaction.
mixture.was.adjusted.to.8.with.triethylamine..After.4.h.of.reaction.at.room.temperature,.
the.mixture.was.centrifuged,.washed.with.DI.water,.and.dried.under.reduced.pressure..
Then.the.product.was.treated.with.triluoroacetic.acid.(TFA).at.0°C.for.30.min.to.remove.
N-Boc.protection..After.evaporation.of.TFA,.the.product.was.washed.with.DI.water.and.
dried.. Trastuzumab. was. then. conjugated. to. the. NPs. by. using. carbodiimide. chemistry..
The.NPs.were.dispersed.in.2.ml.of.0.1.M.PBS.and.placed.in.an.ultrasonic.bath.for.30.min..
Then. 10. mg. of. NHS. and. 50. mg. of. EDC. were. added.. After. that,. 500. µl. of. trastuzumab.
(10.mg/ml).was.added.for.reaction.and.triethylamine.was.used.to.adjust.the.pH.of.the.
reaction.mixture.to.8..After.4.h.reaction.at.room.temperature,.the.resulting.product.was.
