we 
 developed a nanoparticle platform composed of biocompatible and biodegradable 
 polymers ( pla , plga , and pluronic ) already approved by the fda ( and 
 therefore readily applicable to clinical trials ) via a facile single - step 
 nanoprecipitation method . first , the composition ratio of the core 
 and shell layer was optimized for the preparation of tnps with stability 
 and small size ( < 100 nm ) , using plga or pla with carboxy terminals 
 as a core component , and pluronic f127 as a shell component . the tnps 
 were developed in various sizes with core polymer / shell polymer ratios 
 varying from 1:0 to 1:20 . size and zeta potentials ranged from 151 
  4 nm , 31.2  0.6 mv ( in the case of pla - based 
 tnps , 1:0 ) and 137  3 nm , 55.5  3.2 mv ( in the 
 case of plga - based tnps , 1:0 ) , to 51  3 nm , 10.3  
 0.9 mv ( in the case of pla - based tnps , 1:20 ) and 84  1 nm , 23.5 
  3.2 mv ( in the case of plga - based tnps , 1:20 ) ( supporting information , figure s1 ) . notably , 
 in the case of pla - based tnps the nps were more stable than plga - based 
 tnps in pbs with 10% fbs , as well as in a resuspended state after 
 lyophilization , indicating a stronger hydrophobic interaction between 
 the pla and pluronic polymers ( supporting information , figure s3 ) . in addition , the nps showed high encapsulation efficiency 
 ( 90% ) and loading content ( 4.3 wt % ) of lysozyme as a model protein 
 with positive charge and controlled - release kinetics up to a week 
 in pbs as well as pbs ( 10% fbs ) , implying that a sufficient amount 
 of protein drug , could be delivered within a reasonably short period 
 to the target site ( supporting information , figure s4 ) . in the cytotoxicity test , nps ranging from 0.1 to 10 
 mg / ml did not affect the metabolic activity of raw 264.7 macrophage 
 cells for both 24 and 72 h incubation periods ( supporting information , figure s5 ) . therefore , we determined 
 the optimal formulation to be pla as a core material , pluronic f127 
 as a thermosponge shell , and a core / shell ratio of 1:20 for a nanoparticle 
 platform ( with a negative charge or positive charge core ) to deliver 
 various therapeutic proteins with positive charges ( il-10 , epo , etc . ) 
 or negative charges ( insulin , hgh , etc . ) . to analyze 
 the bioactivity of il-10 released from the nps , the intracellular 
 reactive oxygen species ( ros ) generated from lps - stimulated raw 264.7 
 macrophages was measured with a widely used ros detection kit ( h2dcfda ) . before the inhibitory effect of il-10 on the ros formation was checked , 
 lps treatment conditions for cell stimulation were optimized by varying 
 the concentration of lps ( 100 , 300 , and 500 ng / ml ) and incubation 
 time ( 4 and 24 h ) . overproduction of ros increased with stimulation 
 time of lps with macrophages , whereas 
 the lps concentration ( ranging from 100 to 500 ng / ml ) did affect ros 
 generation but not in a dose - dependent manner , implying that lps with 
 100 ng / ml was enough to induce ros ( supporting 
 information , figure s7a ) . the inhibitory effect of il-10 on 
 ros production in lps - induced macrophages was investigated using various 
 concentrations ( 1100 ng / ml ) of il-10 and 100 ng / ml of lps 
 ( figure 3a ) . the dose - dependent inhibition 
 effect of il-10 on ros production was observed more clearly through 
 the pretreatment of cells with il-10 for 24 h and stimulation with 
 lps for 24 h , compared to the shorter induction time of cells pretreated 
 with il-10 and lps . to assess the 
 biological integrity / activity of il-10 released from nps , we evaluated 
 native il-10 , il-10 released from nps at 48 h , and il-10-loaded nps 
 at 50 ng / ml of il-10 and 100 ng / ml of lps . in the case of pretreatment 
 of cells with il-10 ( i.e. , the prophylactic concept ) , there was almost 
 no statistical difference between the released il-10 and native il-10 
 ( # p > 0.05 ) ( figure 3b ) . interestingly , in the case of il-10loaded nps , slightly lower 
 activity than the native and released il-10 indicated that il-10 was 
 still inside the nps based on statistical analysis ( * p < 0.05 ) , suggesting both efficient loading and controlled release . more importantly , in the case of post - treatment of cells with il-10 
 ( i.e. , the therapeutic concept ) , the inhibitory effect on ros production 
 was enhanced with all samples ; most notably , the bioactivity of released 
 il-10 was similar to that of the native protein ( figure 3c ) , indicating that it was maintained during both loading 
 and release . to determine whether the 
 il-10 released from nps inhibits proinflammatory 
 cytokines produced by inflammatory responses , the relative gene expression 
 levels of tnf- and il-12 were compared with the native il-10 
 through qpcr ( figure 3d ) . expression was dramatically 
 increased after lps stimulation and the released il-10 reduced the 
 expression of cytokines ( ca . 2.5-fold ) , suggesting that the released 
 il-10 not only retains bioactivity , but also functions as an anti - inflammatory 
 cytokine , with results similar to native il-10 . in addition , il-10 
 treatment increased sil-1ra promoter activity by 1.5-fold compared 
 to lps alone , and the released il-10 had effects identical to those 
 of the native protein , consistent with the unique response of il-10 
 to the gene expression of the secretory interleukin ( il)-1 receptor 
 antagonist ( sil-1ra ) for which previous studies demonstrate the potential 
 to treat metastatic cancers . the gene 
 expression of several cytokines analyzed in this study suggests that 
 the bioactivity of il-10 released from the nps was successfully maintained 
 inside the hydrophilic shell . western blots were also employed 
 to determine whether the bioactivity of released il-10 is well maintained . 
 because il-10 has been known to signal via the activation of the signal 
 transducer and activator of transcription 3 ( stat3 ) , which is a key 
 mediator of the inflammatory response of macrophages and other immune 
 cell types , the levels of stat3 and phosphorylated 
 stat3 ( p - stat3 at tyr705 ) were measured . compared to the control group , 
 the total levels of stat3 were slightly increased by il-10 treatment , 
 using -actin as a reference protein ( figure 3e ) . moreover , the clear band of p - stat3 was observed in the 
 native il-10 group , indicating the activation of stat3 by il-10 treatment . the levels of stat3 and p - stat3 in the native il-10 and released il-10 
 groups ( respectively ) were almost identical , suggesting that il-10 
 maintained biological integrity throughout the loading process and 
 after release . the 
 bioactivity of insulin released from tnps was analyzed via insulin - dependent 
 proliferation of mcf-7 , as reported previously . it was confirmed that the proliferation of mcf-7 cells 
 was insulin dose - dependent from 1 to 500 nm ( supporting 
 information , figure s8 ) . we compared the enhancement in cell 
 growth ( over the control group ) produced by native insulin and released 
 insulin ( 6 and 48 h postrelease ) at 10 nm concentration in serum - free 
 medium ( figure 3f ) . as expected , the released 
 insulin produced almost the same increase in cellular metabolic activity 
 as native insulin ( no statistical differences , p > 
 0.05 ) , indicating that the bioactivity of proteins released from tnps 
 was well maintained . therefore , based on the results of ros , qpcr , 
 western blot , and protein - dependent cell proliferation assay , the 
 newly developed nanoparticle platforms ( negatively charged and positively 
 charged tnps ) successfully preserve the bioactivity of loaded proteins . bioactivity 
 of proteins released from tnps . ( a ) inhibitory effects 
 on reactive oxygen species ( ros ) production by il-10 at various concentrations 
 ( 1100 ng / ml ) . intracellular ros generated from raw 264.7 macrophage 
 cells by lps stimulation was measured using a ros detection reagent . bioactivity analysis of the inhibitory effects of native il-10 , released 
 il-10 , and loaded il-10 on ros production ( b ) by pretreatment and 
 ( c ) by post - treatment of il-10 ( n = 3 , * p < 0.05 , # p > 0.05 ) . ( d ) relative 
 mrna expression of tnf- , il-12 , and sil-1ra after lps treatment 
 ( 500 ng / ml ) for 4 h , followed by treatment with il-10 ( native il-10 
 or released il-10 at 20 ng / ml ) for 2 h at 37 c ( n = 3 , # p > 0.05 ) . ( e ) western blots were performed 
 to analyze the bioactivity of il-10 released from tnps after treatment 
 with il-10 ( native il-10 or released il-10 at 20 ng / ml ) for 24 h at 
 37 c . # 1 , control ; # 2 , native il-10 ; and # 3 , released il-10 . ( f ) bioactivity analysis of native insulin and released insulin ( 10 
 nm ) on the improved proliferation effect of insulin - dose - dependent 
 human breast cancer cell line mcf-7 ( n = 3 , # p > 0.05 ) . protein - loaded 
 tnps [ il-10@tnps ( 100 g il-10/kg ) and insulin@tnps ( 1 u insulin / kg ) ] 
 were administered intravenously to mice , and blood samples were collected 
 at different time points to analyze the serum concentration of proteins 
 ( figure 4 ) . the mean pharmacokinetic parameters 
 of the proteins were assessed by noncompartmental analysis ( table 1 ) . after intravenous ( iv ) administration of il-10 
 alone , the serum il-10 concentration rapidly decreased until the 8 
 h point , whereas the concentration was maintained up to 24 h when 
 using tnps ( figure 4a ) , increasing the area 
 under the serum concentration  time curve ( auc ) 1.9-fold ( from 
 50.52 to 97.73 nghr / ml ) ( table 1a ) . in 
 addition , il-10loaded tnps reduced the clearance 1.9-fold 
 ( from 1979.57 to 1023.18 mlhr-1kg ) 
 and improved the half - life 5.9-fold ( from 0.25 to 1.48 h ) compared 
 with il-10 alone . similar results were obtained when insulin - loaded 
 tnps were injected into mice ( figure 4b ) . the 
 insulin - loaded tnps produced a remarkable increase in systemic exposure 
 ( 30.9-fold , from 25.97 to 804.99 uhr / ml ) compared with 
 the insulin - alone group ( table 1b ) . the group 
 using tnps also showed significantly reduced clearance ( 31-fold ) and 
 prolonged half - life of insulin ( 1.6-fold ) . our results demonstrate 
 that the tnp platform enhances systemic exposure , reduces clearance , 
 and improves half - life of therapeutic proteins , suggesting that tnps 
 could become standard tools for efficient delivery of proteins for 
 in vivo biomedical applications . pharmacokinetic parameters of 
 ( a ) il-10 and il-10loaded tnp and ( b ) insulin and insulin - loaded 
 tnp administered intravenously to mice . time 
 curve ; vss , volume of distribution at steady state ; mrt , mean residence 
 time . changes in serum protein 
 levels in mice after intravenous administration of ( a ) il-10 and il-10loaded 
 tnp , and ( b ) insulin and insulin  loaded tnp . the serum concentrations 
 of proteins were measured at several time points using elisa kits 
 ( mean  sem , n = 3 ) .
protein therapeutics have gained 
 attention recently for treatment 
 of a myriad of human diseases due to their high potency and unique 
 mechanisms of action . 
 we present the development of a novel polymeric 
 thermosponge nanoparticle for efficient delivery of labile proteins 
 using a solvent - free polymer thermo - expansion mechanism with clinical 
 potential , capable of effectively delivering a range of therapeutic 
 proteins in a sustained manner with no loss of bioactivity , with improved 
 biological half - lives and efficacy in vivo .