chitosan is an abundant 
 linear polysaccharide derived from crustacean shells and is composed 
 of random -(14)-linked d - glucosamines ( deacetylated 
 units ) and n - acetyl - d - glucosamines ( acetylated 
 units ) . it is a biodegradable and biocompatible polymer that has been 
 studied intensively for its therapeutic applications as a drug and 
 gene delivery system . chitosan has garnered interest as a cationic nonviral gene delivery 
 vector because of its ( i ) relative ease of preparation ; ( ii ) low toxicity ; 
 and ( iii ) ability to condense dna which provides protection against 
 nuclease degradation . previous studies have shown 
 that chitosan / pdna polyplexes ( cspp ) are particularly promising as 
 gene delivery formulations for treatment of pulmonary pathologies 
 such as cystic fibrosis , asthma , and lung cancer . although 
 chitosan causes minimal cytotoxicity and is generally noninflammatory , 
 a drawback is its low transfection efficiency when compared to viral 
 vectors and nonviral synthetic vectors such as polyethylenimine ( pei ) . 
 thus , most of the studies involving cspp delivery have been focused 
 on strategies to improve transfection efficiency such as optimizing 
 the molecular weight and the degree of deacetylation of chitosan , as well as establishing appropriate amine to phosphate ratios ( n / p ) 
 of chitosan / dna polyplexes . mixing chitosan 
 with other cationic gene vectors and/or modifying 
 the structure of chitosan has also resulted in enhanced transfection 
 efficiencies . the toxicity of pure or 
 chemically modified chitosan has been investigated in its free form 
 as well as in the form of micro- and nanoparticles . culturing 
 chitosan nanoparticles , prepared by ionotropic gelation with pentasodium 
 tripolyphosphate ( tpp ) , with a cell line derived from human conjuntival 
 epithelium , had no effect on cell viability compared to untreated 
 cells . in addition , it has been reported 
 that cspp have little or no significant impact on cell viability in vitro for a range of cell types . this is in 
 contrast to commonly used transfection reagents such as lipofectamine 
 2000 which can be highly cytotoxic . despite 
 showing promise in cytotoxicity assays in vitro , 
 a thorough characterization of the cytotoxicity and inflammation induced 
 by cspp in vivo , particularly with respect to pulmonary 
 delivery , needs further investigation . aside from chitosan , pdna itself may also play a critical role in the cytotoxicity and 
 inflammation imparted by cspp in vivo . unmethylated 
 5-cytosine - guanosine-3 dinucleotide ( cpg ) sequences 
 in pdna vectors can potentially induce inflammatory responses through 
 the production of proinflammatory cytokines , which , in turn , may result 
 in lowered transfection efficiencies . yew et al . reported that nasal instillation of complexes composed 
 of cationic lipids and cpg - depleted pdna resulted in lower proinflammatory 
 cytokine levels in mouse bronchoalveolar lavage fluid ( bal ) when compared 
 to the complexes comprising cpg - rich pdna . additionally , hyde et al . compared pulmonary inflammatory responses 
 to complexes comprising cationic liposomes and various plasmids containing 
 varying numbers of unmethylated cpg sequences per plasmid . it was 
 found that intranasal administration of complexes made with cpg - free 
 plasmids produced no inflammatory responses while the presence of 
 one unmethylated cpg sequence per plasmid was enough to trigger inflammatory 
 responses . showed that 
 pei complexes with a cpg - free plasmid administered in aerosolized 
 form resulted in 60 times higher luciferase expression in the lung 
 than that of pei complexes containing plasmids with unmethylated cpg 
 sequences . although both types of pei 
 complexes increased cytokine levels at 1 h after administration , the 
 cytokine levels of mice treated with pei complexes with cpg - free plasmids 
 reverted back to normal after 24 h while the others remained elevated . the main goals of this study were to characterize 
 cspp , evaluate their cytotoxicity in vitro , and determine 
 the degree of pulmonary inflammation following nasal delivery 
 to mouse lungs . cpg( ) and cpg(+ ) pdna were compared to determine 
 whether unmethylated cpg sequences in plasmid dna influence transfection 
 efficiency and inflammation . low molecular weight chitosan 
 was purchased from sigma - aldrich , co. ( st . louis , mo ) . briefly , 
 1% ( w / v ) chitosan was dissolved in 1% ( v / v ) acetic acid and filtered 
 with whatman 541 filter paper ( ge healthcare , amersham place little 
 chalfont , buckinghamshire , u.k . ) . the supernatant was titrated with 
 1 n naoh to ph 8.5 and then filtered to collect precipitated 
 chitosan . deproteinization and demetallization were performed 
 by adding 0.5% ( w / v ) sodium dodecyl sulfate ( sds ) and 20 mm ethylenediaminetetraacetic 
 acid ( edta ) disodium salt . insoluble chitosan was filtered , dialyzed 
 ( snakeskin dialysis tubing ; 10k mwco , thermo fisher scientific inc . , 
 rockford , il ) , and lyophilized . purified chitosan tested negative 
 ( < llod , 0.024 eu / ml ) for endotoxin using a modification of the 
 kinetic chromogenic limulus amebocyte lysate ( lal ) assay ( lonza , walkersville , 
 md ) . two types of plasmids used 
 in this study were pcpg - luc ( cpg( ) ) ( invivogen , san diego , 
 ca ) and vr1255 ( cpg(+ ) ) ( vical , san diego , ca ) . all plasmids were 
 purified using a genelute hp endotoxin - free plasmid maxiprep kit ( sigma - aldrich ) , 
 according to the manufacturer s protocol . pdna concentration 
 was determined using a nanodrop 2000 spectrophotometer ( thermo fisher 
 scientific inc . , waltham , ma ) . the cspp were prepared by ionic interaction between 
 positively charged chitosan and negatively charged pdna molecules . in this study , dextran sulfate ( sodium ) ( 5 kda , 
 sigma - aldrich , st . louis , mo ) was introduced into the formulation 
 to weaken the complexation of chitosan and dna in order to limit excessive 
 retention of dna and thus ensure higher transgene expression . cspp were formulated at different ratios of primary amine groups 
 in chitosan to phosphate groups in pdna ( n / p ratios ) . chitosan solutions 
 were prepared by dissolving the lyophilized purified chitosan in 1% 
 ( v / v ) acetic acid solution , then adjusting to ph 5.55.7 using 
 1 n naoh . then 50 mm acetate buffer ( ph 5.5 ) was added to the prepared 
 solution to achieve a final concentration of 5 mg / ml . dextran sulfate 
 stock solution ( 1 mg / ml ) was prepared in ultrapure dnase / rnase - free 
 distilled water ( invitrogen , grand island , ny ) . dextran sulfate : chitosan 
 ( 1:10 ( w / w ) ) was used in this study . plasmid dna ( cpg( ) or 
 cpg(+ ) ) in ultrapure dnase / rnase - free distilled water ( 200 g / ml ) 
 was heated in a water bath to 5055 c prior to mixing . all solutions were sterilized by filtering with a 0.22 m syringe 
 filter ( millex - gs , millipore corporation , billerica , ma ) . an equal 
 volume of the desired concentration of cationic solution was pipetted 
 into the anionic solution and then vortexed immediately for 2030 
 s. the polyplex suspensions were incubated at room temperature for 
 30 min before use . the final concentrations of pdna after polyplex 
 formation were 2.5 g/50 l ( in vitro ) and 12.5 g/50 l ( in vivo ) . as a positive control in the in vitro transfection 
 experiments , branched pei , mw 25 kda ( sigma - aldrich ) , was used to 
 make polyplexes at an n / p ratio of 10 . these types of pei / pdna polyplexes 
 ( peipp ) have previously displayed strong transfection efficiencies 
 generating high luciferase activity in hek293 , cos7 , and hela cell 
 lines . to prepare pei / pdna polyplexes , 
 a stock solution ( 1 mg / ml ) of pei in water was diluted to the desired 
 concentration and mixed with pdna in the same manner as cspp . particle size and zeta potential of cspp ( with varying n / p ratios ) 
 were measured using a zetasizer nano zs particle analyzer ( malvern 
 instrument ltd . , zeta potential measurements 
 were performed on polyplexes that were dispersed in acetate buffer 
 solution , ph 5.46 , at 25 c . cpg(+ ) plasmid 
 and the cspp(cpg(+ ) ) at n / p ratios of 1 , 5 , 10 , 20 , 60 , and 100 were 
 mixed with 2 bluejuice gel loading buffer ( invitrogen ) . the 
 final solutions / suspensions were loaded into the wells of a 1% ( w / v ) 
 agarose gel containing 0.5 g / ml of ethidium bromide in 1 
 tris - acetate - edta ( tae ) buffer . gels were exposed to a constant current 
 of 100 ma for 2 h. dna migration was then visualized with a uv transilluminator 
 ( spectroline , westbury , ny ) . adenocarcinomic 
 human alveolar basal epithelial cells ( a549 ) were maintained in rpmi-1640 
 medium ( gibco , life technologies , grand island , ny ) . human embryonic 
 kidney 293 ( hek293 ) cells were purchased from the american type culture 
 collection ( atcc , rockville , md ) and were maintained in dulbecco s 
 modified eagle medium ( dmem ) ( gibco ) . all of the media were supplemented 
 with 10% fetal bovine serum ( atlanta biologicals , lawrenceville , ga ) , 
 10 mm hepes ( gibco ) , 50 g / ml gentamycin sulfate ( cellgro , manassas , 
 va ) , 1 mm sodium pyruvate ( gibco ) , and 1 mm glutamax ( gibco ) . twenty - four 
 hours prior to treatment , cells were seeded ( 1  10/well ) into a 96-well plate . treatments ( cspp at various n / p ratios , 
 pei / pdna polyplexes , and pdna alone ) were added in a volume of 100 
 l / well ( equivalent to 1 g of pdna ) of serum - free medium . 
 without aspiration , after 48 h , the treatment was aspirated and 
 replaced with 100 l of fresh complete medium and 20 l 
 of the mts tetrazolium compound or celltiter 96 aqueous one solution reagent ( promega corporation , madison , wi ) . the plate 
 was incubated at 37 c at 5% co2 and incubated for 
 14 h , and then the absorbance was recorded at 490 nm using 
 a spectra max plus 384 microplate spectrophotometer ( molecular devices , 
 sunnyvale , ca ) . relative cell viability values are expressed as the 
 percentage of uv absorbance from wells containing treated cells compared 
 to the control wells containing live cells treated with phosphate 
 buffered saline ( pbs ) . a549 or hek293 cells were plated in 60 
 mm dishes ( 2  10 cells / dish ) 24 h prior 
 to treatments . cells 
 were subsequently washed with pbs containing 5 mm pyruvate and incubated 
 in a solution of pbs containing 5 mm pyruvate with 10 m dihydroethidium 
 ( dhe ) for 40 min at 37 c and 5% co2 . the control 
 tubes received 0.1% dimethyl sulfoxide ( dmso ) , and the electron transport 
 chain blocker antimycin a ( ant a ) was used as a positive control . 
 following the incubation the cells were filtered through 35 m 
 mesh , transferred to flow tubes , and placed on ice to stop the reaction . samples were analyzed using a facscan flow cytometer ( becton dickinson 
 immunocytometry systems , inc . , san jose , ca ) using an excitation wavelength 
 of 488 nm and emission of 585 nm . the mean fluorescence intensity 
 ( mfi ) of 10,000 cells was recorded for each sample , and the mean of 
 three samples was calculated for each treatment group . the background 
 fluorescence was subtracted from each sample to generate the net mfi . each sample was normalized to the control group to yield the normalized 
 mfi ( nmfi ) . cells ( hek293 
 and a549 cell lines ) were seeded ( 8  10 cells / well ) 
 into a 24-well plate . after 24 h , 500 l of serum - free medium 
 and 100 l of the treatments ( pdna solution , the cspp and pei / pdna 
 polyplexes ) were added . additional complete medium ( 500 l ) was added 4 h after the 
 treatment . the medium was then replenished every 24 h. at 48 h , the 
 cells were treated with reporter lysis buffer ( rlb ) ( promega corporation ) 
 followed by two freeze  thaw cycles ( frozen at 80 c 
 for 20 min then thawed at room temperature for 30 min ) . cell suspensions 
 were then transferred to microcentrifuge tubes and centrifuged at 
 16100 g for 5 min , after which the supernatants were 
 collected . luciferase expression was measured using a luciferase assay 
 system according to the company protocol ( promega corporation ) . briefly , 
 20 l of each supernatant was added to 100 l of the luciferase 
 assay reagent and luciferase activity was measured for 10 s using 
 a lumat lb 9507 luminometer ( eg&g berthold , wildbad , germany ) . the remaining supernatant was analyzed for protein concentration using 
 a micro bca protein assay kit ( pierce biotechnology inc . , mice ( c57bl/6 , males , 6 weeks old ) were purchased 
 from the jackson laboratory ( bar harbor , me ) . mice were kept in a 
 vivarium in polypropylene , fiber - covered cages in hepa - filtered thoren 
 caging units ( hazelton , pa , usa ) in the pulmonary toxicology facility 
 at the university of iowa . animals were acclimatized in the vivarium 
 for 10 days prior to the instillation exposures . food ( sterile teklad 
 5% stock diet , harlan , madison , wi ) and water ( via an automated watering 
 system ) was provided ad libitum , and a 12 h light all animal protocols used 
 in these studies were approved by the institutional animal care and 
 use committee and complied with nih guidelines . mice were exposed to cspp by nasal 
 instillation ( dose 12.5 g of pdna/50 l ) twice with a 
 1 h interval between each dose . the mice were anesthetized with isoflurane 
 ( 3% ) by inhalation using a precision fortec vaporizer ( cyprane , keighley , 
 u.k . ) . there were three control groups in these studies : ( 1 ) mice 
 without exposure ; ( 2 ) mice exposed to chitosan solution ; and ( 3 ) mice 
 exposed to cpg(+ ) alone . mice were euthanized 24 h after nasal instillation , 
 bronchoalveolar lavage ( bal ) fluid was collected and lungs 
 were harvested as previously described . lungs from euthanized mice were 
 washed with ice - cold pbs , weighed , and homogenized using a tissue 
 tearor ( biospec products , inc . , bartlesville , ok ) in ice - cold reporter 
 lysis buffer ( 4 l / mg of lung tissue ) for 1 min . lysates were 
 then passed through three freeze  thaw cycles ( frozen at 80 
 c for 20 min and thawed in a 37 c water bath for 5 min ) . supernatants were analyzed for luciferase expression in the same manner 
 as described in section 2.3 . the lungs 
 from each euthanized mouse ( n = 6 ) were lavaged via 
 cannulated trachea three times , in situ , with approximately 1 ml / wash 
 of sterile saline ( 0.9% ( w / v ) sodium chloride solution ) . cells were 
 collected from bal fluid by centrifugation at 800 g for 5 min at 4 c . the cell pellets were resuspended in hanks 
 balanced salt solution , and the total white cells were counted using 
 a hemocytometer . the remaining supernatants were stored at 80 
 c and used for measuring total protein , ldh activity , and inflammatory 
 cytokine / chemokine concentrations . different cell types were counted 
 after the bal cells were placed on microscope slides , spun using cytospin 
 4 ( thermo shandon , thermo scientific , waltham , ma ) at 800 g for 3 min , and stained using a hema 3 stain set ( fisher scientific 
 company llc , midland , mi ) . a total of 400 cells ( macrophages , neutrophils , 
 lymphocytes , and eosinophils ) per mouse were counted . the concentration 
 of total protein in bal fluid supernatants was determined using a 
 bradford protein assay ( bio - rad laboratories , inc . , hercules , ca ) . the activity of lactate dehydrogenase enzyme ( ldh ) in bal fluid supernatants 
 was measured using a commercially available cytotoxicity detection 
 kit ( ldh ) ( roche diagnostics , mannheim , germany ) . inflammatory cytokine / chemokine 
 ( tumor necrosis factor [ tnf]- , interferon [ ifn]- , interleukin 
 [ il]-6 , il-12 ( p40 ) , keratinocyte - derived cytokine [ kc ] , and macrophage 
 inflammatory protein [ mip]-1 ) levels in the bal fluid were 
 determined using a multiplexed fluorescent bead - based immunoassay 
 ( bio - rad laboratories , inc . ) . values for data falling below the lower 
 limit of detection ( llod ) for each cytokine were imputed with llod/2 . statistical significance 
 was determined using a one - way anova ( analysis of variance ) with bonferroni s 
 multiple comparison test . a p - value less than 0.05 
 was considered significant . statistical analyses were performed using 
 graphpad prism version 5.02 for windows ( graphpad software , inc . , 
 san diego , ca ) . sizes of the cspp prepared for the in vitro study 
 ( 2.5 g of pdna/50 l ) ranged between 200 and 400 nm for 
 all n / p ratios tested ( figure 1 ) . the sizes 
 of the polyplexes at n / p ratios ranging from 1 to 20 were similar 
 ( 230 nm ) for both cspp(cpg( ) ) and cspp(cpg(+ ) ) while 
 polyplexes progressively increased in size at n / p 60 ( 250300 
 nm ) and n / p 100 ( 300350 nm ) . both types of polyplexes had 
 narrow size distributions with an average polydispersity 
 index ( pdi ) equal to 0.136 . at n / p 1 , the cationic charge contributed 
 by chitosan was lower than the negative charge from pdna . thus , the 
 zeta potential values of both types of polyplex were negative ( -25 
 mv ) . however , when the concentration of chitosan increased ( at n / p 
 ratios of 5 to 100 ) , all polyplexes tested were positively charged 
 ( 2535 mv ) . particle sizes and zeta potentials of cspp(cpg( ) ) 
 ( a ) and cspp(cpg(+ ) ) ( b ) at n / p 1100 as measured by zetasizer 
 nano zs in acetate buffer , ph 5.46 , at 25 c . zetasizer nano zs measurements showed that particle 
 size increased when pdna concentrations were increased above 2.5 g/50 
 l ( data not shown ) . in contrast , concentrations below 2.5 g 
 of pdna/50 l have previously been shown to have no effect on 
 the size of cspp . for the in 
 vitro experiments ( sections 3.3 and 3.4 ) polyplexes were used at a concentration 
 of 2.5 g of pdna/50 l . for the in vivo experiments ( sections 3.5 and 3.6 ) the concentration was increased to 12.5 g 
 of pdna/50 l in order to overcome the limitation of delivery 
 volume . this increase in pdna concentration resulted in an increase 
 in polyplex size as well as a small amount of aggregation of the polyplexes 
 and was consistent with findings from other groups . a bimodal distribution was observed in particle size measurements 
 for cspp with a pdna concentration of 12.5 g/50 l , n / p 
 10 ( 523.0  3.8 nm , 94.6  2.3% by volume and 4888.0  
 355.3 nm , 5.4  2.3% by volume ) . concentrations of greater than 
 12.5 g / ml pdna resulted in substantial precipitation of the 
 polyplexes , rendering them unsuitable for pulmonary delivery . plasmid dna 
 complexed with chitosan did not migrate into an agarose matrix ( 1% 
 ( w / v ) ) when an electric field was applied . the pdna from cspp remained 
 in the loading wells at n / p ratios > 1 ( figure 2 ) . this finding is consistent with the zeta potential data 
 showing a negative charge for cspp n / p = 1 and a positive charge for 
 cspp n / p  5 suggesting that , at n / p ratios 
 5 , chitosan binds to all free pdna molecules , creating polyplexes . gel retardation 
 assay in 1% agarose gel containing ethidium bromide : lane 1 , dna ladder ; 
 lane 2 , naked cpg(+ ) pdna ; lanes 38 , cspp(cpg(+ ) ) with n / p 
 ratios of 1 , 5 , 10 , 20 , 60 and 100 , respectively . these results may vary depending on the characteristics of 
 the chitosan , since the binding strength between chitosan and dna 
 has been shown to be affected by the degree of deacetylation and the 
 molecular weight of the chitosan . chitosan with a high molecular weight 
 and high degree of deacetylation binds to dna more strongly than chitosan 
 with a relatively low molecular weight and low degree of deacetylation . cell viability assays ( see section 2.2.2 ) involving a549 , a human lung epithelial cell line , and hek293 , 
 a human embryonic kidney cell line , commonly used in transfection 
 studies , were used to test cspp at n / p ratios of 1100 using 
 both types of pdna ( cpg( ) or cpg(+ ) ) . all formulations of 
 cspp tested resulted in at least 8590% cell viability in a549 
 ( figures 3a and 3b ) 
 and hek293 ( figures 3c and 3d ) , with no significant differences compared to pbs - treated 
 control cells . in contrast , peipp at n / p 10 showed significantly lower 
 cell viabilities in both a549 ( figures 3a and 3b ) and hek293 ( figures 3c 
 and 3d ) ( 25% , p - value 
 < 0.001 ) . cell viability of a549 ( a , b ) and hek293 ( c , d ) cell lines treated 
 with cspp(cpg( ) ) ( a , c ) and cspp(cpg(+ ) ) ( b , d ) was determined 
 by mts assay . cells were treated with cspp containing n / p ratios ranging 
 from 1 to 100 . cpg( ) represents soluble cpg - free plasmid dna 
 while cpg(+ ) represents soluble cpg - containing plasmid dna . peiss(cpg( ) ) 
 or peipp(cpg(+ ) ) were pei complexes with pdna at n / p 10 . control = 
 cells treated with pbs . the cells were seeded into a 96-well plate 
 at a density of 1  10 cells per well one day prior 
 the experiment and exposed to the treatments for 48 h. each treatment 
 contained 1 g of pdna per well . percent relative cell viability 
 compared to cells treated with pbs . * * * p < 0.001 . in order to determine if treatment 
 with the polyplexes affects intracellular reactive oxygen species 
 ( ros ) levels , superoxide levels were estimated using the oxidation 
 sensitive fluorescent dye , dihydroethidium ( dhe ) . the results indicated 
 that a549 cells treated with cspp , pei polyplexes ( peipp ) , chitosan 
 solution , or pei solution showed significant increases ( 1.52.0-fold ) 
 in dhe oxidation relative to the control group ( p - value < 0.001 and < 0.01 ) ( figure 4a ) . treatment of hek293 cells with the cspp or the chitosan solution did 
 not significantly affect the dhe oxidation levels compared to the 
 control group . however , treatment with peipp , cpg(+ ) alone , or pei 
 solution showed significant increases ( 1.53.0-fold ) in dhe 
 oxidation ( p - value < 0.001 and < 0.01 ) ( figure 4b ) . both a549 and hek293 cells treated with cspp 
 containing either cpg(+ ) or cpg( ) had lower dhe oxidation 
 when compared with cells treated with peipp and pei solution . dhe oxidation 
 levels indicated as normalized mean fluorescence intensity ( normalized 
 mfi ) showing estimates of relative intracellular superoxide levels 
 in a549 cells ( a ) and hek293 cells ( b ) treated with indicated polyplexes . cells were treated with cspp(cpg( ) ) or cspp(cpg(+ ) ) containing 
 n / p 10 . peiss(cpg( ) ) 
 or peipp(cpg(+ ) ) are pei complexes with pdna at n / p 10 . data are 
 expressed as mean  sd ( n = 69 ) . * * * p < 0.001 , * * p < 0.01 . the results from both cell lines showed cspp had 
 a negligible impact on cell viability and cells treated with cspp 
 trended toward lower dhe oxidation when compared to cells treated 
 with peipp . they found that results from 
 the in vitro toxicity assay did not necessarily correlate 
 well with in vivo results . thus , the toxicity , oxidative stress levels , 
 and inflammatory response in an animal model needed to be assessed 
 before cspp delivery systems can be used for clinical applications 
 ( see section 3.5 ) . the effect of the presence of cpg 
 sequences within cspp on transfection efficiencies was examined initially in vitro using a549 and hek293 cell lines . the transfection 
 efficiency of cspp(cpg( ) ) at an n / p ratio of 10 and 20 was 
 significantly higher than that of the control groups for a549 as shown 
 in figure 5a . in contrast , relatively low transgene 
 expression was observed when cspp(cpg(+ ) ) were used to transfect a549 
 cells ( figure 5b ) . in the hek293 cell line , the luciferase expression pattern was similar for both cspp(cpg( ) ) 
 and cspp(cpg(+ ) ) ( figures 5c and 5d ) . it has been reported that a549 cells express the toll - like 
 receptor 9 ( tlr-9 ) , which recognizes cpg sequences in pdna , and we 
 speculate that this may have had a detrimental effect on cspp(cpg(+))-mediated 
 transfection of a549 cells . finally , 
 a549 cells gave higher luciferase expression than hek293 cells when 
 treated with cspp(cpg( ) ) . mao et al . , in studies investigating 
 transfection efficiencies of cspp in cell lines of different lineages , 
 reported differences in luciferase expression levels indicating that 
 the transfection efficiency when using cspp may be dependent on cell - type . transfection efficiency of a549 ( a , b ) and hek293 ( c , 
 d ) cells treated with cspp(cpg( ) ) ( a , c ) or cspp(cpg(+ ) ) ( b , 
 d ) . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . n / p 10 was used as a positive control for transfection 
 ( not shown in graphs ) . the luciferase expression of peipp - transfected 
 a549 cells was equal to 2.32  10  7.45  
 10 rlu / mg ( total protein ) ( for peipp(cpg( ) ) and 
 2.54  10  1.62  10 rlu / mg 
 ( for peipp(cpg(+ ) ) . the luciferase expression in hek293 cells was 
 5.38  10  3.96  10 rlu / mg 
 ( for peipp(cpg( ) ) and 4.23  10  2.92 
  10 rlu / mg ( for peipp(cpg(+ ) ) . the supernatant obtained from homogenized lungs of 
 mice treated with cspp(cpg( ) ) ( at an n / p ratio of 10 ) demonstrated 
 significantly ( p < 0.01 ) higher luciferase expression 
 ( mean , 23846 rlu / mg of total protein ; median , 23043 ) than mice treated 
 with cspp(cpg(+ ) ) ( mean , 1969 rlu / mg of total protein ; median , 777 ) 
 ( figure 6 ) . in addition , only the group in 
 which mice were treated with cspp(cpg( ) ) expressed significantly 
 higher luciferase than the controls ( p < 0.01 ) . these results are consistent with our in vitro data 
 using a549 cells . as such , a possible explanation for our in vivo transgene expression results is that cpg sequences 
 stimulate inflammatory responses that deleteriously affect transfection . each 
 treatment , aside from control and cs , contained 25 g of pdna 
 ( 12.5 g pdna/50 g , 2 nasal instillations ) . * p < 0.01 . the inflammatory response to cspp was investigated by analyzing bal fluids from variously treated mice . acute inflammatory 
 responses were assessed by enumeration of white blood cell types ( macrophages , 
 neutrophils , and lymphocytes ) 24 h subsequent to nasal instillation 
 of cspp formulations . cspp(cpg( ) ) caused a significant 9-fold 
 increase in the total number of white blood cells in the bal fluid 
 compared to control treatment , while cspp(cpg(+ ) ) caused a 7-fold 
 increase ( albeit not statistically significant ) over controls ( figure 7a ) . the majority of these infiltrating white cells 
 proved to be neutrophils ( figure 7b ) , which 
 represents a classical inflammatory response . bal cell counts : total 
 number of cells ( a ) and the number of macrophages , neutrophils , and 
 lymphocytes ( b ) in the bal fluid / mouse after nasal instillation of 
 indicated cspp formulations . each treatment ( aside from 
 control and cs ) contained 25 g of pdna ( 12.5 g of pdna/50 
 g , 2 nasal instillations ) . data are expressed as mean 
  sd ( n = 6 ) . * * p < 0.01 . concentrations of total protein 
 in bal fluid ( an indicator of the increased permeability of the alveolar 
 capillary membrane ) was significantly higher ( compared to control ) 
 in mice treated with cspp(cpg(+ ) ) . total protein concentration was 
 also enhanced ( but not significantly ) in mice receiving cspp(cpg( ) ) 
 ( figure 8a ) . activity of ldh , which denotes 
 cell membrane damage , was not statistically different in bal fluid 
 from mice treated with either cspp(cpg( ) ) or cspp(cpg(+ ) ) 
 ( figure 8b ) . there were no statistically significant 
 differences for both total protein levels and ldh activity between 
 mice treated with cspp(cpg( ) ) and with cspp(cpg(+ ) ) . finally , 
 levels of cytokines ( il-6 , il-12 , kc , and mip-1 ) were consistently 
 significantly higher in the bal fluids from mice treated with cspp(cpg(+ ) ) 
 compared to cspp(cpg( ) ) treated mice ( figure 9 ) . tnf- 
 levels were also higher in the mice receiving cspp(cpg(+ ) ) , however , 
 not significantly . these observed increases in inflammatory cytokines 
 could be due to the presence of cpg since it is well established that 
 triggering tlr-9 can result in a myd88-dependent , nf - kb - mediated upregulation 
 of inflammatory cytokines , including il-12 . we also tested for inf- ; however , levels were below the lowest 
 limit of detection ( 0.95 pg / ml ) in all samples analyzed ( data not 
 shown ) . total protein and ldh activity levels : the concentration of total 
 protein ( a ) and ldh activity ( b ) in bal fluids of mice subsequent 
 to nasal instillation of indicated cspp formulations . each treatment , aside from control 
 and cs , contained 25 g of pdna ( 12.5 g of pdna/50 g , 
 2 nasal instillations ) . data are expressed as mean  sd 
 ( n = 6 ) . * * * p < 0.001 , * * p < 0.01 . the concentration of proinflammatory cytokines / chemokines detected 
 in bal fluid of mice subsequent to nasal instillation of indicated 
 cspp formulations . llod 
 for tnf- = 1.50 pg / ml ( a ) , il-6 = 0.96 pg / ml ( b ) , il-12 = 6.38 
 pg / ml ( c ) , kc = 4.95 pg / ml ( d ) , and mip-1 = 1.3 pg / ml ( e ) . each treatment , aside from control and cs , contained 25 g of 
 pdna ( 12.5 g of pdna/50 g , 2 nasal instillations ) . data are expressed as mean  sd ( n = 6 ) . * * * p < 0.001 , * * p < 0.01 , * p < 0.05 . the results gathered from the bal fluids showed that both 
 cspp(cpg( ) ) and cspp(cpg(+ ) ) induced inflammation , as determined 
 by an increased influx of neutrophils as well as increases in total 
 protein , ldh activity , and inflammatory cytokines . mice treated with 
 chitosan solution alone trended toward increased cytokine levels in 
 their bal fluid compared to untreated mice . these levels were further 
 enhanced for all cytokines tested when chitosan was complexed with 
 cpg(+ ) pdna while cspp(cpg( ) ) treatment resulted in only marginally 
 higher levels ( tnf- , il-6 , mp-1 ) . thus , in terms of 
 cytokine levels , chitosan polyplexes made from plasmids devoid of 
 cpg sequences induced lower inflammatory responses than polyplexes 
 harboring plasmids that contained cpg sequences . the combination of plasmid dna with chitosan 
 results in polyplexes with the potential for use in pulmonary gene 
 delivery systems . in this study , we formulated cspp with 2 different 
 plasmids ( cpg( ) and cpg(+ ) ) at n / p ratios of 1 , 5 , 10 , 20 , 
 and 100 . we found that cspp(cpg( ) ) generated superior transfection 
 efficiencies both in vitro ( using a549 cells ) and in vivo ( mouse nasal instillation ) when compared with cspp(cpg(+ ) ) . in addition , cspp(cpg( ) ) generated lower levels of proinflammatory 
 cytokines ( tnf- , il-6 , il-12 , kc , and mip-1 ) in the 
 bal fluids . our findings suggest that n / p ratios and plasmid cpg sequence 
 content of cspp are important factors to consider in achieving optimal 
 gene expression with minimal toxicity and inflammation .
chitosan 
 polyplexes containing plasmid dna ( pdna ) have significant potential 
 for pulmonary gene delivery applications . 
 however , prior to using 
 chitosan / pdna polyplexes ( cspp ) in clinical applications , their potential 
 cytotoxicity needs to be investigated . in this study 
 , we formulated 
 200400 nm cspp with amine to phosphate ( n / p ) ratios that ranged 
 from 1 to 100 . 
 we compared two types of plasmids within cspp : pdna 
 that was free of cpg sequences ( cpg( ) ) and pdna that contained 
 cpg sequences ( cpg(+ ) ) . 
 both forms of cspp showed low cytotoxicity 
 when cultured with a549 and hek293 cell lines in vitro . 
 cspp(cpg( ) ) 
 generated higher luciferase expression both in vitro , for a549 cells , 
 and in vivo , compared with cspp(cpg(+ ) ) . 
 in addition , 
 cspp(cpg( ) ) 
 elicited milder inflammatory responses in mice one day subsequent 
 to nasal instillation , as determined by proinflammatory cytokine levels 
 within the bronchoalveolar lavage fluid . 
 our findings suggest that 
 to achieve optimal gene expression with minimal cytotoxicity , inflammation , 
 and oxidative stress , the n / p ratios and cpg sequences in the pdna 
 of cspp need to be considered 
 . these findings will inform the preclinical 
 safety assessments of cspp in pulmonary gene delivery systems .