nonviral gene vectors have many advantages such as mass production , easier transportation , less immunogenicity , and being easily targeted to organs [ 1 , 2 ] . among the nonviral vectors , chitosan is known to possess efficient properties owing to their ability to condense nucleic acid into stable complexes , which protects dna from degradation by nuclease . the dna / polymer complexes
are taken up into the cells via endocytosis into the endosomes , following with burst release of complexes fraction in endosomes and the dna translocates into the nucleus .
it is soluble at acidic ph value , and the amino groups carry positive charge in acidic mediums ; it can combine with negatively charged dna .
previous studies have revealed that chitosan , like other cationic polymers , displayed concentration - dependent toxicity toward cells in vitro , although it had many advantages as a gene vector .
magnetic ferriferous oxide nanoparticles possess prominent advantages that might correct the defects of traditional drugs and gene carriers .
whereby numerous dna strands attached to the surface of these ferriferous oxides could reach the desired position with the help of static magnetic field . in order to improve the properties of nanoparticles such as biocompatibility , transfection efficiency , and controlled release
, we embedded the biodegradable polymers on the surface of ferriferous oxide to form a core shell structure .
therefore , the focus of our research was on how to improve the target property and remove the application barriers of nonviral gene vectors in vivo .
the use of a static magnetic field has been shown to result in dramatic increase in transfection efficiency of gene delivery when compared with the conventional transfection system [ 9 , 10 ] .
magnet - assisted transfection is a new , easy - to - handle , very highly efficient technology .
it is a very gentle method with almost no toxicity and has been successfully used on many and also critical cell lines .
all types of nucleic acids from plasmid dna or oligonucleotides to sirna can be used with this approach . in this research ,
the synthesized magnetic nanoparticles have an approximately size of 100  nm and are additionally coated with biodegradable polymers .
we used both of the advantages of magnetic nanoparticles and biodegradable polymers , and the application of the novel polymer - fe3o4 complexes as gene vectors in vitro was then described at length .
these iron oxides can be generated by precipitation from acidic iron - salt solutions upon addition of appropriate bases .
a cts ( mws 45  kda , 20% w / w , ph6.9 ) solution carrying a positive charge or peg ( mws 6  kda , 20% w / w ) solution was prepared .
0.2  ml of this solution was added to 0.8  ml of iron oxide dispersion ( 10% w / w ) for 8  h incubation .
after filtration sterilization with a 0.45  m filter , the nanoparticles were used for the next transfection experiments .
nanoparticles and dna form complexes by ionic interaction of the negatively charged nucleic acid and the positively charged surface of the cts - fe3o4 nanoparticle ( n / p ratio 4  :  1 ) .
the polymer - fe3o4 was analyzed by means of a transmission electron microscope ( tem , hitachi h-700h ) , x - ray diffraction ( xrd , philips x'pert pro ) .
the size and zeta potential of the polymer - fe3o4 were both assessed using the zetasizer nano instrument .
the plasmid pegfp - c1 was propagated in escherichia coli and was purified using an endotoxin - free plasmid maxiprep kit ( qiagen ) . at the ph level of 7.4 the polymer - fe3o4 complexes
were mixed with dna at different volume ratios in a 50  l reaction system .
the final concentration ( fc ) of plasmid dna and polymer fe3o4 was 4  g/l and 1  mm ( concentrations related to fe ) diluted with double - distilled water ( ddh2o ) .
after 1  h incubation at 37c the concentration of dna in the supernatant was measured by uv spectrophotometric absorption at 260  nm . the encapsulation efficiency ( e.e . ) of the process indicates the percentage of dna encapsulated used for the preparation of polymer - fe3o4 complexes . to observe the target distribution of polymer - fe3o4 nanoparticles in different organs of mice , 40 pathogen - free balb / c female mice were purchased from the sichuan industrial institute of antibiotic for the in vivo studies .
the polymer fe3o4 was redispersed as described previously and injected through the caudal vein on the dosage of 1  mm iron oxide in 0.8  ml . a neodymium - iron - boron ( ndfeb ) permanent magnet ( br 1/4 1.5  t )
the mice were sacrificed at different times after the injection ( 2  h , 6  h , 12 , and 24  h ) , and the liver , spleen , lungs , heart , and brain were taken out and made into tissue slices .
the target distribution of polymer fe3o4 was observed by prussian blue and neutral red staining .
preweighed polymer - fe3o4 complexes containing dna were incubated in a test tube with phosphate - buffered saline ( pbs , ph 7.4 ) , for 30  min under moderate stirring at 37c .
dna was reacted with polymer - fe3o4 nanoparticles at three different volume ratios ( 1  :  3 , 1  :  1 , and 3  :  1 ) . at predetermined time intervals
( 12 , 24 , 48 , 72 , and 96  h ) , 50  l of the released medium was collected by centrifugation ( 3,000  g , 1  min ) , and 50  l of fresh pbs was added back into the test tube .
dna release was monitored by uv spectroscopy at 260  nm , and dna integrity was evaluated on a 1% agarose gel .
the amount of released dna was calculated from the free dna concentration in the supernatants , and the curve of dna release in vitro was described . at last , to confirm the functionality of released dna , the discharged dna was applied to the assay of transfection in vitro .
the polymer - fe3o4 complexes ( 1  mm ) were mixed with plasmid dna ( 4  g/l ) according to the optimal e.e .
naked plasmid dna and dna / polymer - fe3o4 complexes were incubated with or without dnasei ( 0.5  u ) in the 30  l reaction system for 1 hour at ph 7.4 . the digestion was stopped by addition of 0.5  m edta .
the product of enzymatic digestion was analyzed by 1% agarose gel electrophoresis , and dna in the gel was visualized by ethidium bromide staining .
naked plasmid dna after being digested by dnasei and naked plasmid dna without digestion were used as controls .
human embryonic kidney 293 cells ( hek-293 ) , human liver carcinoma cells ( hepg2 ) , and mouse myeloma cell line ( sp2/0 ) were maintained in dmem or rpmi-1640 medium ( gibco - brl ) , supplemented with 10% fetal calf serum ( fcs , gibco - brl ) and 1% penicillin / streptomycin .
for the transfection and cytotoxicity test , the cells were grown under standard conditions for 24 hours until 70% to 80% confluency in 96-well flat - bottomed microassay plates before the addition of either the plasmid dna / polymer - fe3o4 complex or only the polymer fe3o4 .
firstly , the precipitate polymer - fe3o4 complexes were resuspended under conditions of ultrasonic agitation for 10  min .
subsequently , the complexes were added into the cell - culture fluid at a different concentration ( 0.2 ~ 1.0  mm , 2 ~ 20  mm ) , diluted with a serum - free medium . at the end of each predetermined time ( 6  h , 12  h , 24  h , and 48  h ) , the polymer - fe3o4 complexes were replaced with 200  l of fresh dmem medium .
then , 20  l of mtt ( 5  g/l ) in dmem was added to each well and incubated for an additional 4 hours .
all mediums were then removed , and 150  l of dmso was added to dissolve the crystals formed by the live cells .
the cell viability was calculated , and the viability of nontreated control cells was arbitrarily defined as 100% .
24-well plates were seeded with 2  10 cells ( hepg2 and sp2/0 cells ) and grown for 24 hours to obtain 7080% confluence .
prior to transfection , the medium was removed , and the cells were rinsed once with pbs ( ph 7.4 ) , then supplied with serum - free medium . the plasmid dna was mixed with cts - fe3o4 and peg - fe3o4 as described previously and incubated for 30 minutes at 37c .
dna / polymer - fe3o4 complexes were suspended in a serum - free medium to get the final concentrations of 2  g/l and 1.5  mm , respectively . to verify the short exposure to a static magnetic field
would improve transfection efficiency ; the cells were placed on a ( ndfeb ) magnet for 30  min at a distance of 3  mm from the magnet surface , which leads to a magnetic flux density of 340  mt and a magnetic field gradient perpendicular to the well plate of 14  t / m . after a further incubation of 4  h
the cells were incubated with plasmid dna alone and dna / polymer - fe3o4 complexes under standard conditions and grown in culture medium for 24 hours to allow for egfp expression .
chitosan ( mws 45  kda ) , lipofectamine ( bestbio ) , and pbs were added to an equal volume of dna as controls .
tem images showed that most of the iron oxide complexes were approximately spherical ( unpublished data ) .
the xrd measurements also indicated that the samples had a cubic crystal system and magnetite fe3o4 was the dominant body of the polymer - fe3o4 complexes .
the size and zeta potential showed the two samples to have a uniform size of 100  nm ( figure 1(a ) ) and almost the same distribution .
the sizes of 10100  nm in diameter are desirable since they are too small not to be eliminated by the reticuloendothelial system ( res ) but too large to be filtered out by the kidneys .
cts - fe3o4 had a positive charge of about 20  mv ( figure 1(b ) ) , and the zeta potential of peg - fe3o4 was 0  mv .
it has been reported that surface charge plays an important role in determining the efficiency and mechanism of cellular uptake .
it is also an important factor to improve stability of polymer - fe3o4 complexes and to prevent from further aggregation in aqueous solution via electrostatic repulsion .
the polymer - fe3o4 complexes were mixed with plasmid dna according to different volume ratios ( 1  :  3 , 1  :  2 , 1  :  1 , 2  :  1 , and 3  :  1 ) in a 50  l reaction system .
increased along with the proportion of the magnetic materials mainly because of the electrostatic interactions , surface energy of nanoparticles , and branched structures of polymers .
the optimal e.e emerged when the iron oxide complexes were mixed with dna at 3  :  1 volume ratio , and the final concentration of dna and iron oxide was 2  g/l and 1.5  mm respectively .
of peg - fe3o4 was inferior to cts - fe3o4 notably for the lack of electrostatic attraction .
the different organs from the mice injected with polymer - fe3o4 were taken out and made into tissue slices .
target distribution of polymer fe3o4 in vivo was demonstrated with the help of outer static magnetic field .  
figure 2(b ) shows a large number of iron particles scattered in the hepatic tissue ; many of them were distributed along the hepatic sinusoid 2  h after injection .
the iron particles decreased gradually over time and disappeared 24  h after injection ( data not shown ) .
the shape of the liver cells was seen under a high - power microscope to be integrated .
there was no iron staining in the other organs , such as the lungs ( figure 2(d ) ) , the spleen , and the heart .
naked pegfp - c1 without digestion and naked pegfp - c1 following digestion by dnasei were used as controls .
we could evidence partial protection of dna coated by polymer fe3o4 from nuclease - mediated dna degradation ( unpublished data ) .
it was assumed that dna degradation occurs in several layers ; external layers will be degraded easily but not internal layers .
furthermore , cts - fe3o4 nanoparticles offered higher protection for dna than peg - fe3o4 , as the dna chains could be attached more strongly to the former .
in addition , dnasei digestion resulted in a shift in the most distribution of the dna isoforms : supercoiled plasmid in nontreated samples was replaced by the open loop form in treated samples .
the in vitro release rates of dna from polymer - fe3o4 complexes were studied at different volume ratios . a significant proportion ( 30% ) of the adsorbed dna was released very rapidly from the cts - fe3o4 nanoparticles in the initial 12 hours . after 48  h , the amount of released dna reached 55% at the optimal e.e . and
the remainder of the adsorbed dna was released slowly , reaching 70% at 96  h ( figure 3(a ) ) .
compared to dna release from cts - fe3o4 , a burst release phase of more than 61% from peg - fe3o4 was observed .
the release curve showed that the dna was released more rapidly ; more than 80% of dna was discharged from peg - fe3o4 after 24  h at the optimal e.e . , and the entire release was mostly completed at 72  h ( figure 3(b ) ) .
the dna integrity test at predetermined time points was assessed by agarose gel electrophoresis ( data not shown ) .
no differences were observed between egfp expression from the released dna and the controlled plasmid pegfp - c1 , indicating that adsorption and release from the polymer - fe3o4 do not alter the functionality of plasmid dna .
overall , the controlled release effect of cts - fe3o4 complexes was relatively obvious compared with peg - fe3o4 .
the speed of dna release was inversely proportional to the volume ratios of nanoparticles .  
the n / p ratio ( the ratio of negatively charged dna to positively charged chitosan ) is a key factor to determine the optimal complexation conditions .
the difference ph and counterions in the medium might directly affect the binding between cts and dna .
it could be inferred that the burst release was induced by the dna degradation in the external layers .
the results showed that the controlled - release effect of cts - fe3o4 was more obvious , and the unsteady binding power made the efficient binding with dna and peg - fe3o4 impossible .
in addition , the small proportion of chitosan in the polymer - fe3o4 complexes actually hindered the effect of controlled release .
increasing the proportion of chitosan would slow down the dna release but augment the particle size and positive charge of the complexes .
it has been reported that positively charged nanoparticles exhibited dose - dependent hemolytic activities and cytotoxicities .
in addition , most of the larger nanoparticles ( > 150  nm ) are trapped by the liver and lung where many macrophages are located . for the drug and gene target delivery application , the nonspecific uptake of nanoparticles by macrophages in the res should be minimized .
the contradictory issue of controlled - release and particle size needs to be resolved urgently by carrying out a further study .
it has been reported that chitosan derivatives are less toxic than other cationic polymers such as pei in vitro and in vivo .
evaluation of cell viability was conducted on hek-293 and hepg2 cells using a 0.220  mm concentration gradient of polymer - fe3o4 complexes for different incubation periods .
more than 90% cell viability of both polymer - fe3o4 complexes was obtained after 24  h of incubation with a concentration of 2  mm or less , and apparent cytotoxicity emerged when the concentration of polymer fe3o4 was more than 10  mm ( data not shown ) .
this result showed that both cts - fe3o4 and peg - fe3o4 had low cytotoxicity .
the security application could therefore be deduced according to the previously mentioned data and the optimal e.e .
hepg2 and sp2/0 cells were transfected as described previously with either dna / cts - fe3o4 or dna / peg - fe3o4 , with dna / chitosan , dna / lipofectamine , and naked plasmid as controls .
exposure to a permanent magnetic field ( magnet ) for 30  min was followed by 4  h incubation .
the highest transfection rates were achieved in hepg2 cells corresponding to 67.2% and 45.8% after transfected with cts - fe3o4 and peg - fe3o4 complexes .
significantly lower transfection rates of 14.3% , 8.7% , and 0.4% resulted from transfection with lipofectamine , chitosan , and naked plasmid , respectively .
in addition , the transfection rates were significantly increased by 4.1- and 3.2-fold in hepg2 and sp2/0 cells , when compared to cells not exposed to the magnetic field .
similar transfection results were also obtained with sp2/0 cells , and lower rates of 43.7% and 32.5% treated with cts - fe3o4 and peg - fe3o4 complexes were achieved .
compared with conventional transfected methods , the results were still statistically significant ( figure 4 ) . thus , the transfections rates enhanced by the assistance of magnetic field were verified again in hepg2 and sp2/0 cells .
it seems that the use of a static magnetic field can improve the translocation of the particles across the cell membrane .
it has been reported that the higher transfection rates with magnetic nanoparticles were mainly attributed to their size surface charge , since the larger nanoparticles faster sedimentated on the surfaces of the cells , and this resulted in higher endocytic uptake , and positively charged nanoparticles were more easily taken up by cells .
the chosen cells used in our study were malignant cells from human and mice , and these cells differed in characteristics used as models for different human diseases .
thus , they were good representative samples for enhancement of delivery and effective targeting of gene expression . furthermore , the egfp expression was strong in transfected cells indicating that the function of dna was kept and no fragmentation occurred .
magnetic materials modified by biodegradable polymers as gene carriers possess many merits . for examples , simple manufacturing operation , arriving at the target point with the help of an outer magnetic field ; a powerful surface energy effect and a small size effect are their outstanding characters .
moreover , it is easy to modify all kinds of multifunctional groups or targeting molecules to form the structure of the core shell , such as cts , pei , specific ligands , and monoclonal antibodies , since the complexes have multiple binding sites on their surface , and dna attaches itself to them in sizeable amounts either through an electrostatic effect or by chemical bond coupling . in order to improve the e.e . of the polymer - fe3o4 complexes and realize the controlled release of the dna , we modified the fe3o4 with multifunctional groups cts and peg .
in addition , the process of linking polymeric groups did not utilize organic solvent extraction , and the iron content used does not surpass the acceptable daily intake .
furthermore , some of the novel nanoparticles could improve the antigen presentation effect , show a better adjuvant effect , and make a long - term , single - immunization vaccine possible .
there are likely to be further applicative studies of polymer - fe3o4 complexes as gene delivery systems .
preliminary data from our studies suggest that fe3o4 nanoparticles when decorating with positive - charged polymer cts exhibit preferential gene delivery .
dna encapsulation efficiency increased with the proportion of polymer - fe3o4 nanoparticles , and the optimal e.e .
simultaneously , the attachment of dna to polymer - fe3o4 complexes did provide protection against cleavage by nuclease .
the target distribution of polymer - fe3o4 complexes with an outer magnetic field was demonstrated in vivo .
the controlled - release effect of cts - fe3o4 complexes was more apparent than peg - fe3o4 , and the dna binding and release from the polymer - fe3o4 do not alter its functionality . both cts - fe3o4 and peg - fe3o4 had low cytotoxicity to hek-293 and hepg2 cells .
the concentration of 2  mm or less in an in vitro application was shown to be absolutely safe .
in addition , the magnetic forces lead to an accelerated sedimentation of polymer - fe3o4 complexes on the cell surface and do directly enhance the transfection efficiency in hepg2 and sp2/0 cells compared with conventional transfection methods .
the novel gene delivery system proved to be an effective tool for future , and it is promising in targeting expression and delivery of therapeutic genes in in vivo studies .
we will continue to do research in this field to provide a more detailed evaluation about the transfer of dna .
 new approaches to improve the traditional gene carriers are still required . here 
 we explore fe3o4 modified with degradable polymers that enhances gene delivery and target delivery using permanent magnetic field . 
 two magnetic fe3o4 nanoparticles coated with chitosan ( cts ) and polyethylene glycol ( peg ) were synthesized by means of controlled chemical coprecipitation . 
 plasmid pegfp was encapsulated as a reported gene . 
 the ferriferous oxide complexes were approximately spherical ; surface charge of cts - fe3o4 and peg - fe3o4 was about 20  mv and 0  mv , respectively . the controlled release of dna from the cts - fe3o4 nanoparticles was observed . 
 concurrently , a desired fe3o4 concentration of less than 2  mm was verified as safe by means of a cytotoxicity test in vitro . 
 presence of the permanent magnetic field significantly increased the transfection efficiency . 
 furthermore , the passive target property and safety of magnetic nanoparticles were also demonstrated in an in vivo test . 
 the novel gene delivery system was proved to be an effective tool required for future target expression and gene therapy in vivo . 