Biomedical Engineering Reference
In-Depth Information
nanotechnology. for. cells. used. speciically. in. bioprocessing.. Microbial. and. mammalian.
cell.culture.for.production.of.recombinant.protein.therapeutics,.industrial.enzymes,.and.
diagnostic.proteins.comprises.the.core.of.the.biotechnology.and.biopharmaceutical.indus-
try..Therapeutic.proteins.range.from.small.peptides.such.as.insulin.to.large.antibodies.and.
enzymes. (e.g.,. tissue. plasminogen. activator).. There. are. nearly. 200. recombinant. proteins,.
monoclonal. antibodies,. and. nucleic-acid-based. drugs. currently. approved. (Walsh. 2006,.
2007).with.a.market.greater.than.$50.billion.dollars.a.year.and.growing.at.a.rate.of.∼20%.
annually.(Heffner.2006)..As.described.in.Section.15.3.1,.industrial.enzymes,.which.are.also.
produced. recombinantly. or. isolated. from. their. native. host,. also. comprise. a. multibillion.
dollar.a.year.industry..The.majority.of.cell.culture.and.fermentation.processes.are.carried.
out.in.suspension,.in.large.(10,000-20,000).liter.vessels,.which.does.not.present.an.obvious.
role. for. nanotechnology.. However,. nanotechnology. can. play. an. important. role. in. deliv-
ery.of.nucleic.acids,.proteins,.and.small.molecules.into.cells,.selection.of.hybridoma.cells.
producing.the.correct.monoclonal.antibody,.selection.of.high.yielding.cell.clones,.and.in.
developing.substrates.for.cells.requiring.adherent.culture.
15.3.3.1 Delivery of Nucleic Acids, Proteins, and Small Molecules to Cells
Delivery. of. nucleic. acids. into. cells. is. a. critical. irst. step. in. recombinant. protein. produc-
tion..In.this.step,.an.exogenous.gene.is.introduced.into.the.cell.where.it.is.either.incorpo-
rated. into.the.chromosomal. DNA,. in. the.case. of. mammalian.cell. cultures,. or. replicated.
extra-chromosomally,.in.the.case.of.microbial.systems..The.application.of.interfering.RNA.
(RNAi).has.permitted.short-term.gene.editing.of.cell.cultures.to.determine.the.function.
of. endogenous. genes. and. identify. genes. whose. knockouts. would. be. beneicial. for. bio-
processing.applications..As.nucleic.acids.are.large,.hydrophilic,.negatively.charged.mol-
ecules,.they.cannot.easily.penetrate.the.negatively.charged,.hydrophobic.cell.membrane..
To.cross.the.cell.membrane,.they.are.generally.complexed.with.cationic.compounds,.often.
either. cationic. polymers. or. cationic. lipids.. While. these. compounds. are. generally. effec-
tive,. on. some. occasions,. they. will. not. provide. suficient. transfection. eficiency.. In. those.
cases,. magnetic. nanoparticles. can. be. used. as. an. alternative.. Several. magnetic. nanopar-
ticles.that.are.complexed.to.proprietary.polymers.are.commercially.available..While.these.
have.been.used.in.a.variety.of.cell.lines,.we.attempted.to.use.them.to.deliver.nucleic.acids.
into. Chinese. hamster. ovary. cells. (the. workhorse. of. the. biotechnology. industry). grown.
in.a.3D.collagen.hydrogel.as.a.precursor.to.high-throughput.screening.of.silencing.RNA.
(siRNA).or.short.hairpin.RNA.(shRNA)..When.we.found.that.these.complexes.had.insuf-
icient. transfection. eficiencies. in. our. system,. we. hypothesized. that. this. was. due. to. the.
large. magnetic. core. size. (250.nm).. To. alleviate. this. problem,. we. synthesized. novel. mag-
netic.nanoparticles.with.a.5.nm.iron.oxide.core.using.a.ligand.exchange.reaction.to.coat.the.
iron.oxide.particles.with.low-molecular-weight,.branched.polyethyleneimine.(PEI)..These.
coated.superparamagnetic.nanoparticles.were.then.used.to.condense.siRNA.or.plasmid.
DNA,.forming.small.particle-size.magnetic.complexes.(Zhang.et.al..2010a).
An.alternative.to.gene.delivery.is.the.delivery.of.proteins.or.peptides..This.approach.has.
been.particularly.advocated.for.cellular.reprogramming.of.cells.to.create.stem.cells;.how-
ever,. one. could. envision. similar. approaches. for. modifying. cells. used. for. bioprocessing.
if.they.could.effect.a.long-term.change.in.cellular.physiology..Kam.et.al..(2004).reported.
the.uptake.of.SWNTs.by.a.variety.of.cell.types.including.HL60.cells,.Jurkat.cells,.Chinese.
hamster.ovary.cells.(CHO),.and.3T3.ibroblast.cell.lines..They.were.then.able.to.biotinylate.
the. SWNTs. using. EDC. and. biotin-LC-PEO-amine,. and. inally. to. create. a. SWNT-biotin-
streptavidin. complex. which. could. then. be. taken. up. by. the. cells.. The. presence. of. the.
