pbs buffer solutions with different ph values can change

Dental Materials Journal 24(3) : 414-421, 2005

PBS Buffer Solutions with Different pH Values Can Change Porosity of

DNA-chitosan Complexes

Tadao FUKUSHIMA1, Tohru HAYAKAWA2, Minoru KAWAGUCHI1, Rieko OGURA3, Yusuke INOUE4, Koichiro MORISHITA5 and Koji MIYAZAKI1 'Department of Dental Engineering, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 2Department of Dental Materials, Research Institute of Oral Science, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho Nishi, Matsudo, Chiba 271-8587,Japan 3Department of Functional Bioscience, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 4Fukuoka College of Health Sciences, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 5Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan

Corresponding author, E-mail : [email protected]

Received June 20, 2005/Accepted July 26, 2005

We examined the effect of phosphate-buffered saline (PBS) solution with different pH values on the formation of porosity in the DNA/chitosan complexes, and evaluated the intercalation behavior of the complexes . Four different PBS solutions with pH = 6.0, 7.0, 7.4, and 7.8 were used for rinsing water-insoluble DNA-chitosan complexes . All complexes showed high porosities ranging from 83 to 95%. Rinsing with PBS at pH 7.0, 7.4, and 7.8 reduced the porosity of the DNA-chitosan com-plexes. Re-rinsing with PBS at pH 7.4 reduced the porosity of the DNA-chitosan complex rinsed with PBS at pH 6.0. The appearances for porous formation were influenced by the differences in pH of PBS . Daunorubicin hydrochloride intercalated and bound in the grooves of DNA within all of the DNA-chitosan complexes , indicating that DNA in the complexes main-tained its double-stranded helical structure. These results suggested that PBS-rinsed DNA -chitosan complex is promising as a scaffold material in tissue engineering.

Key words : Porosity, DNA-chitosan, PBS

INTRODUCTION

Deoxyribonucleic acid (DNA) is an important com-

pound not only as a source of biological information, but can also be regarded as a naturally occurring and highly specific functional biopolymer1) . It is ex-pected that DNA has some favorable characteristics for use as a biomaterial — as a scaffold or drug de-livery material. For example, DNA has phosphate

groups — which have a high affinity for calcium ions, and it is less antigenic2). Moreover, DNA can inter-act with antibiotics and proteins3,4) and can be hydro-lyzed by enzymes such as Dnase I 5) .

Okahata and Tanaka6) synthesized water-insoluble DNA-cationic lipid complexes from interaction be-tween DNA and cationic lipids, and then prepared DNA-lipid films by casting DNA-cationic lipid com-

plexes. We also prepared water-insoluble DNA-lipid complexes from the reaction between DNA and newly synthesized cationic lipids — which were derived from amino acids — in order to provide DNA-lipid films as biomaterials7,8) . We found that DNA-lipid films showed no inflammation reactions in the subcutane-ous tissues of rats and in their intercalation with ethidium bromide (EB) . These results indicated that DNA-cationic lipid complexes have a potential to be scaffold or drug delivery materials because they maintained a double helical structure and showed wa-ter-insoluble property.

Based on the above findings, we recently pre-

pared DNA-chitosan complexes from anion-cation re-action with a view to evaluating their potential as biomaterials9) . Chitosan is a nontoxic , biodegradable, and cationic polymer10-12) which also contains ammo-nium groups, and which exhibits beneficial biological activities such as accelerating wound healing10) and enhancing the appearance of healed skin") . DNA in the complexes maintained a double helical structure , and as expected, DNA-chitosan complexes showed

good biocombability9) . Moreover, we found that the porosity of DNA-chitosan complexes could be con-trolled by rinsing them with different buffer solu-tions at pH 7.2, including phosphate-buffered saline (PBS) , Tris-HC1, boric acid, and N- (2-hydroxyethyl)

piperazine-N'- (2-ethanesufonic acid) (HEPES) buffer9) . For example, rinsing the complexes with PBS re-sulted in 84% porosity, whereas rinsing with Tris-HC1 produced 94% porosity.

Prerequisites for a scaffold material included ade-

quate porosity for cell seeding, cell growth, and extracellular matrix productionl3-15) . Since scaffolds are exposed to a variety of buffers or body fluids during application, understanding the effect of buffer conditions on the formation of porosity in the DNA-chitosan complexes is absolutely important. Yao et al.16) reported that the swelling of glutaraldehyde-crosslinked chitosan/gelatin hydrogel rapidly de-creased between pH 6 and 7. Therefore , we predicted

FUKUSHIMA et al. 415

that alternation in pH might affect the porosity of DNA-chitosan complexes.

In the current study, we examined the effect of PBS with different pH values on the formation of po-rosity in the DNA-chitosan complexes, and evaluated the intercalation behavior of the DNA-chitosan com-

plexes.

MATERIALS AND METHODS

Preparation of DNA-chitosan complexes Chitosan (500 mg; average molecular weight =129,000; Nichro Co., Ltd., Tokyo, Japan) was dissolved in 40 ml of 0.2 N HC1. NaOH solution (0.2 N) and dis-tilled water were gradually added to the chitosan to bring the pH to 5.0 and the volume to 100 ml. Salmon testes DNA (500 mg; 300 base pairs (bps) ; Nichro Co., Ltd., Tokyo, Japan) was dissolved in 100 ml of distilled water. DNA aqueous solution was added to the chitosan-NaOH solution and the mix-ture was stirred at 20C for one hour. The pH of the reaction mixture was essentially constant during complex formation, and was approximately 5.2 after the reaction. The DNA-chitosan complexes (Fig. 1) were collected by centrifugation at 9000 rpm for 10 minutes and washed with distilled water. This proc-ess was repeated two more times. After which, the DNA-chitosan complexes were frozen in liquid nitro-

gen and dried for 24 hours in a freeze-dryer (FD-5N, Eyela, Tokyo, Japan) .

To measure intercalation and groove binding, the frozen DNA-chitosan complex was cracked with a frozen cell crasher (CRYO-PRESS, Microtec Nition, Funabashi, Japan) and then freeze-dried for 24 hours.

Rinsing of DNA-chitosan complexes with phosphate-buffered salines (PBS) having different pH Four different PBS solutions with pH = 6.0, 7.0, 7.4, and 7.8 were used for rinsing the DNA-chitosan com-

plexes. Collected DNA-chitosan complexes were

rinsed for 30 seconds with one of the four types of PBS (10 mM) , and then collected by centrifugation. This procedure was repeated twice. Collected DNA-chitosan complexes were then washed with distilled water and collected by centrifugation. Washing with distilled water was repeated twice. Finally, the buffer-rinsed DNA-chitosan complexes were freeze-dried. The un-rinsed DNA-chitosan complex was used as a control.

Re-rinsing of DNA-chitosan complexes with phos-

phate-buffered saline at pH 7.4 Freeze-dried DNA-chitosan complexes were first rinsed with one of the three PBS at pH = 6.0, 7.0, and 7.8 as described above. Then, they were re-rinsed for 30 seconds with PBS at pH 7.4 for 30 seconds. After which, they were collected by centrifugation. This

procedure was repeated twice. Collected DNA-chitosan complexes were then washed with distilled water and collected by centrifugation. Washing with distilled water was repeated twice. Finally, the PBS re-rinsed DNA-chitosan complexes were freeze-dried.

Determination of the binding of DNA to chitosan in the DNA-chitosan complexes The binding ratio of DNA to chitosan in the un-rinsed and PBS/pH7.4 rinsed DNA-chitosan com-

plexes were estimated from the amount of incorpo-rated phosphate. Phosphate was determined using the molybdenum blue method17) and measured using a U/2001 spectrophotometer (Hitachi Ltd., Tokyo, Japan) . Measurements were performed in triplicate. The DNA-chitosan binding ratio was calculated based on the following formula for the incorporated phos-

phate in percentage:

where P (%) is the phosphate percentage measured,

p is the total weight of phosphate in DNA, D is the molecular weight of DNA, c is the molecular weight

Fig. 1 Schematic illustration of the DNA-chitosan complex.

416 POROSITY OF DNA-CHITOSAN COMPLEX IN PBS BUFFER WITH DIFFERENT PH

of chitosan, and N is the number of chitosan mole-

cules bound to each molecule of DNA. To solve for N, the equation is given as follows:

Finally, the DNA-chitosan binding ratio=1/N.

Measurement of porosity of DNA-chitosan complexes The porosity of un-rinsed, PBS-rinsed, and PBS re-rinsed DNA-chitosan complexes were estimated from the true density as measured with a gas pycnometer

(AccuPyc 1330, Micromeritics Instrument Corp., Norcross, GA, USA) and from bulk density as meas-ured with a mercury porosimeter (AutoPore 9200, Micromeritics Instrument Corp.) . All measurements were performed in triplicate. The volume change ratio of DNA-chitosan complex was determined based on the following equation for porosity:

where Ps is the porosity, V is the volume of voids in the DNA-chitosan complex, and C is the volume of the DNA-chitosan complex. Reduction in porosity

(Rps) of the DNA-chitosan complex is then calcu-lated as follows:

where 95 is the volume of voids in the un-rinsed DNA-chitosan complex.

Field emission-scanning electron microscopic (FE-SEM) observation of porosity in DNA-chitosan com-

plexes PBS-rinsed and PBS re-rinsed DNA-chitosan com-

plexes and un-rinsed complex were freeze-fractured to observe their interior structures. The fractured sur-

faces were coated with evaporated carbon and then observed with a field emission-scanning electron mi-croscope (JSM-6330F, JEOL, Tokyo, Japan) .

Intercalation and groove binding of daunorubicin hy-drochloride (DH) within DNA-chitosan complexes Pulverized buffer rinsed and buffer re-rinsed DNA-chitosan complexes (10 mg) , and un-rinsed complex were immersed for 30 hours at 20 °C in 5 mL of 7.1 X 10-4 M DH. Absorbance from 400 nm to 600 nm by each solution was measured before and after the reaction using a spectrophotometer (V-560 , Jasco Corp., Tokyo, Japan) . The amount of intercalated and bound DH in the complexes was calculated from the difference in the total peak area for absorbance from 400 to 580 nm before and after the reaction . All experiments were performed in triplicate.

RESULTS

Preparation of DNA-chitosan complexes Water-insoluble DNA-chitosan complexes were ob-tained from the reaction of native DNA with chitosan, and the efficiency of complex formation was approximately 60%.

The amount of incorporated phosphate in the un-rinsed and PBS/pH7.4 rinsed complexes were 5.7% and 5.6% respectively. The molar binding ratios of the un-rinsed and PBS/pH7.4 rinsed complexes , which were calculated from the analysis of phosphate con-tent, were 1.01 and 0.96 respectively.

Measurement of porosity of DNA-chitosan complexes The true density, bulk density , and porosity of the DNA-chitosan complexes, and the change in porosity of the buffer rinsed complexes are shown in Tables 1 and 2. The true density of the un-rinsed complex was 1.75 g/cm3, and the bulk densities of the com-

plexes ranged from 0.08 to 0.29 g/cm3. All complexes

Table 1 Densities and porosities of DNA-chitosan complexes rinsed with and without PBS

Table 2 Densities and porosities of DNA-chitosan complexes , which had been rinsed with PBS at pH 6.0, pH 7.0, and pH 7.8, and re-rinsed with PBS at pH 7.4


showed high porosities from 83 to 95%. The un-rinsed complex and the complexes rinsed with PBS at

pH 6.0 had higher porosities than the others. Rins-ing with PBS at pH 7.0, 7.4, and 7.8 reduced the po-rosity of the DNA-chitosan complexes.

Re-rinsing with PBS at pH 7.4 reduced the po-rosity of the DNA-chitosan complex rinsed with PBS at pH 6.0, but showed little change in porosity of the complex rinsed with PBS at pH 7.0 or pH 7.8.

Observation of the porosity of DNA-chitosan com-

plexes by FE-SEM Fig. 2 show the FE-SEM views of the fractured sur-faces of the un-rinsed and PBS rinsed DNA-chitosan complexes. The DNA-chitosan complexes rinsed with PBS at pH 6.0 appeared to be almost the same as the un-rinsed complex, while the complexes rinsed with the other PBS solutions appeared to be very similar to each other.

The un-rinsed and PBS/pH6.0 rinsed complexes appeared like stacks of dead leaves, and areas with large pores were visible. In contrast, the complexes

rinsed with PBS — except for PBS at pH 6.0 — looked like cobwebs, and the mean pore size of these com-plexes was smaller than that of the un-rinsed and PBS/pH6.0 rinsed complexes.

Fig. 3 shows the FE-SEM views of the fractured surfaces of PBS re-rinsed DNA-chitosan complexes. The re-rinsed complexes with PBS at pH 7.4 appeared to be very similar to each other, despite the differ-ences in pH value at first rinsing.

The appearances of the re-rinsed complexes were similar to those of the complexes rinsed with PBS at

pH 7.0 or 7.8. The appearance of PBS/pH6.0 rinsed DNA-chitosan complex was changed by re-rinsing with PBS at pH 7.4.

Intercalation and groove binding of DH by DNA in the DNA-chitosan complexes Fig. 4 shows the intercalation and groove binding of DH within the PBS rinsed and un-rinsed DNA-chitosan complexes, whereas Fig. 5 shows the results of PBS re-rinsed DNA-chitosan complexes.

DH intercalated and bound in the grooves of

Fig. 2 SEM

rinsedphotomicrographs of the

with PBS at pH 6.0, pH

fractured surfaces of un-rinsed DNA-chitosan

7.0, pH 7.4, and pH 7.8. Bar= 10um.

complex, and the complexes


Fig. 3 SEM photomicrographs of the

rinsed with PBS at pH 6.0, pH

fractured surfaces of

7.0, and pH 7.8, then

DNA-chitosan

re-rinsed with

complexes,

PBS at pH

which had been

7.4. Bar=10 um.

Fig. 4 Intercalation

plexes rinsed and pH 7.8,

and groove binding of DH with corn-

with PBS at pH 6.0, pH 7.0, pH 7.4,

and with un-rinsed complex.

DNA within all of the DNA-chitosan complexes. The

amount of intercalated and groove-bound DH in un-

rinsed complex was 0.8 mg, and those of the rinsed

complexes were 0.70 to 1.02 mg after 24 hours. In

the re-rinsed complexes, the amount of intercalated

and groove-bound DH was 0.99 to 1.22 mg after 24

hours.

These results suggested that in all of the rinsed

complexes, one molecule of DH intercalated and

groove-bound to approximately 4.2 to 6.1 bps of DNA. Overall, re-rinsing allowed more DH intercala-

tion and groove binding than the original rinsed

complexes within 24 hours. In the re-rinsed com-

plexes, one molecule of DH intercalated and groove-

bound to approximately 3.5 to 4.3 bps of DNA.

Fig. 5 Intercalation and groove binding of DH with com-

plexes which had been rinsed with PBS at pH 6.0, pH 7.0, and pH 7.8, followed by re-rinsing with

PBS at pH 7.4. pH 7.4 refers to complex rinsed with PBS at pH 7.4.

DISCUSSION

DNA-chitosan complexes were prepared from the re-action between native DNA and chitosan in an aque-ous solution as previously described9). Porous white DNA-chitosan complex was obtained in about 60%

yield; molar ratio of DNA to chitosan was 1.01; and porosity was high at 95% (Fig. 2). These results agreed with our previous results9).

One critical factor for a scaffold material is its

porosity. This is because cells require sufficient free space in a scaffold to form new tissue in vitro or in vivo — although there are different views on the optimal pore size for scaffolds used for cell seeding, cell growth, and extracellular matrix production13-15).


The un-rinsed DNA-chitosan complex had a high po-rosity of 95%, and the pores in the complex were uniformly distributed. Chen et al.18) reported that

poly (DL-lactic-co-glycolic acid [PLGA]) -collagen hy-brid sponges with a porosity of approximately 90% show good cell attachment. Further, Wei and Li19) reported that a nano-hydroxyapatite and polyamide biocomposite with a porosity of 80% could be used as a scaffold material. On these grounds of porosity, the porosity of un-rinsed DNA-chitosan complex made it a suitable scaffold material.

However, Chu et al.20) described that some hydrogels swelled or shrank as a result of various factors such as solvent composition, pH, and ionic strength. In particular, the volume of chitosan-xanthan gel was influenced by differences in osmotic

pressure between the ionic solutes in the gel and those in the ambient solution. Yao et al.16) also re-

ported that the volume change of chitosan-gelatin complexes depended on the dissociation of hydrogen bonds. Previously, we reported that the volume of DNA-chitosan complex could be controlled by rinsing with different types of buffer solution such as PBS, HEPES, boric acid, or Tris-HC1 (pH 7.2 and 10 mM) 9) . We found that the porosity in DNA-chitosan com-

plexes was greatly reduced by rinsing with PBS, and hypothesized that the reduction in volume was mainly due to the integration of hydrogen bonds. If this assumption is correct, then the volume change of DNA-chitosan complex is expected to occur when the latter is rinsed in PBS buffer solutions with different

pH. This is because the complex contains many amino and phosphate groups that can form intra-complex hydrogen bonds. Therefore, in the present study, we investigated the volume change of the DNA-chitosan complex in PBS buffer solutions with different pH. As shown in Table 1, the porosity of the un-rinsed DNA-chitosan complex was reduced after rinsing with PBS. In particular, PBS of pH 7.0 or more greatly decreased the porosity of the un-rinsed complex (95%) , such that the porosity of the complexes rinsed with PBS at pH 7.0, 7.4, and 7.8 were 85%, 86%, and 83% respectively. Indeed, rins-ing with PBS at pH more than 7.0 could greatly shrink an un-rinsed DNA-chitosan complex. As shown in Table 1, the reduction in porosity was 67% and 87% when DNA-chitosan complex was rinsed at

pH 7.4 and pH 7.8 respectively. Further, SEM micrographs in Fig. 2 showed that the un-rinsed com-

plex was very different from those of the complexes rinsed with PBS at pH 7.0 or more, and the mean

pore size of these rinsed complexes was smaller than that of the un-rinsed complex. Yao et al.16) reported that the swelling degree of chitosan-gelation gel in sodium acetate and potassium dihydrogen phosphate buffer solutions — of various pH values — decreased as the pH value increased. At an acidic medium of

pH 7.0, the swelling degree decreased sharply, and

twin peaks — attributed to -NH3+ — were detected by means of infrared spectroscopy. They explained that this phenomenon occurred due to hydrogen bonds within the gel dissociating in acidic medium. There-fore, we suspected that volume changes were mainly due to the dissociation or integration of hydrogen bonds — a result of the ionization function of the amino group.

Apart from water, PBS has been used to rinse scaffolds and drug delivery materials to control their

porosity or for the latter to be bound with drug and cytokines. Eventually, these biomaterials will be ex-

posed to body fluids. Against this background, we measured the change in porosity of the rinsed DNA-chitosan complexes after re-rinsing with PBS at pH 7.4. As shown in Table 2, when PBS/pH7.0 and PBS/pH7.8 rinsed complexes were re-rinsed, their

porosities of 85% and 86% respectively were close to that of PBS/pH7.4 rinsed complex (86% . Moreover, their SEM micrographs in Fig. 3 showed them to be similar to that of PBS/pH7.4 rinsed complex (Fig. 2) . On the other hand, for PBS/pH6.0 rinsed complex, the porosity was greatly decreased by re-rinsing and the SEM micrograph became similar to that of PBS/

pH7.4 rinsed complex. These results suggested that the PBS/pH6.0 rinsed complex have a possibility to shrink after implantation in the living tissue.

Maintaining the double stranded structure of DNA in the complexes is very important because

polycyclic aromatic cations, such as DH or adriamycin, can be intercalated between stacked base

pairs of DNA or bound within the groove of DNA strands4) . The formation of double-stranded DNA in DNA films was confirmed by the binding studies of ethidium bromide (EB) 8,21) . However, we described in our previous study9) that DH, rather than EB, more accurately reflects the presence of double-stranded DNA in the DNA-chitosan complexes. This is be-cause EB binds DNA at the dinucleotide level, whereas DH and DNA can occur by either intercala-tion or binding in the major or minor groove. Therefore, in the present study, we measured the amount of intercalated and bound DH in the com-

plexes. We found that DH was bound in un-rinsed, rinsed, and re-rinsed complexes (Figs.2 and 3) . In the un-rinsed and rinsed complexes, one molecule of DH was bound to approximately 4.2 to 6.1 bps of DNA in the complexes within a 24-hour immersion, and complexes rinsed with alkaline PBS had higher intercalation and groove-binding rates of DH. Remeta et al.22) reported an exclusion number (bp/ drug) of 3.7 for the binding of DH by native salmon sperm DNA, and we confirmed that chitosan alone did not bind with DH in the previous study9) . We suspected that either steric hindrance by chitosan or NH3 + of chitosan in the DNA-chitosan complex resulted in the slightly lower intercalation and

groove-binding rate of DH in the complexes. On the


other hand, re-rinsing increased the amount of inter-calated and bound DH in the complexes, whereby one molecule of DH was bound to approximately 3.5 to 4.3 bps of DNA in the complexes. Moreover, the in-tercalation and groove-binding rate of DH in the re-rinsed complexes was faster than in the original rinsed complexes. We expected the amounts of inter-calated and bound DH and the intercalation and

groove-binding rates of DH in the re-rinsed com-plexes to be close to those of PBS/pH7.4 rinsed com-plex because the porosity of the re-rinsed complexes was almost close to that of PBS/pH7.4 rinsed com-

plex. However, for PBS/pH7.0 and PBS/pH7.8 rinsed complexes after re-rinsing, the amount of interca-lated and bound DH was higher than that of PBS/

pH7.4 rinsed complex and the intercalation and groove-binding rate was faster too. Although we suspected that re-rinsing converted residual NH3+ to NH2 in the complexes and hence obtaining these re-sults, the reason is not yet clear. Nonetheless, the

present results suggested that the intercalation and groove binding of DH with DNA-chitosan complex will be favorably applicable in the drug delivery sys-tems for oral cancer.

In conclusion, we demonstrated that the porosity of DNA-chitosan complexes and the intercalation and

groove-binding rate of DH in the complexes could be controlled by rinsing and re-rinsing with PBS of dif-ferent pH values. The DNA in PBS-rinsed and PBS re-rinsed DNA-chitosan complexes maintained their double-stranded helical structure. These results sug-

gested that the PBS rinsed DNA-chitosan complex is promising as a scaffolding material for tissue engi-neering, and that these pH-sensitive complexes may be useful for the controlled release of drugs or cytokines. Leveraging on the results of the current study, DNA-chitosan complex should be further in-vestigated in vivo.

ACKNOWLEDGEMENTS

This study was partly supported by Grants-in-Aid for Scientific Research, (B) (1) (16390574) , (C) (1) (15592091) , and (C) (2) (17592046) , from the Japan Society for the Promotion of Sciences. We are also

grateful to Nichiro Co., Ltd. for their supply of chitosan and salmon testes DNA.

REFERENCES

1) Blackburn GM, Gait MJ. Nucleic acids in chemical and biology, 2nd ed, Oxford University Press, Oxford,

1996, pp.15-81. 2) McGee JO'D, Isaacson PG, Wright NA. Oxford text-

book of pathology, Oxford University Press, Oxford, 1992, pp.198-205.

3) Werner MH, Gronenborn AM, Clore GM. Intercala- tion, DNA kinking, and the control of transcription.

Science 1996; 271: 778-784. 4) Glumoff T, Golgman A. Interaction of proteins wit]

nucleic acids. In: Nucleic acids in chemical and biology 2nd ed, Blackburn GM, Gait MJ editors, Oxford Uni

versity Press, Oxford, 1996, pp.375-441. 5) Stryer L. Eucaryotic chromosomes and gene expres

sion. In: Biochemistry, 4th ed, WH Freeman and Corn

pany, New York, 2000, pp.975-1008. 6) Okahata Y, Tanaka K. Oriented thin films of a DNA

lipid complex. Thin Solid Films 1996; 284-285: 6-8. 7) Fukushima T, Inoue Y, Hayakawa T, Taniguchi K

Miyazaki K, Okahata Y. Preparation of and tissue re sponse to DNA-lipid films. J Dent Res 2001; 80: 1772

1776. 8) Fukushima T, Hayakawa T, Inoue Y, Miyazaki K

Okahata Y. Intercalation behavior and tensile strengtl of DNA-lipid films for the dental application

Biomaterials 2004; 25: 5491-5497. 9) Fukushima T, Hayakawa T, Okamura K, Takeda S

Inoue Y, Miyazaki K, Okahata Y. Buffer solution car control the porosity of DNA/chitosan complexes.

Biomed Mater Res (Part B) : Applied Biomaterials (ir. press) .

10) Morimoto M, Saimoto H, Shigemasa Y. Control of functions of chitin and chitosan by chemical modifica-

tion. Trends Glycosci Glycotechnol 2002; 14: 205-222. 11) Rikimaru S, Wakabayashi Y, Nomizu M, Nishi N,

DNA-chitosan bilayer membrane as a bi-functional bio- medical adhesive. Polym J 2003; 35: 255-260.

12) Kato Y, Onishi H, Machida Y . Application of chitin and chitosan derivatives in the pharmaceutical field. Curr Pharm Biotechnol 2003; 4: 303-309.

13) Nakao H, Hyon SH, Tsutsumi S, Matsumoto T, Takahashi J. Control of pore size in L-lactide/ E -

caprolactone copolymer foarms for tissue regeneration by the freeze-drying method. Dent Mater J 2003; 22:

262-271. 14) Schoof H, Apel J, Heschel I, Rau G . Control of pore

structure and size in freeze-dried collagen sponges. J Biomed Mater Res 2001; 58: 352-357.

15) Itala A, Ylanen HO, Ekholm C, Karlsson KH , Aro HT. Pore diameter of more than 100,u m is not requisite for bone ingrowth in rabbits. J Biomed Mater Res 2001;

58: 679-683. 16) Yao KD, Yin YJ, Xu MX, Wang YF . Investigation of

pH-sensitive drug delivery system of chitosan/gelatin hybrid polymer network. Polym Int 1995; 38: 77-82.

17) Boltz DF, Mellon MG. Spectrophotometric determination of phosphorus as molybdiphosphoric acid . Anal

Chem 1948; 20: 749-751. 18) Chen G, Ushida T, Tateishi T. A biodegradable hybrid

sponge nested with collagen microsponges. J Biomed Mater Res 2000; 51: 273-279.

L9) Wei J, Li Y. Tissue engineering scaffold material of

nano-apatite crystals and polyamide composite. Eur Polym J 2004; 40: 509-515.

?0) Chu CH, Sakiyama T, Yano T . pH-sensitive swelling of a polyelectrolyte complex gel prepared from xanthan

and chitosan. Biosci Biotechnol Biochem 1995; 59: 717- 719


21) Yamada M, Kato K, Nomizu M, Sakairi N, Ohkawa K, Yamamoto H, Nishi N. Preparation and characteriza-

tion of DNA films induced by UV irradiation. Chem Eur J 2002; 8: 1407-1412.

22) Remeta DP, Mudd CP, Berger RL, Breslauer KJ. Ther-

modynamic characterization of Daunomycin-DNA

teractions: Comparison of complete binding profiles

a series DNA host duplexes. Biochemistry 1993;

5064-5073.

in-

for

32:

pbs buffer solutions with different ph values can change

Documents