collagen is a major connective tissue protein that plays an important role in the extracellular matrix in animals . as such atelocollagen is a type of soluble collagen produced from tropocollagen , the collagen molecule that makes up collagen fibrils , via the elimination of the telopeptide moieties , which are considered to account for most of collagen 's antigenicity [ 1 , 2 ] . thus , atelocollagen is considered to have little immunogenicity , which makes it a safe biomaterial . for example , a minipellet atelocollagen formulation has been demonstrated to sustain the release and maintain stable blood concentrations of protein drugs for more than 1 week . many kinds of protein drugs such as interferon- , interleukin-2 , nerve growth factor , and bone morphogenetic protein , and so forth , have been administered using this drug delivery system , and interferon- and interleukin-2 showed strong antitumor activities in animal models when administered in this manner [ 3 , 4 ] . in the past decade , as well as being used as a solid substrate , dissolved atelocollagen has been used as a drug delivery vehicle for nucleic acid - based medicines for gene conversion , inflammatory disease [ 8 , 9 ] , and tumor therapy . atelocollagen can be used to deliver most kinds of nucleic acid - based medicines including plasmid dna , antisense oligodeoxynucleotides ( odn ) [ 1113 ] , short interference rna ( sirna ) [ 1420 ] , and micro rna ( mirna ) [ 2123 ] . it is also capable of delivering oligonucleotides to subcutaneous xenografts and metastatic tumors after its local and/or systemic administration . many studies , including some involving in vivo tumor models , have evidenced the contribution of atelocollagen to the enhancement of drugs ' antitumor activities , and some of them described the mechanisms . for example , nucleic acids delivered by atelocollagen are protected against degradation by host nucleases [ 8 , 14 , 24 ] , and it has also been shown to improve the delivery efficiency of oligonucleotides to tumors [ 15 , 16 ] . however , the biological functions of atelocollagen and the mechanism by which it enhances delivery efficiency are still not fully understood . it is essential to reveal the biological characteristics of atelocollagen in order to be able to fully exploit its drug delivery potential . while we were studying the basic properties of atelocollagen , we discovered another of its functions : it increases endothelial permeability . here , we describe the results of a study of the effects of atelocollagen on intercellular sealing function . we measured transendothelial electrical residence ( ter ) in order to estimate intercellular barrier function and performed an immunohistochemical examination to see whether any cellular morphological changes were induced . rhodamine red - conjugated atelocollagen was prepared in accordance with the manufacturer 's instructions ( fluoreporter rhodamine red - x protein labeling kit ; life technologies japan , tokyo , japan ) . the oligodeoxynucleotides ( odn ) and double stranded rna ( dsrna ) were synthesized by eurogentec ( seraing , belgium ) . the sequences of the oligonucleotides are listed in table 1 [ 20 , 25 , 26 ] . each atelocollagen - oligonucleotide formulation ( ac formulation ) was prepared by gently mixing aqueous atelocollagen with a solution containing a defined concentration of oligonucleotides . the final oligonucleotide concentration was usually 5  m and that of atelocollagen was 0.1%  w / v unless otherwise stated in the text , tables , and/or figures . normal human dermal microvascular endothelial cells ( hmvec ) were purchased from eidia ( tokyo , japan ) . these cells were cultured in egm ( endothelial growth media ; eidia , tokyo , japan ) until they reached confluence on 12  mm transwell filters with a 0.4  m pore size ( corning glass works ; corning japan , tokyo , japan ) coated with rat tail collagen . porcine brain microvascular endothelial cells ( bmvec ) were purified and maintained according to the method described in a previous study . ter was determined using an evom voltohmmeter and an endohm-12 chamber ( world precision instruments , sarasota , fl ) at 37c . cell growth was monitored by measuring ter . once stable intercellular seals had formed ; that is , at confluence , the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30%  v / v of ac formulation . one hour after treatment with the ac formulation , which was performed as described in section 2.2 , 100  l of 0.36%  w / v texas red - conjugated dextran ( mw : 40  kda ; life technologies japan , tokyo , japan ) were added to the inner chamber . one hour later , the dextran concentration of the medium in the outer chamber was analyzed by measuring its fluorescence . to investigate the enhancement of paracellular transport by atelocollagen , solute transportation was compared among the ac formulation , bovine serum albumin , and dextran . once stable intercellular seals had formed , the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30%  v / v of the ac formulation , which had been produced using 0.1 or 0.3%  w / v rhodamine red - conjugated atelocollagen ( i.e. , approximately 0.03 or 0.1%  w / v atelocollagen was added ; molecular weight ( mw ) : 300  kda ) ; 0.1%  w / v of fluorescein conjugated dextran ( mw : 70  kda ; life technologies japan , tokyo , japan ) ; 0.1%  w / v of alexa fluor 594 conjugated bovine serum albumin ( bsa ; mw : 66  kda ; life technologies japan , tokyo , japan ) . the solute concentrations of the outer chambers were analyzed by measuring their fluorescence at 1 and 2 hours after the medium exchange . after treatment for 1  hr with the ac formulation , oligonucleotide alone , atelocollagen alone , or phosphate - buffered saline ( pbs ) as a control , hmvec cells were fixed with 1% paraformaldehyde for 10  min and then treated with 0.2% triton x-100 for 10  min . after preincubation with 5% skimmed milk , they were incubated for 1  hr at room temperature with rabbit or mouse antibodies against vascular endothelial ( ve)-cadherin ( bd biosciences , san diego , ca ) , zonula occludens-1 ( zo-1 ) ( zymed laboratories , san francisco , ca ) , claudin-5 ( zymed laboratories , san francisco , ca ) , and -tubulin ( amersham , poole , uk ) . then , the samples were incubated for 1  hr with appropriate secondary antibodies labeled with alexa fluor-488 or alexa fluor-596 ( life technologies japan , tokyo , japan ) . actin filaments were labeled with alexa fluor-546 phalloidin ( life technologies japan , tokyo , japan ) . the expression of each protein was examined using a laser scanning confocal microscope ( mrc 1024 ; bio - rad , hercules , ca ) . western blotting was performed according to the method described in a previous report . for western blotting of the total cell lysates , the dishes were washed with pbs , and then 300  l of sample buffer ( 1  mm nahco3 and 2  mm phenylmethylsulfonyl fluoride ) was added to 60  mm culture dishes . the cells were scraped and collected in microcentrifuge tubes and then sonicated for 10  sec . the protein concentrations of the samples were determined using a bca ( bicinchoninic acid ) protein assay reagent kit ( pierce chemical , rockford , il ) . for each sample , aliquots of protein ( 15  g per lane ) were separated by electrophoresis in 4/20% sodium dodecyl sulfate polyacrylamide gels ( cosmo bio , tokyo , japan ) ( sds page ) . after being electrophoretically transferred to nitrocellulose membranes ( immobilon ; millipore , billerica , ma ) , the membranes were saturated with blocking buffer ( trisbuffered saline [ tbs ] supplemented with 0.1% tween 20 and 4% skimmed milk ) for 30  min at room temperature and incubated with antiactin , anti - zo-1 , anti - ve - cadherin ( bd biosciences , san diego , ca ) , anticlaudin-5 ( zymed laboratories , san francisco , ca ) , anti - p38 mitogen - activated protein kinase ( map kinase or mapk ) ( santa cruz biotechnology , santa cruz , ca ) , antiphospho - p38 mapk ( cell signaling , beverly , ma ) , anti - p42/44 mapk ( promega , madison , wi ) , antiphospho - p42/44 mapk ( cell signaling , beverly , ma ) , anti - rho - a , and anti - cdc42 ( santa cruz biotechnology , santa cruz , ca ) antibodies ( 1:1000 ) for 1  h at room temperature . the membranes were then incubated with horseradish peroxidase - conjugated anti - rabbit or mouse igg ( dako a / s , copenhagen , denmark ) at room temperature for 1  h. the immunoreactive bands were detected using an ecl western blotting analysis system ( ge healthcare , little chalfont , uk ) . paracellular flux is dependent on the function of tight junctions [ 30 , 31 ] . we assessed the effects of an ac formulation on the ter of hmvec to evaluate their tight junction function . as shown in figure 1 and table 1(a ) , the odn containing ac formulation caused a time - dependent reduction in ter , while ter was hardly affected by treatment with odn or atelocollagen alone . as for the type of oligonucleotide in the formulation , phosphorothioate odn produced a more significant reduction in ter than phosphodiester odn , which only produced slight alterations . various formulations containing different ratios of odn and atelocollagen were examined in order to understand which parameters have the greatest effect on the change in ter . as a result , we found that the ter change was dependent on the size of the odn and the composition of the formulation , but not the odn sequence , as shown in tables 1(b ) , 1(c ) , and 1(d ) . specifically , odn composed of 15 or more bases were effective and those containing around 30 bases were the most effective , but 10-base - long odn were not effective . the change in tight junction function was also dependent on the concentrations of odn and atelocollagen in the formulation . to verify that the ac formulation increased paracellular flux , the amount of texas red - labeled dextran ( molecular weight : 40  kd ) transported across an endothelial cell layer was examined . as shown in figure 2(a ) , dextran transport was increased approximately twofold in the cell cultures incubated with the ac formulation . next , the paracellular transport of atelocollagen was analyzed and compared with those of bsa and dextran . only very small amounts of bsa and dextran penetrated the cell sheet during the 2-hour study period ; on the other hand , much more atelocollagen passed through , even though the molecular weight of atelocollagen is 4 - 5 times higher than those of bsa and dextran ( figure 2(b ) ) . an examination using bmvec   was performed to determine whether the effect of the ac formulation was specific to hmvec . as a result , we found that the ter value of the bmvec was also reduced by the ac formulation ( and only the ac formulation ) , as shown in figure 3 . bmvec forms the blood - brain barrier ( bbb ) , where intercellular sealing function is strictly maintained . these results showed that the ac formulation is able to affect the paracellular flux of endothelial barriers . it is well known that increased endothelial permeability is associated with impaired intercellular contact [ 3235 ] . we carried out an immunohistochemical analysis of the cells treated with the ac formulation to clarify how their intercellular sealing was affected . as shown in figure 4(a ) , treatment with the ac formulation markedly reduced the degree of intercellular contact , as shown by intercellular gap formation , actin stress fiber formation , cellular contraction , and a lack of ve - cadherin . adequate expression of claudin-5 , one of the key components of the endothelial barrier , was noted at the cell periphery . however , zo-1 protein expression was absent from the intercellular gaps . on the contrary , western blotting revealed that treatment with the ac formulation did not affect the expression of these proteins ( figure 4(b ) ) . although the ter value remained low as long as the ac formulation was present in the culture medium , the treatment did not cause toxicity . the cells survived well for at least 24  hrs , and both the ter and morphology of the cells could be recovered by removing the formulation ( data not shown ) . no such morphological changes were induced by treatment with odn or atelocollagen alone ( figure 4(a ) ) . microtubules play an important role in regulating actin formation and hence , endothelial barrier function [ 36 , 37 ] . as shown in figure 5 , -tubulin formed a fine network in the blank control . however , treatment with the ac formulation caused the peripheral fine structure of the -tubulin network to be lost . many studies have shown that increased endothelial permeability and impaired intercellular contact can be induced by signal transduction , mainly that of rho a [ 32 , 38 ] and p38 map kinase [ 36 , 3942 ] . thus , we investigated the effects of the ac formulation on signal transduction . as a result , no differences were found in the expression levels of rho - a , cdc42 , or p42/44 map kinases or their phosphorylated forms . regarding p38 map kinase , although no changes were noted among the control , oligonucleotide alone , or atelocollagen alone groups , the levels of phosphorylated p38 map kinase were markedly increased in the cells treated with the ac formulation ( figure 6 ) , which indicates that the impact of the ac formulation on tissue permeability is associated with the activation of p38 map kinase . collagen plays an important role in the extracellular matrix by supporting cells so that they can form tissues and organs . atelocollagen is produced from type i collagen and is widely used in its solid state as a biomaterial for medical and surgical products because of its biocompatibility and workability . however , the kinetics , dynamics , and biological functions of atelocollagen after its injection into the living body are still poorly understood , and it is essential to elucidate the characteristics of atelocollagen in order to fully exploit its potential . here , we demonstrated a novel biological function of atelocollagen . when endothelial cell sheets were treated with atelocollagen or oligonucleotides alone , the intercellular structure of the sheet was not changed . however , when the atelocollagen and oligonucleotides were administered together , intercellular gaps formed and consequently the paracellular flux of the sheet was elevated . the abovementioned changes were elicited via the activation of p38 map kinase , a signal - transduction - related molecule . in addition , the changes observed in this study are similar to those triggered by thrombin , histamine , tnf- [ 34 , 36 ] , and vegf ( vascular endothelial growth factor ) , and so forth . as shown in section 3.1 , the degree to which endothelial function was affected was dependent on the molecular structure of the oligonucleotides including their size and chemical modifications , suggesting that the three - dimensional structure of the oligonucleotide and atelocollagen complex stimulates a signal transduction pathway that acts as a permeability modulator , although the specific pathway that it stimulates remains unknown . to date , no severe systemic edema or side effects of the ac formulation have been noted , even after the intravenous administration of atelocollagen as an oligonucleotide drug carrier . these findings indicate that atelocollagen could be used as a permeability enhancer at local treatment sites without the adverse systemic effects that cytokines and chemokines sometimes provoke . since tight junction modulators are regarded as practical drug delivery enhancer candidates [ 4446 ] , the function of atelocollagen demonstrated in the present study should be thoroughly investigated . the unique biological functions of atelocollagen have led to the development of unique antitumor therapies and products , such as surgical products ; formulations that sustain the release of antitumor proteins [ 24 ] ; treatments that enhance the antitumor activities of various molecules including antisense odn [ 1113 ] , sirna [ 1420 , 24 ] , and mirna [ 2123 ] . obtaining more information about atelocollagen would allow us to develop the next generation of atelocollagen - mediated drug delivery systems .
atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial . to date 
 , atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models . 
 however , the biological effects of this technology are not fully understood . in the present study , we investigated the effects of atelocollagen on endothelial paracellular barrier function . 
 an atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells , and the change was dependent on the molecular size , structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation . 
 an immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins . 
 these changes were induced through p38 map kinase signaling . 
 it is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties .