Biomedical Engineering Reference
In-Depth Information
Polymers.SucPG.having.56%.Suc.residues.combined.with.long.alkyl.chains.as.anchors.
to eggPC. liposomes. remain. stable. at. neutral. pH. but. induce. strong. fusion. at. pH. 4.0..
Liposomes. bearing. the. polymer. fuse. more. intensively. with. decreasing. pH. and. with. an.
increasing.amount.of.bound.polymer.
136
.A.high.percentage.(early.70-80%).of.Suc.COO
-
.must.
lose.their.negative.charges.by.protonation.to.induce.the.content.release.of.liposomes.
133
EggPC. liposomes. can. be. associated. with. a. maximum. proportion. of. 30%. w/w. of.
SucPG.(PG.mean.Mw.=.4,600.and.calculated.Mw.of.SucPG.=.ca..10,000)..The.uptake.of.
SucPG.liposomes.by.CV-1.cells.is.inversely.proportional.to.the.SucPG.content,.but.the.
calcein.release.into.cytoplasm.is.directly.proportional.to.the.SucPG.content.(30.>.20.>.
10%).
137
.Fusion.assay.(FRET
NBD.to.Rho).suggests.the.occurrence.of.fusion.between.the.
polymer-modiied.liposomes.and.endosomal.or.lysosomal.membranes..Liposomes.with.
a.higher.SucPG.content.show.a.higher.percent.fusion.after.internalization..Hence,.the.
high. cytoplasmic. delivery. at. a. higher. content. of. pH-sensitive. polymer. overcomes. its.
low.rate.of.uptake.
PH. sensitivity. of. SucPG. liposomes. (eggPC/SucPG,. 20%. w/w,. 130. nm). coupled. to. ∼20.
anti-BCG.monoclonal.antibody.molecules.per.liposome.showed.≥90%.calcein.release.trig-
gered. between. pH. 4.0. and. 4.5;. it. decreases. below. 30%. above. pH. 5.. Fluorescence. micro-
scopic. observation. indicates. that. BCG-sucPG. immunoliposomes. bind. to. colon. 26. tumor.
cells. and. induce. receptor-mediated. endocytosis. at. 37°C.. Fusion. assay. (FRET. NBT-PE/
Rh-PE). suggests. that. fusion. between. BCG-SucPG. immunoliposomes. and. endosomal. or.
lysosomal.membrane.does.occur,.without.impairment.caused.by.the.presence.of.anti-BCG.
monoclonal.antibodies.
138
pH-sensitive. SucPG. imunoliposomes. (eggPC/SucPG,. 30%. w/w. in. 5%. glucose. solu-
tion).were.recently.employed.as.a.nonviral.vector.of.transfection.in.vitro.
139
.SucPG.lipo-
somes. are. negatively. charged. and. form. electrostatic. complexes. with. positively. charged.
lipoplexes. (cationic. liposomes. made. of. 3,5-dipentadecyloxybenzamidine. hydrochloride.
(TRX-20)/dilauroylphosphatidylcholine. (DLPC)/DOPE,. 0.26:0.26:0.52). plus. plasmid. DNA.
encoding.the.irely.luciferase.sequence,.imparting.fusion.ability.to.them
.
.Complexes.pre-
pared. in. an. aqueous. glucose. solution. have. small. diameters. and. much. higher. transfec-
tion. activity. toward. HeLa. cells. than. do. complexes. prepared. in. phosphate. buffer. saline.
(PBS).
140
. Transferrin. (t),. a. glycoprotein. transporting. iron. into. cells,. is. incorporated. into.
the. surface. of. SucPG. liposomes. to. overcome. the. low. afinity. by. cell. membranes. caused.
by. the. shielding. of. positive. charges. upon. complexation.. Transferrin. receptors. (TRs). are.
expressed.on.the.cell.surface.and.are.constitutively.taken.up.through.clathrin-dependent.
endocytosis.
141,142
.After.internalization,.t.and.its.TR.are.recycled.to.the.cellular.surface.via.
the.recycling.pathway.
143
.The.combination.of.cytoplasmic.delivery.of.DNA.after.complex.
fusion. with. the. endosomes,. and. the. addition. of. t. results. in. a. higher. eficiency. of. trans-
fection..However,.the.extent.of.the.t-induced.enhancement.is.dependent.on.the.cell.line..
Conjugation.of.t.increases.the.transfection.activity.on.HeLa.(human.cervical. cancer.cell.
line). and. KB. (human. epidermoid. adenocarcinoma. line). cells,. although. it. only. slightly.
enhances. transfection. on. HT1080. (human. ibrosarcoma. line),. HepG2. (human. hepatoma.
line),. and. K562. (human. leukemia. line). cells.
144,145
. Remarkably,. TR. in. HeLa. and. KB. cells.
are. internalized. quickly. and. actively,. whereas. those. in. HT1080,. HepG2,. and. K562. cells.
are. internalized. slowly.. Hence,. the. transfection. mediated. by. the. ligand-attached. hybrid.
complex.does.not.correlate.with.the.amount.of.TR.in.the.cell.surface,.but.correlates.with.
the. activity. of. internalization. of. TR. into. the. cells.. Toward. further. improvement. of. their.
transfection.activity,.selection.of.the.ligand.might.be.important.to.facilitate.cellular.inter-
nalization.and.acid-triggered.activation.of.SucPG.complexes.
