Preparation of Hydrogen Peroxide Sensitive Nanofilms by a Layer-by-Layer Technique

H2O2-sensitive nanofilms composed of DNA and hemin-appended poly(ethyleneimine) (H-PEI) were prepared by a layer-by-layer deposition of DNA and H-PEI through an electrostatic interaction. The (H-PEI/DNA)5 film was decomposed by addition of 10 mM H2O2. H2O2-induced decomposition was also confirmed in the hemin-containing (PEI/DNA)5 in which hemin molecules were adsorbed by a noncovalent bond to the nanofilm. On the other hand, the (PEI/DNA)5 film containing no hemin and the (H-PEI/PSS)5 film using PSS instead of DNA did not decompose even with 100 mM H2O2. The mechanism of nanofilm decomposition was thought that more reactive oxygen species (ROS) was formed by reaction of hemin and H2O2 and then the ROS caused DNA cleavage. As a result (H-PEI/DNA)5 and hemin-containing (PEI/DNA)5 films were decomposed. The decomposition rate of these nanofilms were depended on concentration of H2O2, modification ratio of hemin, pH, and ionic strength.

In this work we report the H 2 O 2 -induced decomposition of LbL nanofilms composed of hemin-appended poly(ethyleneimine) (H-PEI) and DNA. PEI and DNA have positive and negative charge, respectively, and could form LbL multilayer film through electrostatic interaction. Hemin is iron porphyrin molecule and the active cofactor for various enzymes, such as catalase and peroxidase [28]. It is known that the iron porphyrin produces more reactive oxygen species (ROS) such as hydroxy radical (·OH) from reaction with H 2 O 2 according to literatures [29,30]. The ROS causes non-specific DNA cleavage [31][32][33][34]. Consequently, H-PEI/DNA nanofilm was expected to decompose by addition of H 2 O 2 as illustrated in Figure 1. On the other hand, oxidative stress of H 2 O 2 related to various diseases caused [35,36]. Therefore, H 2 O 2 -sensitive gel and nanoparticles were used for the drug delivery system (DDS) [37][38][39][40]. It was thought that the H-PEI/DNA nanofilm is also used for DDS for delivery system (DDS) [37][38][39][40]. It was thought that the H-PEI/DNA nanofilm is also used for DDS for this purpose by enclosing the drug inside the nanofilm. Actually, (H-PEI/DNA)5 film was decomposed by addition of H2O2 and the decomposition ratio depended on concentration of H2O2, modification ratio of hemin, pH, and ionic strength.
Hemin-appended PEI (H-PEI) was synthesized as follows. PEI (100 mg) and hemin (37.9 mg) were dissolved in 0.1 M HEPES buffer (pH 7.0), then N-hydroxysuccinimide (8.02 mg) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (13.4 mg) were added in the solution at 4 °C. After 24 h, reaction mixture was purified by dialyzing to water for three days and freeze-dried. The content of hemin residues in PEI was 8.7% (molar ratio of hemin to primary amine), as determined by UV-visible absorption spectroscopy at 390 nm using a molar extinction coefficient of 42,000 M −1 cm −1 for hemin. Other modification ratio of 1.8%, 4.1%, and 6.5% H-PEI and hemin-appended PAH (H-PAH: modification ratio of 8.0%) were synthesized by similar manners and hemin was added 9.52 mg, 18.98 mg, 28.4 mg and 69.72 mg hemin to 100 mg each polymers, respectively. Chemical structures of hemin, H-PEI, H-PAH, and PSS were shown in Figure 2.
Hemin-appended PEI (H-PEI) was synthesized as follows. PEI (100 mg) and hemin (37.9 mg) were dissolved in 0.1 M HEPES buffer (pH 7.0), then N-hydroxysuccinimide (8.02 mg) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (13.4 mg) were added in the solution at 4 • C. After 24 h, reaction mixture was purified by dialyzing to water for three days and freeze-dried. The content of hemin residues in PEI was 8.7% (molar ratio of hemin to primary amine), as determined by UV-visible absorption spectroscopy at 390 nm using a molar extinction coefficient of 42,000 M −1 cm −1 for hemin. Other modification ratio of 1.8%, 4.1%, and 6.5% H-PEI and hemin-appended PAH (H-PAH: modification ratio of 8.0%) were synthesized by similar manners and hemin was added 9.52 mg, 18.98 mg, 28.4 mg and 69.72 mg hemin to 100 mg each polymers, respectively. Chemical structures of hemin, H-PEI, H-PAH, and PSS were shown in Figure 2. delivery system (DDS) [37][38][39][40]. It was thought that the H-PEI/DNA nanofilm is also used for DDS for this purpose by enclosing the drug inside the nanofilm. Actually, (H-PEI/DNA)5 film was decomposed by addition of H2O2 and the decomposition ratio depended on concentration of H2O2, modification ratio of hemin, pH, and ionic strength.
Hemin-appended PEI (H-PEI) was synthesized as follows. PEI (100 mg) and hemin (37.9 mg) were dissolved in 0.1 M HEPES buffer (pH 7.0), then N-hydroxysuccinimide (8.02 mg) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (13.4 mg) were added in the solution at 4 °C. After 24 h, reaction mixture was purified by dialyzing to water for three days and freeze-dried. The content of hemin residues in PEI was 8.7% (molar ratio of hemin to primary amine), as determined by UV-visible absorption spectroscopy at 390 nm using a molar extinction coefficient of 42,000 M −1 cm −1 for hemin. Other modification ratio of 1.8%, 4.1%, and 6.5% H-PEI and hemin-appended PAH (H-PAH: modification ratio of 8.0%) were synthesized by similar manners and hemin was added 9.52 mg, 18.98 mg, 28.4 mg and 69.72 mg hemin to 100 mg each polymers, respectively. Chemical structures of hemin, H-PEI, H-PAH, and PSS were shown in Figure 2.

Apparatus
A quartz crystal microbalance (QCM, QCA 917 system, Seiko EG & G, Tokyo, Japan) was used for gravimetric analysis of the LbL films. A 9-MHz AT-cut quartz resonator coated with a thin Au layer (surface area; 0.2 cm 2 ) was used as a probe, in which the adsorption of 1 ng of substance induces a −0.91 Hz change in the resonance frequency. Atomic force microscope images were recorded on SPM-9600 (SIMADZU). UV-VIS spectrum was recorded on a Shimadzu 3100PC spectrophotometer (Kyoto, Japan).

Preparation of Nanofilms
H-PEI/DNA nanofilms were prepared on 9 MHz Au-coated quartz resonator for QCM analysis.

Decomposition of Nanofilms
The H 2 O 2 -induced decomposition of the nanofilms was studied by monitoring the resonance frequency change (∆F) of the 5-bilayer film-coated quartz resonator in a flow-through cell of QCM. The H 2 O 2 solution was injected to the flow-through cell to monitor the decomposition of the nanofilm and the decomposition characteristic was evaluated by ∆F after 1 h. All experiments were carried out at room temperature (ca. 20 • C).

Observation of Nanofilms with AFM
For AFM observation, (H-PEI/DNA) 5 nanofilm was prepared on a circular glass slide (diameter was 1.5 cm) which was cleaned using fuming nitric acid and thoroughly rinsed in distilled water. The glass slide was immersed in distilled water two times of 30 min to desalt and dried in a desiccator for three days. Then the film was cut with an ultrasonic cutter (USW-333, Honda Electronics Co., LTD., Aichi, Japan) and the surface and thickness of the film were observed. In addition, the (H-PEI/DNA) 5 nanofilm was immersed in 100 mM of H 2 O 2 solution one hour to disintegrate and observed by a similar manner. All AFM images were taken in contact mode using Olympus microcantilevers (OMCL-TR800PSA-1, Olympus Co., Tokyo, Japan) at room temperature in air. Figure 3 shows the change in the resonance frequency (∆F) of a QCM observed on depositing H-PEI and DNA on the quartz resonator. The ∆F values decreased on deposition of both H-PEI and DNA, showing that H-PEI and DNA could form a LbL nanofilm. It is reasonable that H-PEI and DNA form the H-PEI/DNA film, because PEI and DNA could bind through the electrostatic interaction (PEI and DNA are polycation and polyanicon, respectively). Deposition amount of H-PEI and DNA were 1.33 µg/cm 2 and 12.8 µg/cm 2 in (H-PEI/DNA) 5 film. Hemin was appended to PEI at 8.7% for primary amin of PEI (PEI contains primary, secondary, and tertiary amines at 1:2:1 [41]). It was estimated that (H-PEI/DNA) 5 film contained 6.17 µmol/cm 2 of hemin molecules. The H2O2-induced decomposition of a (H-PEI/DNA)5 film was investigated on QCM ( Figure 4). ΔF has not changed in buffer solution, suggesting the (H-PEI/DNA)5 film was stable in this condition. By contrast, the ΔF increased when a 100 mM H2O2 solution was applied. In the case of exposing (H-PEI/DNA)5 film to 100 mM H2O2, ΔF was increase 1500 Hz after 1 h, and it showed that ca. 90% weight of (H-PEI/DNA)5 film exfoliated from the quartz slide. The increase of ΔF depended on concentration of H2O2. When (H-PEI/DNA)5 films were exposed to 10, 20, 50, and 100 mM H2O2 solutions, these decomposition ratios were 18.7, 22.0, 44.2, and 95.1%, respectively. The surface morphology and thickness of (H-PEI/DNA)5 film was observed by AFM ( Figure 5). The surface of (H-PEI/DNA)5 film was rough and average thickness was estimated at 27.7 nm from cross section (Figure 5a). On the other hand, the surface of (H-PEI/DNA)5 film was smooth and average thickness was decreased at 2.7 nm after exposure to H2O2 (Figure 5b). The thickness of (H-PEI/DNA)5 film was decreased by H2O2 processing. It was suggested that (H-PEI/DNA)5 film was decomposed by H2O2.  The H2O2-induced decomposition of a (H-PEI/DNA)5 film was investigated on QCM ( Figure 4). ΔF has not changed in buffer solution, suggesting the (H-PEI/DNA)5 film was stable in this condition. By contrast, the ΔF increased when a 100 mM H2O2 solution was applied. In the case of exposing (H-PEI/DNA)5 film to 100 mM H2O2, ΔF was increase 1500 Hz after 1 h, and it showed that ca. 90% weight of (H-PEI/DNA)5 film exfoliated from the quartz slide. The increase of ΔF depended on concentration of H2O2. When (H-PEI/DNA)5 films were exposed to 10, 20, 50, and 100 mM H2O2 solutions, these decomposition ratios were 18.7, 22.0, 44.2, and 95.1%, respectively. The surface morphology and thickness of (H-PEI/DNA)5 film was observed by AFM ( Figure 5). The surface of (H-PEI/DNA)5 film was rough and average thickness was estimated at 27.7 nm from cross section (Figure 5a). On the other hand, the surface of (H-PEI/DNA)5 film was smooth and average thickness was decreased at 2.7 nm after exposure to H2O2 (Figure 5b). The thickness of (H-PEI/DNA)5 film was decreased by H2O2 processing. It was suggested that (H-PEI/DNA)5 film was decomposed by H2O2.  The surface morphology and thickness of (H-PEI/DNA) 5 film was observed by AFM ( Figure 5). The surface of (H-PEI/DNA) 5 film was rough and average thickness was estimated at 27.7 nm from cross section (Figure 5a). On the other hand, the surface of (H-PEI/DNA) 5 film was smooth and average thickness was decreased at 2.7 nm after exposure to H 2 O 2 (Figure 5b). The thickness of (H-PEI/DNA) 5 film was decreased by H 2 O 2 processing. It was suggested that (H-PEI/DNA) 5 film was decomposed by H 2 O 2 . To investigate the mechanism of the (H-PEI/DNA)5 film decomposition, various polymers were used for H2O2-sensitive LBL film preparation and these films were tested ( Figure 6). (H-PEH/DNA)5 film was decomposed at 96% by addition of 100 mM H2O2 solution after 1 h. By contrast, the decomposition rate of (PEI/DNA)5 which did not contain hemin molecules was 3.6%. A hemincontaining (PEI/DNA)5 film in which hemin was adsorbed by a noncovalent bond after preparation of film was decomposed 85.5% by H2O2. These results suggested that hemin molecules were necessary for H2O2 decomposition of nanofilms for producing of ROS from reaction with H2O2. The decomposition rate of the (H-PEI/PSS)5 film prepared using PSS instead of DNA and (H-PAH/DNA)5 film prepared using PAH instead of PEI were 2.6% and 8.4% under same conditions. It was thought that LbL nanofilms composed of PEI and DNA were convenient for H2O2 response. The ROS effectively cleaved DNA more than PSS. In addition, PEI was branched polycation and its electric charge density is lower than that of liner polycation of PAH. Low charge density of PEI was useful for degradation. Porphyrin molecules such as hemin could interact with a major groove of DNA [42,43]. It was known that PEI binding porphyrin could interact with DNA effectively than PAH binding one [43]. Therefore, (H-PEH/DNA)5 and hemin-containing (PEI/DNA)5 film were decomposed by H2O2 more effectively than other nanofilms.  To investigate the mechanism of the (H-PEI/DNA) 5 film decomposition, various polymers were used for H 2 O 2 -sensitive LBL film preparation and these films were tested ( Figure 6). (H-PEH/DNA) 5 film was decomposed at 96% by addition of 100 mM H 2 O 2 solution after 1 h. By contrast, the decomposition rate of (PEI/DNA) 5 which did not contain hemin molecules was 3.6%. A hemin-containing (PEI/DNA) 5 film in which hemin was adsorbed by a noncovalent bond after preparation of film was decomposed 85.5% by H 2 O 2 . These results suggested that hemin molecules were necessary for H 2 O 2 decomposition of nanofilms for producing of ROS from reaction with H 2 O 2 . The decomposition rate of the (H-PEI/PSS) 5 film prepared using PSS instead of DNA and (H-PAH/DNA) 5 film prepared using PAH instead of PEI were 2.6% and 8.4% under same conditions. It was thought that LbL nanofilms composed of PEI and DNA were convenient for H 2 O 2 response. The ROS effectively cleaved DNA more than PSS. In addition, PEI was branched polycation and its electric charge density is lower than that of liner polycation of PAH. Low charge density of PEI was useful for degradation. Porphyrin molecules such as hemin could interact with a major groove of DNA [42,43]. It was known that PEI binding porphyrin could interact with DNA effectively than PAH binding one [43]. Therefore, (H-PEH/DNA) 5 and hemin-containing (PEI/DNA) 5 film were decomposed by H 2 O 2 more effectively than other nanofilms. To investigate the mechanism of the (H-PEI/DNA)5 film decomposition, various polymers were used for H2O2-sensitive LBL film preparation and these films were tested ( Figure 6). (H-PEH/DNA)5 film was decomposed at 96% by addition of 100 mM H2O2 solution after 1 h. By contrast, the decomposition rate of (PEI/DNA)5 which did not contain hemin molecules was 3.6%. A hemincontaining (PEI/DNA)5 film in which hemin was adsorbed by a noncovalent bond after preparation of film was decomposed 85.5% by H2O2. These results suggested that hemin molecules were necessary for H2O2 decomposition of nanofilms for producing of ROS from reaction with H2O2. The decomposition rate of the (H-PEI/PSS)5 film prepared using PSS instead of DNA and (H-PAH/DNA)5 film prepared using PAH instead of PEI were 2.6% and 8.4% under same conditions. It was thought that LbL nanofilms composed of PEI and DNA were convenient for H2O2 response. The ROS effectively cleaved DNA more than PSS. In addition, PEI was branched polycation and its electric charge density is lower than that of liner polycation of PAH. Low charge density of PEI was useful for degradation. Porphyrin molecules such as hemin could interact with a major groove of DNA [42,43]. It was known that PEI binding porphyrin could interact with DNA effectively than PAH binding one [43]. Therefore, (H-PEH/DNA)5 and hemin-containing (PEI/DNA)5 film were decomposed by H2O2 more effectively than other nanofilms.  The decomposition properties of (H-PEI/DNA) 5 film were investigated under various conditions. Figure 7A shows the effect of hemin modification ratio in PEI on decomposition ratio of (H-PEI/DNA) 5 films. The (H-PEI/DNA) 5 film which used modification ratio of 6.5% and 4.1% H-PEI film were more sensitive than 1.8% of one, because these films could react effectively with H 2 O 2 by lot of hemin molecules in film. However, decomposition of modification ratio of 8.7% hemin film was decreased. We thought that the interaction between hemin and DNA was excessively formed in nanofilm and disturbed the decomposition of the film. Effects of reaction solution pH and ion strength were studied ( Figure 7B,C). The pH 9 solution decomposed (H-PEI/DNA) 5 film effectively more than pH 4 and pH 7 solutions. The interaction for (H-PEI/DNA) 5 film formation weakened, because positive charge of PEI was decreased in pH 9 solution. Thus, H-PEI and DNA interaction for composition of LbL film was decreased and LbL film was decomposed low concentration of H 2 O 2 . Similarly, high ion strength reduced electrostatic interaction of PEI and DNA for LbL film. Therefore, (H-PEI/DAN) 5 film was decomposed by low concentration of H 2 O 2 in 300 mM NaCl solution ( Figure 7C). The decomposition properties of (H-PEI/DNA)5 film were investigated under various conditions. Figure 7A shows the effect of hemin modification ratio in PEI on decomposition ratio of (H-PEI/DNA)5 films. The (H-PEI/DNA)5 film which used modification ratio of 6.5% and 4.1% H-PEI film were more sensitive than 1.8% of one, because these films could react effectively with H2O2 by lot of hemin molecules in film. However, decomposition of modification ratio of 8.7% hemin film was decreased. We thought that the interaction between hemin and DNA was excessively formed in nanofilm and disturbed the decomposition of the film. Effects of reaction solution pH and ion strength were studied ( Figure 7B,C). The pH 9 solution decomposed (H-PEI/DNA)5 film effectively more than pH 4 and pH 7 solutions. The interaction for (H-PEI/DNA)5 film formation weakened, because positive charge of PEI was decreased in pH 9 solution. Thus, H-PEI and DNA interaction for composition of LbL film was decreased and LbL film was decomposed low concentration of H2O2. Similarly, high ion strength reduced electrostatic interaction of PEI and DNA for LbL film. Therefore, (H-PEI/DAN)5 film was decomposed by low concentration of H2O2 in 300 mM NaCl solution ( Figure  7C).  The decomposition properties of hemin-containing (PEI/DNA) 5 films were investigated similarly. Figure 8A shows the decomposition ratio of hemin-containing (PEI/DNA) 5 films prepared by immersing in 0.1 mg/mL and 0.01 mg/mL hemin solutions to absorb on nanofilm and these films contained 2.27 nmol/cm 2 and 1.09 nmol/cm 2 hemin molecules, respectively. The decomposition ratio of hemin-containing (PEI/DNA) 5 film increased with increasing hemin content in film. However, hemin-containing (PEI/DNA) 5 film (1.0 mg/mL) and (H-PEI/DNA) 5 (6.5%) film were 62% and 85%, respectively. It seems that introduce hemin molecules into the LbL film by covalent bond was effective for the decomposition of the LbL film. Effects of reaction solution pH and ion strength were studied ( Figure 8B,C). The pH 9 solution decomposed hemin-containing (PEI/DNA) 5 films film effectively more than pH 4 and pH 7 solutions from same reason of (H-PEI/DNA) 5 film. However, the ionic strength did not effect on the collapse of the film. When hemin molecules adsorbed into (PEI/DNA) 5 film, counter ions were also adsorbed. The effect of the ion strength of the reaction solution was reduced. The decomposition properties of hemin-containing (PEI/DNA)5 films were investigated similarly. Figure 8A shows the decomposition ratio of hemin-containing (PEI/DNA)5 films prepared by immersing in 0.1 mg/mL and 0.01 mg/mL hemin solutions to absorb on nanofilm and these films contained 2.27 nmol/cm 2 and 1.09 nmol/cm 2 hemin molecules, respectively. The decomposition ratio of hemin-containing (PEI/DNA)5 film increased with increasing hemin content in film. However, hemin-containing (PEI/DNA)5 film (1.0 mg/mL) and (H-PEI/DNA)5 (6.5%) film were 62% and 85%, respectively. It seems that introduce hemin molecules into the LbL film by covalent bond was effective for the decomposition of the LbL film. Effects of reaction solution pH and ion strength were studied ( Figure 8B,C). The pH 9 solution decomposed hemin-containing (PEI/DNA)5 films film effectively more than pH 4 and pH 7 solutions from same reason of (H-PEI/DNA)5 film. However, the ionic strength did not effect on the collapse of the film. When hemin molecules adsorbed into (PEI/DNA)5 film, counter ions were also adsorbed. The effect of the ion strength of the reaction solution was reduced.

Conclusions
The (H-PEI/DNA) 5 and hemin-containing (PEI/DNA) 5 nanofilms were prepared by LbL technique and were decomposed by addition of H 2 O 2 . The decomposition depended on modification rate of hemin, concentration of H 2 O 2 , pH and ion strength of reaction solution. We have previously reported H 2 O 2 sensitive nanofilms using the reaction of phenylboronic acid to phenol by H 2 O 2 [44,45]. We also developed glucose and lactate stimuli responsive nanofilm combining this thin film with the enzymatic reaction of glucose oxidase [46] and lactate oxidase [47] (These enzymes produce H 2 O 2 from each substrate). These nanofilms responded to each stimulus and decomposed within several minutes under physiological conditions, suggesting the application of insulin and anticancer drugs to DDS. However, there are some drugs that sustained release is desirable for DDS applications. The (H-PEI/DNA) 5 and hemin-containing (PEI/DNA) 5 nanofilms showed sustained degradation against low concentration H 2 O 2 . If the carrier containing drugs can be degraded persistently, it may be achieved as sustained drug release carrier by using this hemin base nanofilm.
Author Contributions: K.S. designed the work. K.Y., Y.K., and K.S. collected the materials. K.Y., T.D., T.O., and K.S. were involved in the experimental works. The manuscript was prepared by K.Y. and K.S.