Dental Materials Journal 26(6)：854－860, 2007
Drug Binding and Releasing Characteristics of DNA/Lipid/PLGA Film Tadao FUKUSHIMA1, Minoru KAWAGUCHI1, Tohru HAYAKAWA2, Shoji TAKEDA3, Yusuke INOUE4, Jun OHNO5 and Kunihisa TANIGUCHI5
Department of Dental Engineering, Bioengineering Section, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 2 Department of Dental Biomaterials, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho Nishi, Matsudo, Chiba 271-8587, Japan 3 Department of Biomaterials, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata, Osaka 573-1121, Japan 4 Fukuoka College of Health Sciences, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan 5 Department of Morphological Biology, Section of Pathology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan Corresponding author, Tadao FUKUSHIMA; E-mail:
[email protected] 1
Received May 24, 2007/Accepted June 27, 2007 We evaluated the blended compound of DNA/lipid complexes and PLGA (poly(D,L-lactide-co-glycolide)) as a carrier material for drug delivery system (DDS). Transparent, self-standing DNA/lipid/PLGA films were prepared by casting from an organic solvent such as DMSO/chloroform. Daunorubicin hydrochloride (DH) could intercalate and groove bind into DNA in the films, whereby the amount of DH bound to the films was controlled by the latter’ s immersion period in DH aqueous solution. DH was released from DH films after immersion in PBS solution, whereby release rate was dependent on the chemical structure of lipids. Released DH caused reduction of cell viability during the cell culture of L929 mouse fibroblasts. These results suggested that DNA/lipid/PLGA film was a promising useful material for DDS. Keywords: Intercalation, DDS, DNA/lipid/PLGA film
INTRODUCTION In recent years, strategies to apply deoxyribonucleic acid (DNA) as a biomaterial in tissue engineering have been developed and carried out1-5). Such immense interest in DNA has arisen because of the latter’ s favorable properties as a biomaterial6-8). For example, antibiotics or cytokines can be intercalated between stacked base pairs of DNA or bound within the grooves of DNA strands7,8). DNA can also be hydrolyzed by enzymes such as DNase9), and is less antigenic than other macromolecules such as proteins or polysaccharides6). In particular, a useful characteristic of DNA is that it can be intercalated and groove bound by natural or synthetic drugs, such as adriamycin, amsacrine, and daunorubicin hydrochloride (DH). On this account, DNA is expected to be very useful as a drug carrier in drug delivery system (DDS)8). However, native DNA alone is limited in its use as a carrier because of its water-soluble property10). To overcome this limitation, several techniques of forming water-insoluble DNA complexes have been reported4,11,12). Amongst which, Tanaka and Okahata11) developed a technique to form DNA/cationic lipid complexes. Water-insoluble DNA/cationic lipid complexes, which are soluble in organic solvents, were prepared from the interaction between DNA and cationic lipids. Then, with a view to investigating the potential of DNA-lipid films as a biodegradable material, novel cationic lipids were synthesized from
the reaction of n-alkyl alcohol and L-alanine1,13). Consequently, self-standing, water-insoluble DNA/lipid films were obtained by casting DNA/lipid complexes from organic solvent1,13). The DNA/lipid films did not cause any inflammatory reaction in the subcutaneous tissues of rats, and that they degraded approximately within three days1). Moreover, the DNA/lipid films could form intercalation complexes with ethidium bromide (EB)13). In another study5,14), water-insoluble DNA/chitosan complexes were prepared from the reaction between DNA and chitosan as scaffolds for tissues engineering. DNA/chitosan complexes could easily bind daunorubicin hydrochloride (DH), an anticancer drug itself and an intercalator like EB, in aqueous solutions. Indeed, one molecule of DH was bound to 4.5－6.5 base pairs (bps) of DNA in these complexes5). These binding ratios were close to an exclusion number (bps/drug) of 3.7 for the binding of DH by native salmon DNA15). Anticancer drugs such as DH are water-soluble. The intercalation of water-soluble compounds into water-insoluble DNA/cationic lipid films can take place in an aqueous solution. Then, by controlling the immersion time of films in the aqueous solution containing the anticancer drug, the amount of intercalated anticancer drug into DNA/cationic lipid films can be adjusted accordingly. In the context of DDS, it is very important to control the amount of drug release in order to be aligned with its therapeutic objective. However, if
FUKUSHIMA et al. the degradation of DNA/lipid films is too fast, then it will not be appropriate or advisable to use DNA/ lipid films alone for DDS. In light of this concern, it was proposed that if DNA/lipid could be blended with a biodegradable compound such as PLGA (poly(D,L-lactide-co-glycolide)), the release rate or amount of intercalated drug could be controlled. Therefore, we decided to evaluate the blended compound of DNA/lipid complexes and PLGA as a carrier material for DDS. The objectives of the present study were: (1) to synthesize two types of artificial lipids from the reaction of n-alkyl (n-decyl and n-laury) alcohols and Lalanine; (2) to prepare DNA/lipid/PLGA films; (3) to measure the amount of DH released from DNA/lipid/ PLGA films bound with DH; and (4) to evaluate the cell viability of DNA/lipid/PLGA films bound with DH. MATERIALS AND METHODS Synthesis of artificial lipids Two types of lipids were prepared from the reaction of n-alkyl alcohols (n-octyl alcohol and n-dodecyl alcohol) and L-alanine in the presence of p-toluenesulfonic acid in toluene according to a previous report1). The chemical structures (Fig. 1) of the synthesized lipids (C8-Ala/pts: L-alanine octyl ester p-toluenesulfonic acid salt; C12-Ala/pts: L-alanine dodecyl ester ptoluenesulfonic acid salt) were identified by elemental analysis, infrared absorption spectral analysis, and 1H nuclear magnetic resonance spectral analysis based on a previous report1). Preparation of DNA-lipid complexes Salmon testes DNA (300 mg; Nichiro Co. Ltd., Tokyo, Japan), which was cleaved with protease P3 into 300bp fragments, was dissolved in 100 ml of distilled water. Two molecules of C8-Ala/pts or 1.2 molecules
of C12-Ala/pts per bps of DNA were dissolved in 100 ml of distilled water. Two types of DNA/lipid (DNA/C8-Ala and DNA/C12-Ala) complexes were prepared from the mixture of DNA and lipid solutions according to previous reports1,13). Preparation of DNA/lipid/PLGA blended films Figure 2 shows the preparation procedure of DNA/ lipid/PLGA films. DNA/C8-Ala (13 mg) or DNA/ C12-Ala (15 mg) complex powder (Fig. 2a-A) was dissolved in 0.4 ml of dimethyl sulfoxide (DMSO). Chloroform (CHCl3, 0.6 ml) was added to DMSO solution after complexes were completely dissolved in DMSO. Then, PLGA powder (Fig. 2a-B, 45 mg; RESOMER® RG 504, Boehringer Ingelheim GmbH Co., Ingelheim, Germany) was added to DMSO/CHCl3 solution. The mixture (Fig. 2b) was stirred at 20℃ until PLGA was completely dissolved. The resulting solution was spread evenly on a Teflon plate of 0.1×1×1.5 cm dimensions (Fig. 2c), and all solvent was evaporated at 55℃ under slightly reduced pressure. Then, the DNA/lipid/PLGA films were carefully removed from the Teflon plate (Fig. 2d). PLGA film without DNA/lipid complexes was prepared for use as a control. Intercalation and groove binding of daunorubicin hydrochloride (DH) within DNA/lipid/PLGA films DNA/C8-Ala/PLGA or DNA/C12-Ala/PLGA films were immersed in 1 mL of 0.88×10－3 M DH for three days. Absorbance from 400 to 600 nm of each solution was measured using a visible spectrophotometer (V-560, Jasco Corp., Tokyo, Japan). The amount of intercalated and groove bound DH in the films was calculated from the difference in total peak area for absorbance from 400 to 580 nm before and after the reaction. The time for binding one molecule of DH to 10
Fig. 2 Fig. 1
Schematic illustration of DNA/lipid complex and poly(D,L-lactide-co-glycolide).
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DNA/lipid/PLGA film formation procedure: a) DNA/lipid complex powder (A), PLGA powder (B); b) DNA/lipid complex and PLGA in DMSO/CHCl3; c) Casting on Teflon plate; d) DNA/lipid/PLGA film.
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Drug binding and releasing of DNA/lipid/PLGA film
bps of DNA in DNA/lipid/PLGA films was also determined. DNA/C8-Ala/PLGA and DNA/C12-Ala/ PLGA films were immersed in 1 mL of 0.5×10－3 M DH and 1.04×10－3 M DH at 20℃, respectively. The films were washed with distilled water after immersion, and then dried under vacuum. The amount of intercalated and groove bound DH in the films was measured by the same method as that described above. Measurement of amount of DH released from films Three DNA/C8-Ala/PLGA films with intercalated and groove bound DH, and likewise three of such for DNA/C12-Ala/PLGA (otherwise known as DH films) were prepared for measurement of DH amount to be released from films. Each DH-intercalated-and-groove-bound film was immersed in 3 mL of phosphate buffered saline (PBS, 10 mM, pH 7.4) solution at 20℃. One DH film with DNA/C8-Ala or DNA/C12-Ala complex was immersed in PBS solution for three hours and 24 hours respectively. For each respective immersion period, the PBS solution was changed six times. For DH films with DNA/C8-Ala complex, PBS solutions were changed at 1 minute, 3 minutes, 5 minutes, 10 minutes, 30 minutes, and 1 hour after first immersion. For DH films with DNA/C12-Ala complex, PBS solutions were changed at 1 minute, 30 minutes, 1 hour, 3 hours, 6 hours, and 12 hours after first immersion. For each PBS eluting solution, visible absorption from 400 to 600 nm was scanned with a V-560 spectrophotometer. To obtain the amount of released DH, it was determined from the total peak area for absorbance from 400 to 580 nm of the visible spectrum. The amount of DH released into PBS solutions was determined by calibration with DH in native DNA solutions. The DH solutions with DNA for calibration were prepared by adding native DNA to DH solutions with different concentrations. The DNA/ DH molar ratio in each solution for calibration was 0.025. Following which, visible absorption from 400 to 600 nm of PBS solutions containing DH alone or DH+native DNA was scanned with a V-560 spectrophotometer. Cell viability assay All PBS eluting solutions with released DH were diluted with a medium to adjust the DH concentration in PBS eluting solutions. Adjusted concentrations were 0.03, 0.07, 0.15, 0.3, 0.6, 1.3, 2.5, 5.0 and 10 (×10－4 mM). L-929 mouse fibroblast cells were cultured with Eagle’ s minimum essential medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10％ fetal bovine serum (MP Biomedicals, OH, USA). A 0.1-ml suspension of 5,000 cells/ml was poured into each well of 96-well multiplates and incubated at 37ºC in CO2.
After 24-hour incubation, the medium was removed and replaced with 0.1 ml of PBS eluting solution or their serial dilutions. The cells were cultured for an additional three days at 37ºC. Cell viability was examined using a tetrazolium compound, 3-(4,5dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt (CellTiter 96 Aqueous, Promega, WI, USA). Experimental viability results were expressed as a percentage of the untreated control. Cell viability for DH alone or DH+native DNA in PBS solution was obtained by the same method as that described above. DH-alone PBS solution and DH+native DNA in PBS solution were prepared by diluting 10 mM DH-alone solution with water or/and adding native DNA. RESULTS Preparation of DNA/lipid complexes Water-insoluble DNA/lipid complexes were obtained from the reaction of native DNA with synthesized lipids. However, these DNA/lipid complexes were soluble in organic solvents such as chloroform and DMSO. The DNA/lipid/PLGA films were easily obtained by casting from DMSO/CHCl3 solution of DNA/lipid/ PLGA, as shown in Fig. 2. The self-standing DNA/ lipid/PLGA films (10 mm×15 mm×approximately 0.1 mm) were transparent. Intercalation and groove binding of daunorubicin hydrochloride (DH) within DNA/lipid/PLGA films The color of DNA/lipid/PLGA films became red immediately upon immersion in red DH solution. On the other hand, the control was not colored after immersion. The red color could not be removed from
Fig. 3
Typical visible spectra of: a) PBS solution with DH; b) PBS solution with DH and native DNA; and c) PBS eluting solution.
FUKUSHIMA et al.
Fig. 4
Amount of DH released in PBS eluting solution.
Fig. 5
Relationship between concentration of DH released in PBS eluting solution and cell viability.
all films by washing. In DNA/C8-Ala/PLGA and DNA/C12-Ala/PLGA films, one molecule of DH was bound to approximately 6 and 8 bps of DNA in each film respectively within three-day immersion. As for the time durations required for binding one molecule of DH to 10 bps of DNA in DNA/C8Ala/PLGA and DNA/C12-Ala/PLGA films, they were approximately five minutes and three hours respectively. Amount of DH released from films Color of PBS became red after colored DH film was immersed in it, while red color of DH film became transparent after final immersion. Figure 3 shows the visible spectra obtained after immersion. Figure 3a shows the visible spectrum of DH alone in PBS solution, while Fig. 3b shows the visible spectrum of PBS solution containing DH and native DNA. Figure 3c shows the visible spectrum of PBS eluting solution with red color. Its pattern differed from that of DH in PBS solution (Fig. 3a), but was very similar to that of PBS solution containing DH and DNA (Fig. 3b). Figure 4 shows the amounts of DH released from DH films. Both DH films released DH continuously, and the total amount of DH released from each film
Fig. 6
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Typical morphologies of cells exposed to: a) PBS buffer solution (1 mM); and b) DH (1×10－3 mM) in PBS (1 mM) eluting solution.
was almost equal to the initial amount of DH intercalated into the DH film. DH film containing C8-Ala with shorter alkyl group released DH faster than DH film containing C12-Ala with longer alkyl group. Cell viability assay Figure 5 shows the relationship between cell viability and the concentration of DH released from DH film for C8-Ala immersed in PBS eluting solution for the first one minute, as well as that for C12-Ala immersed in PBS eluting solution for the first 30 minutes. In addition, the relationship between cell viability and the concentrations of DH and DH+native DNA in PBS solutions were also shown for comparison with those of PBS eluting solutions. For both DH films, cell viability decreased with increase of DH concentration in PBS eluting solution. Moreover, cell viability was not dependent on the chemical structure of lipids in DH films. Both PBS eluting solutions showed the same relationship between cell viability and DH concentration, and they did not differ much from the PBS solutions containing DH+native DNA and DH alone. Cell viabilities in different PBS eluting solutions, which were obtained from different immersion
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Drug binding and releasing of DNA/lipid/PLGA film
periods ― except for 1 minute for DH film with C8Ala and 30 minutes for DH film with C12-Ala ― were also investigated. Curves plotted according to data obtained were almost the same as those in Fig. 5, which showed the relationship between cell viability and DH concentration (no data shown here). Figure 6 shows the typical morphologies of cells exposed to PBS buffer solution (1 mM) (Fig. 6a) and DH (1×10－3 mM) in PBS eluting solution (Fig. 6b). The cells had a round cell shape, and dead cells were observed after exposure to DH in PBS eluting solution. DISCUSSION DNA/lipid complexes and PLGA are soluble in chloroform/ethanol. On this basis, we initially prepared DNA/lipid/PLGA films by casting mixtures of DNA/lipid complexes and PLGA from chloroform/ ethanol. With chloroform/ethanol solution as a casting solution, transparent films were easily formed through this method. However, colorless areas in the films were visible after immersing the films in DH aqueous solution ― a consequence of the heterogeneous dispersion of DNA/lipid complexes in the films. The latter phenomenon arose because of differences in precipitation rate between DNA/lipid complexes and PLGA during solvent vaporization ― which occurred very quickly. In the present study, therefore, DMSO/ chloroform was used instead as a casting solvent for film formation. Transparent films with uniform dispersion of DNA/lipid complexes in the films were thereby obtained by this casting method with DMSO/ chloroform. It is very important to maintain the doublestrand structure of DNA in the films. This is because double-strand DNA has specific functions such as the accumulation of intercalating and groove-binding compounds like DH9). However, the double-strand structure of DNA is unstable against heat and organic solvents. In the present study, DNA/lipid complexes were exposed to heat and organic solvents during film formation. To confirm that the double-strand structure of DNA remains intact after such exposure, the conventional method is to measure the amount of EB or DH bound to DNA. It has been established that the exclusion parameters for the interaction of EB or DH with native DNA are 2.0－2.84) and 3.715) respectively. Iwata et al.16) measured the amount of EB bound to DNA-alginic acid films immersed in EB aqueous solution for 50 hours. They reported that one molecule of EB intercalated into five base pair of DNA. Moreover, a circular dichroism study confirmed that DNA in the films maintained the native doublestrand structure. On the other hand, Fukushima et al.5) measured the amount of DH bound to DNA-chito-
san complexes immersed in DH aqueous solution for 50 hours. They reported that one molecule of DH was bound to 4.5－6.2 bps of DNA in the complexes, and that the complexes maintained a double-helical structure similar to that of native DNA. In the present study, it was found that DH could be easily bound to DNA/lipid/PLGA films by immersing the latter in DH aqueous solutions. A three-day immersion resulted in one molecule of DH bound to approximately 6 and 8 bps of DNA in the films. These reaction ratios were slightly low in comparison with that of native DNA. In a study on DNA/chitosan complexes, Fukushima et al.5) reported that lower intercalation and groove binding of DH were due to steric hindrance by chitosan. Similarly for Iwata et al.16), their study on DNA/alginic acid films revealed that DNA bps/EB ratio was lower than that of native DNA. In the present study, it was thus presumed that either steric hindrance by lipid or PLGA in the DNA/lipid/PLGA films resulted in the slightly lower DNA base pair/DH ratios. At any event, DNA in both films maintained a double-helical structure similar to that of native DNA even after forming DNA/lipid/PLGA films. The concentration of DH in DH aqueous solutions was adjusted to 0.5×10－3 M for DNA/C8-Ala/PLGA film and 1.04×10－3 M for DNA/C12-Ala/PLGA film. This was done so that we could easily control the amount of DH that reacted with the films as DH intercalated and groove bound into the films. To bind one molecule of DH to 10 bps of DNA in DNA/C8Ala/PLGA and DNA/C12-Ala/PLGA films, the durations required were approximately 5 minutes and 3 hours respectively. The difference in immersion time was probably due to the different alkyl chain lengths in lipids. Fukushima et al.13) reported that the intercalation rate of EB within DNA/lipid films was influenced by the hydrophobicity of DNA/lipid films and
Fig. 7
Schematic illustrations of: a) DH intercalation and groove binding into the films; and b) DH releasing from the films.
FUKUSHIMA et al. the steric factors of artificial lipids. The results of the present study further showed that the amount of drug bound to DNA in the films could be easily controlled by adjusting the initial drug concentration in the aqueous solution or by designing the chemical structure of lipids. The bond between DNA and lipid in DNA/lipid/ PLGA films was mainly that of electrostatic force between anions in DNA phosphate groups and cations in lipid amino groups. On this basis, Okahata and Tanaka17) used quaternary ammonium salts to synthesize lipids for DNA film formation. Further on this, Morimoto et al.18) reported that quaternary chitosan derivatives retained their electrical properties. However, polycationic materials are generally known to exhibit cytotoxicity in cell cultures19,20). Therefore, in the present study, primary ammonium salts were used to synthesize lipids for reducing the cationic property of DNA film ― as it was done previously in a series of studies related to DNA/lipid complexes1,2,13). Fukushima et al.1) reported that DNA/C12-Ala film degraded approximately within three days in the subcutaneous tissues of rats. From this result, it was suggested that lipid could be gradually removed from DNA in DNA/lipid complexes by means of external interventions such as ions. Moreover, it was expected that this characteristic could be leveraged for drug release. Indeed, as expected (Fig. 7), DH was released from DH films in PBS solution where red films changed to white films. In a preliminary investigation, the amounts of DH released from DNA films with C8-Ala or C12-Ala including DH were not observed with the elapse of time because these films shortly decomposed in PBS buffer solution. Release rate of DH from DNA/C8-Ala/PLGA film was very fast as compared to that of DNA/C12Ala/PLGA film. It was thought that the hydrophobicity of DNA/lipid/PLGA films and the steric factors of artifical lipids affected the release rate of DH. It was also thought that DH released from the films might bind to DNA without lipids in the PBS solution. This was because the visible spectra and color of PBS eluting solutions after the DH films were immersed were very similar to those of PBS solution containing DH+native DNA. Di Marco et al.21) reported that the inhibitory effect of DH on nucleic acids and mitotic activity of HeLa cells was related to the binding of DH to DNA in cells. Therefore, the presence of DNA in PBS eluting solutions could have inhibited DH activity. However, as shown in Fig. 5, all PBS eluting solutions induced cell death. Moreover, cell viabilities in all PBS eluting solutions used for DH release test were very close to that of PBS solution with DH alone. Based on the morphology results, it was clear that cells exposed to PBS eluting solution were dead (Fig. 6). For the PBS solution in which films without
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DH were immersed, it did not induce any cell death. Relationship between cell viability and DH concentration in PBS solution with native DNA was very similar to those of PBS solutions in which DH films were immersed. These results suggested that DH could induce cell death even if DNA were present in the form of a mixture. To date, the binding and release studies of drugs and cytokines (such as BMP) are still underway. Based on the results of the present study, their potential to form complexes with DNA/lipid/PLGA films should also be further investigated. Nonetheless, the present results augered well for DNA/lipid/PLGA films to be used as a drug carrier material for DDS. PLGA is a known biodegradable compound22). In the context of DNA/lipid/PLGA films, their degradation rate could be controlled by some factors such as the mixing ratio of each compound or the molecular weight of PLGA. In view of the need for further clarif ication in these areas, an in vivo degradation study should be our next study subject. In conclusion, we demonstrated that transparent DNA/lipid/PLGA films could be prepared by casting from an organic solvent such as DMSO/chloroform. We also demonstrated that DH could easily intercalate and groove bind into DNA in the films, whereby the amount of DH bound to the films was controlled by the latter’ s immersion period in DH aqueous solution. DH was released from DH films after immersion in PBS eluting solution, whereby the release rate was dependent on the chemical structure of lipids. These results clearly suggested that DNA/ lipid/PLGA film was a promising useful material for drug delivery systems. ACKNOWLEDGEMENTS This study was supported in part by Grants-inaid for Scientific Research, (B) (19390505) and (C) (19592277) (19592260), from the Japan Society for the Promotion of Science. REFERENCES 1)
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