Chitosan-DNA nanoparticles as gene carriers-synthesis,characterization and transfection efficiency

H.-Q.Mao et al./Journal of Controlled Release70(2001)399–421405

Scheme3.PEGylation of chitosan-DNA nanoparticles and conjugation of transferrin through a PEG spacer.

medium containing50m g/ml ampicillin,and grown 2.14.In vitro transfection of HEK293cells with for an additional 1.5h at378C.The culture was chitosan-DNA nanoparticles

induced with0.5mM isopropyl-b-thiogalac-

topyranoside(IPTG)and subsequently incubated for Cells were seeded24h prior to transfection into a 5h in a378C incubation shaker.The cells were12-well plate(Becton-Dickinson,Lincoln Park,NJ)

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harvested by centrifugation and resuspended in10ml at a density of8310cells per well in1ml of of suspension buffer(20mM sodium phosphate complete medium(MEM containing10%FBS, buffer containing500mM NaCl,pH7.8).The supplemented with2mM L-glutamate,50U/ml suspension was treated with lysozyme(100m g/ml)penicillin and50m g/ml streptomycin).At the time on ice for15min,sonicated for80s on ice using a of transfection,the medium in each well was re-Branson B15soni?er operating in pulse mode(20-s placed with1ml of fresh complete medium.The pulse)at the microtip limit,and then subjected to amount of nanoparticles equivalent to0.1–2m g of three freeze–thaw cycles.The cell debris was re-DNA were added to each well and incubated with moved by centrifugation.The crude extract was the cells for48–72h.Transfection with calcium ?

puri?ed using ProBond resin(Invitrogen,Carlsbad,phosphate-DNA complexes or Lipofect AMINE E-DNA CA)according to the manufacture’s protocol to yield complexes was performed as positive controls ac-the PolyHis tagged KNOB.KNOB protein was used cording to the reported method[31]or the manufac-directly for conjugation without removing the Poly-ture’s protocol.Brie?y,cells were incubated with His tag.KNOB protein was conjugated to the calcium phosphate-DNA or Lipofect AMINE E-DNA nanoparticle surface via a PEG spacer according to complexes in0.5ml of Opti-MEM or low serum Scheme3as described above.medium(1%FBS)for4h,and further incubated

408H.-Q.Mao et al./Journal of Controlled Release70(2001)399–421

Fig.2.Ternary phase diagram of complex coacervation between

pRE-luciferase plasmid and chitosan at558C in50mM Na SO.

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Sodium sulfate solution was regarded as one component,since the

concentration change in the experiment range was minimal.The

region to the right of line ABC depicts the conditions under which

phase separation occurred.The concentration ranges in the small

grid area yielded distinct particles as observed under a phase

contrast microscope.

system.Concentrations in the shadowed area would

yield large aggregates other than uniform particles.

In a typical preparation using chitosan C390(100

m g/ml)and pRE-luciferase plasmid(50m g/ml),

nanoparticles obtained under these conditions

showed a spherical and polydisperse nature as re-

?

vealed by their TEM image(Fig.3A).Zetasizer

measurement of these particles showed a fairly

unimodal distribution from150to300nm.The zeta

potential at the complexation pH(|5.7)was approxi-

mately112to118mV.

The effect of charge ratio on the particle size and

Fig.3.Physical characterization of chitosan-DNA nanoparticles. surface charge was examined within the concen-

(A).SEM image of nanoparticles.(B)Effect of N/P ratio on the tration range stated above.PCL113was used with a mean size(?)and zeta potential(h)of the nanoparticles.(C)?xed concentration of100m g/ml in 2.5mM Effect of the pH of the nanoparticle suspension on the zeta

potential of the nanoparticles.

Na SO,while the plasmid concentration was varied

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to yield different N/P ratios.Because only|90%of

the amino groups in chitosan were protonated at pH have higher thermal dynamic stability with an aver-5.5–5.7,an N/P ratio,instead of charge ratio,was age size between150and250nm.Interestingly,the used to represent a more accurate quanti?cation,zeta potential of PCL113-DNA nanoparticles only although these two ratios should be very close at pH increased slightly with the N/P ratio(Fig.3B). 5–https://www.360docs.net/doc/2e17803964.html,rge aggregates formed at N/P ratios around Results from the C390-DNA nanoparticle system 1(Fig.3B).An N/P ratio below0.75and above2were similar,except the zeta potential value and yielded submicron size particles.Nanoparticles pre-average size were both marginally higher than that of pared with an N/P ratio between3and8tended to PCL113-DNA nanoparticles(data not shown).

60.6%

DNA and chitosan contents were40.2an

(w/w),respectively.The small sample size p us from determining accurately the in v release kinetics.Most likely the hydrophob

of the nanoparticles at neutral pH would p from quickly leaching out until the nano were degraded in the lysosomes.

The transfection ef?ciency of CQ inc

C390-p43-hGFP nanoparticles is shown in

At the dose of1m g of DNA/well,nano containing CQ yielded three times as many cells as the nanoparticles without CQ.Perc

cells transfected by the CQ incorporated n

cles reached17%,a similar level achi Lipofect