drug delivery systems ( ddss ) are useful for reducing drug side effects and maximizing drug action .
the design of drug carriers for dds is the most important activity in this area .
the advent of molecular biology studies has enabled the identification of many disease - causing proteins . because some of these are effective as drugs ,
a variety of nanoparticles such as liposomes , micelles , and polymers have been adopted as drug carriers [ 14 ] . because polymers are similar in size to proteins , they are not suitable as protein carriers . whereas liposomes and micelles are larger than polymers and proteins , they can be used as protein carriers . however , because these are self - assembled nanoparticles , some treatments are necessary for their preparation as carriers .
since most proteins are sensitive to temperature , ph , and organic solvents , it is possible that such treatments induce protein denaturation .
hydroxyapatite ( ha ) , ca10(po4)6(oh)2 , is a major component of hard tissues such as bones and teeth and has been used as a biomaterial [ 5 , 6 ] . because it has been reported that some proteins , such as bovine serum albumin ( bsa ) and lysozyme , can bind to ha just by mixing ,
it is a good candidate for a protein carrier [ 79 ] . in this study
, we investigated the association and dissociation behavior of two bioactive proteins , cytochrome c and insulin , to ha .
it is known that the release of cytochrome c from mitochondria to the cytosol induces apoptosis .
therefore , the delivery of cytochrome c into the cytosol of cancer cells should induce apoptosis , which may be useful for cancer therapy .
insulin , a key protein of diabetes , is commonly injected into diabetic patients to suppress blood sugar levels , and its controlled release can markedly improve their quality of life . because the delivery of these two proteins is important , we attempted to use ha for a delivery system .
the absorption and desorption on ha were affected by the surface conditions dependent on the preparation procedure of ha .
considering universal use of ha for protein delivery , commercially available ha was used as a carrier in this study .
ha nanoparticles were purchased from sigma - aldrich ( mo , usa ) . according to the material data sheet ( no .
677418 ) , the size and surface area were smaller than 200  nm and larger than 9.4  m / g , respectively .
cytochrome c is cationic , and insulin is anionic at physiological ph , and the molecular weight of cytochrome c is larger than that of insulin .
adsorption experiments with cytochrome c were performed by mixing ha ( 0 , 10 , 20 , and 30  mg ) with an aqueous solution ( 2.5  mg / ml , 200  l ) .
after 4  h mixing with a rotator , centrifugation ( 15,000  g , 20c , 60  min ) was performed to collect the supernatants .
these were analyzed by reversed phase high performance liquid chromatography ( hplc ) to estimate the residual concentration of cytochrome c. the hplc system was equipped with a cosmosil 5c18-ms - ii column ( nacalai tesque , inc . , kyoto , japan ) and a uv detector ( 220  nm ; uv-2075plus , jasco inc . , tokyo , japan ) .
samples ( 5  l ) were injected with an autosampler ( as-2057plus , jasco inc . , tokyo , japan ) and eluted with acetonitrile/0.05% trifluoroacetic acid = 20/80 ( a ) and acetonitrile/0.05% trifluoroacetic acid = 60/40 ( b ) at 1.0  mlmin by pu-2089plus ( jasco inc . ,
a linear gradient elution was performed over 20  min from an initial state ( a ) 100% to the final state ( b ) 100% . in the case of insulin adsorption ,
the same experimental procedures were performed except the insulin solution was prepared by dissolving it in 0.01  n hcl and adjusting to ph 3 .
association ratio ( % ) was calculated as [ ( c0c)/c0 ] 100 , at which c0 and c are the initial concentration and the supernatant concentration of proteins , respectively . during desorption experiments ,
ha ( 10 and 20  mg ) absorbing cytochrome c and insulin was transferred into 400  l of phosphate buffer saline ( pbs ; 8  mm na2hpo4 , 2  mm kh2po4 , 137  mm nacl , 3  mm kcl ) , and rotated . after predetermined incubation times , centrifugation ( 15,000  g , 20c , 60  min ) was performed to collect the supernatants .
the residual concentrations of cytochrome c and insulin were estimated by hplc . in the case of insulin ,
dissociation ratio ( % ) was calculated as [ c / c0 ] 100 , at which c0 and c are the total concentration of the associated proteins and the supernatant concentration , respectively .
therefore , insulin was dissolved in an acidic solution ( ph 3 ) . after the incubation and subsequent centrifugation
, the residual cytochrome c and insulin in the solution were estimated from the hplc analysis .
cytochrome c and insulin were eluted after 10  min and 13  min , respectively , under the running conditions ( figure 1(a ) ) , and the peak areas were proportional to the protein concentrations ( figure 1(b ) ) .
thus , the protein concentrations in the supernatants were evaluated by hplc analysis and the adsorbed amounts were calculated by subtracting the concentrations in the supernatant from the initial ones .  
the adsorption efficiency of insulin was higher than that of cytochrome c. as less as 10  mg ha was sufficient to load almost 0.5  mg insulin , but 30  mg ha is necessary to load the same amount of cytochrome c under this condition .
this may be because cytochrome c is larger than insulin . from the surface area of the ha ( > 9.4  m / g ) and the protein diameters ( d ) of insulin ( 3  nm ) and cytochrome c ( 4  nm ) , the percent of occupied area can be estimated . in the experiment ,
0.5  mg of proteins , that is 9  10mol ( 5  10 molecules ) of insulin and 4  10mol ( 2  10 molecules ) of cytochrome c , were used .
the projected area of proteins may be approximated as (d/2 ) , assuming the protein to be spherical .
given that all molecules were absorbed , the occupied areas of insulin and cytochrome c were 0.4  m and 0.3  m , respectively .
the surface area of ha ( 10  mg , 20  mg , and 30  mg ) can be calculated as > 0.094  m , > 0.19  m , and > 0.28  m , respectively .
even though the absorption amount of cytochrome c could be correlated to the total surface area of ha , that of insulin could not .
long - term incubation contributed to the efficient loading of cytochrome c even with a small amount of ha .
the initial absorption occurred in less than 1  h , and the subsequent absorption occurred more slowly , in the range of an hour .
the first absorption phase might be attributed to surface absorption and the latter by penetration into the pores .
the release profiles also occurred over two phases , in less than 1  h and over the hour range ( figure 3 ) , similar to the adsorption profile .
this result suggests that absorbed proteins at the surface were released very fast and those within the pores were released more slowly .
thus , regulation of release can be achieved by the control of protein size and the pore size of ha .
it is possible that step - by - step protein release can be performed in an ha - based delivery system . 

less than 40% of the bound insulin was released from ha , even though 70% of cytochrome c was released after 24  h. insulin is smaller than cytochrome c and readily bound to ha ( table 1 and figure 2 ) , but the release of insulin is slower than that of cytochrome c ( figure 4 ) .
it is likely that the slow release may be caused by its poor solubility in pbs , compared to cytochrome c. the release of insulin was next examined at ph 3 , because insulin is easily soluble in acidic solution , which is a condition of the association .
however , the release at ph 3 was slower than that at ph 7.4 and was perhaps affected by the charge of insulin .
insulin has an isoelectric point ( pi ) of 5.3 so is positively charged at ph 3 and negatively at ph 7.4 .
a decrease in insulin release was observed , especially at ph 7.4 after more than 5  h. the readsorption of the released insulin to ha might have occurred , because the desorption conditions differed from the absorption condition .  
our results suggest that the association and dissociation properties to ha were affected by both the charge and size of proteins .
ha has a hexagonal structure , in which the c ( ca - rich ) site is arranged in the a 
c and b  c planes and the p ( ca - deficient ) site is in the a 
it was reported that anionic molecules bind to the c site and cationic ones to the p site .
therefore , ha - based protein delivery is suitable for ph - dependent controlled release .
cationic cytochrome c and anionic insulin at the physiological ph were absorbed and desorbed in different manners .
because the charge of insulin was changed with decreasing ph , it markedly influenced the adsorption and desorption behaviors .
the absorption behavior may be very complex , because large protein molecules bind to ha at multiple points .
therefore , the regulation of controlled release of protein is still to be investigated ( for further information , see supplementary material available online at doi:10.1155/2012/932461 . ) .
in conclusion , we prepared protein - associated ha and characterized its association and dissociation properties .
. however , their association and dissociation behaviors differed , depending on the size and charge of the proteins .
 hydroxyapatite ( ha ) is a precursor of bone and has been studied as a biomaterial . 
 we attempted ha to apply to protein delivery systems . in this study , 
 the association and dissociation properties of two types of bioactive proteins , cytochrom c and insulin , to ha were investigated . 
 cytochrom c was less associated with ha than insulin , which was easily released from it . 
 however , the release of insulin from ha was slow . 
 insulin was released from ha at ph 7.4 more rapidly than at ph 3 . 
 the association and dissociation properties might be influenced by the size , solubility and net charge of protein . 
 ha is a potential protein carrier with controlled release . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions