2017 Volume 65 Issue 8 Pages 718-725
Reduction-sensitive nanogel was developed by including dithiodipropionic acid (DTPA) in tripolyphosphoric acid (TPPA) cross-linked poly(ethylenimine) (PEI) nanogel. According to the light scattering measurement, DTPA (a disulfide compound) seemed to cross-link PEI chains in a cooperative manner with TPPA (a multi-valent anion). Nanogels composed of TPPA, PEI, and DTPA exhibited negative zeta potential and the absolute value increased with the amount of TPPA and DTPA. TPPA and DTPA were found to be contained in the nanogel, evidenced by Fourier transform (FT)-IR spectroscopy and Raman spectroscopy, respectively. 1H-NMR spectroscopy also revealed DTPA was contained in the nanogel. The DTPA content in the nanogel was determined colorimetrically to be 7.14 and 9.4%, depending on the DTPA content in the raw mixture for the preparation of nanogel. On the transmission electron microscopy (TEM) micrographs of the negatively stained nanogel, the diameter was about 20–30 nm. The specific loading of carboxylic fluorescein (CF) in the nanogel was around 1.8%, determined by fluorometric analysis, and it was not affected by the DTPA content. The maximum release degree of CF loaded in nanogel containing no DTPA was less than 10% and it was almost the same regardless of dithiothreitol (DTT) concentration. Whereas, the release of the dye loaded in nanogel containing DTPA was markedly promoted by DTT, possibly because the disulfide bond can be broken by DTT and the diffusivity of the dye through the nanogel matrix can increase.
Reduction-sensitive carriers have been of great interest to drug delivery scientists because they can be used as an intracellular delivery carrier.1) Glutathione is much more abundant in the intracellular space than the extracellular one, and reductive compounds are readily reduced in the intracellular space. Compounds having disulfide bond have been used as a key component of the reduction-sensitive carriers.2) Once the disulfide compounds are internalized into cells, they are cleaved because the disulfide bond is reduced to the thiol groups. Therefore, the reduction-sensitive carriers can be subjected to structural change and disintegration, leading to an extensive intracellular release of their cargo. An amphiphilic polymer was prepared by copolymerizing an alkyl chain-attached monomer (interconnected by disulfide bond) and a polar monomer, and it was used for the preparation of a reduction-sensitive micelle.3) The amphiphilicity disappears and the micelle is disassembled upon the cleavage of disulfide bond in the intracellular space. Disulfide bond cross-linked poly(ethylenimine) (PEI) was prepared for the intracellar delivery of DNA.4) Poly(ε-caprolactone) was linked to poly(ethyl ethylene phosphate) via disulfide bond and the copolymer was used for the preparation of nanoparticles which could facilitate the intracellular delivery of an anti-cancer drug (i.e., doxorubicin).5) Poly(ethylene glycol) was interconnected to poly(propylene sulfide) through disulfide bond and the copolymer was used as an amphiphile for the preparation of polymersome.6) When the disulfide was broken down in a reducing environment, the amphiphilicity was lost, and the polymer vesicle was destabilized. PEI and dithiodipropionic acid (DTPA) were included in the water channel of monoolein cubic phase for the reduction-responsive release of its payload.7) PEI chains could be cross-linked by DTPA molecules via the electrostatic interaction, and the release through the water channel was suppressed. When placed in a reductive environment, the release was promoted, possibly because the PEI network could be broken down due to the cleavage of the disulfide bond of the cross-linker (i.e., DTPA). Recently, a reduction-responsive liposome was developed by including benzyl disulfide in the egg phosphatidylcholine liposomal bilayer.8) If benzyl thiols are formed upon the reduction of benzyl disulfide, they could reorient and perturb the liposomal membrane, leading to a triggered release. In this study, reduction-sensitive nanogel was developed by including DTPA in tripolyphosphoric acid (TPPA) cross-linked PEI nanogel. TPPA can physically cross-link PEI chains because the phosphoric acids of TPPA electrostatically interact with the amino groups of PEI. In addition, DTPA can also electrostatically cross-link PEI chains because it has two carboxyl acids. If the disulfide bond of DTPA is broken down in a reducing environment, the cross-linking density of nanogel can decrease and the diffusion through the network of the nanogel would increase, resulting in a promoted release (Fig. 1). TPPA cross-linked PEI nanogels having different amounts of DTPA were prepared and their reduction-sensitive release property was observed at the different concentrations of dithiothreitol (a reducing agent) using carboxylic fluorescein as a dye.
TPPA can physically cross-link PEI chains because the phosphoric acids of TPPA electrostatically interact with the amino groups of PEI. DTPA can also electrostatically cross-link PEI chains because it has two carboxyl acids at its terminals. If the disulfide bond of DTPA is broken down in a reducing environment, the cross-linking density of nanogel can decrease, resulting in a promoted release.
Poly(ethylenimine) (PEI molecular weight (MW) 25000, branched form), dithiodipropionic acid (DTPA), sodium tripolyphosphate (TPPA), chloroform-d (CDCl3, 99.8 atom % D), phosphotungstic acid, carboxylic fluorescein (CF), and dithiothreitol (DTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Phosphorus pentoxide (P2O5) was obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). All other reagents were in analytical grade.
Preparation of PEI Nanogels Bearing DTPAVariable amounts of DTPA solution (0.99 mg/mL, in distilled water) was added to 1 mL of PEI solution (3 mg/mL, in distilled water) contained in a 10 mL vials so that the molar ratio of the amino group of PEI to the carboxyl group of DTPA was 1 : 0, 1 : 0.05, and 1 : 0.1. Variable amounts of TPPA solution (19.34 mg/mL, in distilled water) was added to the mixture solution of PEI/DTPA so that the molar ratio of the phosphoric group of TPPA to the amino group of PEI was 0 : 1, 0.5 : 1, 1 : 1, and 2 : 1. After the mixture solution of TPPA/PEI/DTPA was made up to 4 mL with distilled water, it was stirred using a magnetic stirrer at room temperature for 4 h then dialyzed for 3 d in 2.5 L of distilled water using a dialysis bag (MWCO 1000) with the dialysis medium being exchanged with fresh distilled water every 6 h. Nanogel formed in the TPPA/PEI/DTPA aqueous mixture whose phosphoric group/amino group/carboxyl group molar ratio was x/y/z was termed as nanogel(x/y/z).
Measurement of Light Scattering Intensity and Zeta PotentialThe light scattering intensity of nanogel(x/y/z) formed in TPPA/PEI/DTPA mixture solution was measured at room temperature (22–25°C) on a dynamic light scattering equipment (ZetaPlus 90, Brookhaven Instrument Co., U.S.A.) to investigate the effect of TPPA and DTPA on the degree of the nanogel formation. In parallel, the zeta potential of the nanogel(x/y/z) was measured at room temperature using the same equipment to observe the effect of the acidic components (i.e., TPPA and DTPA) on the surface potential of the nanogel.
SpectroscopyFourier Transform (FT)-IR SpectroscopyEach of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) was mixed with KBr in a mortar, the mixture was grinded to powder using a pestle, and the powder was molded into a pellet using a press. The FT-IR spectra were obtained on a FT-IR spectrophotometer (FT-3000-Excalibur, Varian Inc., U.S.A.).
Raman SpectroscopyRaman spectroscopy was employed to verify the disulfide compound (i.e., DTPA) was contained in TPPA cross-linked PEI nanogel. The Raman spectrum of nanogel suspension (2 mg/mL) contained in a glass cuvette was taken on a Raman spectrometer (ARAMIS, HORIBA JOBIN, France, located in the Central Laboratory Center of Kangwon National University). The parameters used for the Raman spectroscopy were as follows. The exposure time was 30 s, the radiation wavelength was 532 nm, the grating was 1800 lines/mm, and the accumulation number was 3.
1H-NMR SpectroscopyFreeze-dried nanogel (i.e., nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1)) was further dried in an oven thermostated at 36°C using P2O5 as a water-absorbing agent. The nanogel was dissolved in CDCl3 and it was subjected to 1H-NMR spectroscopy on a Bruker Avance 400 spectrometer (Karlsruhe, Germany, installed in the Central Laboratory of Kangwon National University).
Determination of DTPA Content in NanogelThe concentration of nanogel suspension was adjusted to 2 mg/mL with distilled water and the absorbance of DTPA was measured on a UV spectrophotometer (6505 UV/Vis. Spectrophotometer, JENWAY, U.K.) at 230 nm where no substantial absorbance of PEI and TPPA took place. The amount of DTPA was calculated by determining the concentration using the absorbance and the calibration curve.
Transmission Electron Microscopy (TEM)Negative staining technique was employed to take the TEM micrograph of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1). 0.1 mL of the nanogel suspension (2 mg/mL) and the same amount of phosphotungstic acid solution (2% (w/v)) were mixed in an Eppendorf tube and the mixture was left at room temperature for 3 h for the negative staining. The formvar/copper-coated grid (mesh size 200, Electron Microscopy Science) was dipped into the nanogel suspension for the deposition of nanogel onto the grid. The wet grid was dried at room temperature and the TEM micrograph was taken on a transmission electron microscope (JEM-2100F, JEOL, Japan, installed in the Central Laboratory of Kangwon National University).
Loading of Fluorescence Dye in NanogelTwo hundred milligrams each of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) was put in 4 mL of CF solution (1 mg/mL, in distilled water) contained in a 15 mL conical tube. It was rolled on a roller mixer (205RM, Hwashin Technology Co., Korea) at room temperature for 24 h and centrifuged at 3000 rpm for 30 min in a centrifuge (rotor 13, NB-550, N-BIOTEK, Korea). The supernatant was decanted, the sediment was washed with distilled water, and the washed sediment (CF-loaded nanogel) was freeze-dried. The amount of CF loaded in the nanogel was determined by subtracting the amount of dye in the supernatant from the total amount of dye in the nanogel suspension. The amount of CF was determined by measuring the solution fluorescence at 512 nm on a fluorescence spectrophotometer (Hitachi F2500, Hitachi, Japan) with the solution being excited at 494 nm. The specific loading was reported as a measure of how much the dye was loaded in nanogel and it was defined as the mass of dye loaded in nanogel per the unit mass of nanogel.
Reduction-Sensitive Release StudyDTT was dissolved in distilled water so that the concentration was 0, 2, 5, and 10 mM, and the DTT solution was used as a release medium. Five milligrams each of dye-loaded nanogel was dispersed in 1 mL of DTT solution and the nanogel suspension was put in a dialysis bag (MWCO: 10000). After tightly closed, the dialysis bag was put in a 50 mL conical tube containing 40 mL of release medium. After tightly sealed, the conical tube was gently rolled on a roller mixer for 24 h at room temperature. Two millilters of release medium was taken out to measure the fluorescence intensity at a certain time. The fluorescence intensity was measured at 512 nm on a fluorescence spectrophotometer (Hitachi F2500) with the release medium being excited at 494 nm. After fluorescence intensity was measured, the release medium was put back to the conical tube to keep the volume of release medium constant throughout the release experiment. The release degree was reported as the percent of the amount of dye released, based on the initial amount of dye loaded.
The light scattering intensity and the zeta potential of nanogel(x/y/z) was summarized in Table 1. When the relative mole of phosphoric group of TPPA was 0.5, the light scattering intensity of nanogel suspension increased from 2.6 to 62.5 kcps as the relative mole of the carboxyl group of DTPA increased from 0 to 0.1. The light scattering intensity is a measure of the degree of nanogel formation because the nanogel particles in suspension can scatter the incident laser light. Without DTPA, the light scattering intensity was low (i.e., 2.6 kcps), indicating that the relative amount of TPPA (i.e., 0.5) was not high enough to cross-link PEI chains and to give a rise to the nanogel formation effectively. The addition of DTPA led to a marked increase in the light scattering intensity. One molecule of DTPA has two carboxyl groups at its terminals. DTPA can cross-link PEI chains because its carboxyl groups electrostatically interact with the amino groups of PEI chains thus it can bridge PEI chains. When the relative mole of phosphoric group of TPPA was 1.0, the light scattering intensity was still low (i.e., 3.4 kcps) without DTPA, implying that the relative amount of TPPA (i.e., 1) was not high enough either for the effective nanogel formation. The light scattering intensity of nanogel suspension was also proportional to the relative mole of the carboxyl group of DTPA, possibly because DTPA could cross-link PEI chains through the salt bridging. When the relative mole of phosphoric group of TPPA was 2.0, the light scattering intensity of nanogel suspension was relatively high (i.e., 125.1 kcps) regardless of the amount of DTPA, indicating that the relative amount of TPPA (i.e., 2) was high enough for the effective nanogel formation. One molecule of TPPA has five phosphoric acids and it can cross-link PEI chains through the electrostatic interaction between the phosphoric acids and the amino groups. In fact, it was used as a cross-linker for PEI chains.9) Since the amount of phosphoric group of TPPA was stoichiometrically two times excess with respect to the amount of amino group of PEI, it was thought that most of the amino groups of PEI chains participated in the electrostatic interaction with the phosphoric groups of TPPA thus PEI chains could be effectively cross-linked by TPPA. The excess amount of TPPA could account for why the light scattering intensity was constantly high regardless of the amount of DTPA. Since the light scattering intensity was almost constant with respect to the amount of DTPA and the value was relatively high, nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) was chosen for further examination including the spectroscopy, the TEM and the reduction-sensitive release study. The zeta potential was measured for all the nanogels to investigate the effect of the acidic components (i.e., TPPA and DTPA) on the surface charge. When the relative mole of phosphoric group of TPPA was 0.5, the zeta potential of all the nanogels was negative. PEI is strongly positively charged due to its amino group. But the positive charge would be neutralized by the phosphoric group of TPPA. Thus, the negative value of the zeta potential could be ascribed to the electrostatic attachment of TPPA to PEI chains. The zeta potential decreased from −1.91 to −7.38 mV when the relative amount of the carboxyl group of DTPA increased from 0 to 0.1. DTPA can also be electrostatically attached to PEI chains due to its carboxyl groups, leading to an increase in the negative potential value of the nanogel. When the relative mole of phosphoric group of TPPA was 1, the zeta potential of all the nanogels was also negative. The negative value was greater than that of nanogel whose the relative TPPA amount was 0.5, possibly because nanogel(1 : 1 : z) contained more TPPA than nanogel(0.5 : 1 : z). The negative value of zeta potential was almost constant with respect to the amount of the carboxyl group of DTPA. This is possibly because most of the amino groups of PEI could electrostatically interact with the phosphoric groups of TPPA and the amount of DTPA was relatively small compared with that of TPPA. When the relative mole of phosphoric group of TPPA was 2, the zeta potential of all the nanogels was also negative, the absolute value was greater than that of nanogel whose relative mole of phosphoric group was 1, and it was almost constant with respect to the amount of the carboxyl group of DTPA. The explanation for the zeta potential values of nanogel(1 : 1 : z) would be also applicable to the zeta potential values of nanogel(2 : 1 : z).
| Nanogel(x/y/z) | Light scattering intensity (kcps) | Zeta potential (mV) |
|---|---|---|
| Nanogel(0.5 : 1 : 0) | 2.6 | −1.91 |
| Nanogel(0.5 : 1 : 0.05) | 36.7 | −3.04 |
| Nanogel(0.5 : 1 : 0.1) | 62.5 | −7.38 |
| Nanogel(1 : 1 : 0) | 3.4 | −12.10 |
| Nanogel(1 : 1 : 0.05) | 66.9 | −12.25 |
| Nanogel(1 : 1 : 0.1) | 111.4 | −12.36 |
| Nanogel(2 : 1 : 0) | 125.1 | −15.52 |
| Nanogel(2 : 1 : 0.05) | 128.6 | −15.75 |
| Nanogel(2 : 1 : 0.1) | 127.2 | −15.82 |
Figure 2 shows the FT-IR spectrum of PEI, nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1). In the spectrum of PEI, the –C–N– signal was found at 1117 cm−1, and the –N–H signal was found at 3289 cm−1 and 1571 cm−1. In the spectrum of nanogel(2 : 1 : 0), the P–O–P signal of TPPA was found at 899 cm−1, and the –N–H signal of PEI was found at 3414 cm−1 and 1639 cm−1. So, it could be said that TPPA was included in nanogel(2 : 1 : 0). One molecule of TPPA has five phosphoric groups thus it can cross-link PEI chains through the electrostatic interaction.7,8) In the spectrum of nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1), the P–O–P signal of TPPA and the –N–H signal of PEI appeared at the same position as they was shown in the spectrum of nanogel(2 : 1 : 0). But the signal of DTPA was not found, possibly because the amount of DTPA was relatively small compared with the amount of TPPA and PEI. The –N–H signal of PEI contained in the nanogels (i.e., 3414, 1639 cm−1) appeared at higher wavenumbers than the signal of free PEI (i.e., 3289, 1571 cm−1) did. That is, the –N–H signal of PEI was shifted to higher wavenumbers by addition of TPPA and DTPA. Their electrostatic interactions with PEI chains were thought to be responsible for the wavenumber shift. Figure 3 shows the Raman spectrum of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1). It was reported that the disulfide signal appeared in 425 to 550 cm−1.10) No appreciable signal of disulfide bond was found in the spectrum of nanogel(2 : 1 : 0). The signal of disulfide bond was found around 485 cm−1 in the spectrum of nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1), indicating DTPA (a disulfide compound) was contained in those nanogels. DTPA would cross-link PEI chains through the electrostatic interaction between the carboxyl groups of DTPA and the amino groups of PEI because one molecule of DTPA has two carboxylic groups at its terminals. Figure 4 shows the 1H-NMR spectrum of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1). In the spectrum of nanogel(2 : 1 : 0), the ethylene group adjacent to the primary amine of PEI was found at 2.8 ppm (a), the methylene group next to the secondary amine was found at 3.15 ppm (c), and the methylene group next to the tertiary amine was found at 3.4 ppm (b). In the spectrum of nanogel(2 : 1 : 0.05), the methylene group next to the disulfide bond of DTPA was found at 2.4 ppm (d) and the signals of PEI chains appeared at the same position as they did in the spectrum of nanogel(2 : 1 : 0). In the spectrum of nanogel(2 : 1 : 0.1), the methylene group next to the disulfide bond of DTPA was found at 2.54 ppm (d) and the methylene group next to the carboxyl group of DTPA was found at 2.4 ppm (e), along with the signals of PEI chains. Accordingly, it was concluded that DTPA was included in nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1).
The calibration curve of DTPA could be expressed by the equation y=1.14x−0.01 (R2=0.9993), where x is the concentration of DTPA in mg/mL and y is the absorbance at 230 nm. Using the absorbance of DTPA contained in the nanogel, the DTPA content in nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1) was calculated to be 7.14 and 9.4%, respectively. Even if the amount of DTPA used in the preparation of nanogel(2 : 1 : 0.1) was two times higher than the amount in the preparation of nanogel(2 : 1 : 0.05), the content of DTPA in nanogel(2 : 1 : 0.1) was slightly higher than the content in nanogel(2 : 1 : 0.05). The amount of TPPA used for the cross-linkage of PEI chains was stoichiometrically excessive. In this circumstance, most of the amino groups of PEI would electrostatically interact with the phosphoric groups of TPPA, and the amount of amino group available for the interaction with DTPA would be limited. This would be a reason why the content of DTPA in nanogel(2 : 1 : 0.1) was only slightly higher. On the other hand, the DTPA content in the TPPA/PEI/DTPA mixture used for the preparation of nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1) were 4.2 and 8.1%, respectively. The DTPA content in the nanogel (7.14, 9.4%) was higher than the content in the raw mixture used for the preparation of the nanogel (4.2, 8.1%). During the nanogel preparation, the TPPA/PEI/DTPA mixture solution was dialyzed to remove free TPPA molecules and it would be a reason why the DTPA content in the nanogel was lower than in the raw mixture.
TEMFigure 5 shows the TEM micrograph of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1). Agglomerated nanoparticles were found and the diameter was 20 to 30 nm on the micrograph. There was no marked difference in the size and the shape among three kinds of nanogel, indicating that DTPA had little effect on the size and the shape. DTPA would act as a cross-linker to cross-link PEI chains because its two carboxyl groups can electrostatically interact with the amino groups of the cationic polymer, thus it might affect the size and the shape. However, the amount of DTPA seemed not to be enough to affect them. According to the colorimetric analysis, the DTPA content in nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1) was found to be only 7.14 and 9.4%, respectively.
The calibration curve of CF could be expressed by the equation y=251290x+31 (R2=0.9921), where x is the concentration of CF in mg/mL and y is the fluorescence intensity. The specific loading of CF in nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) was calculated to be 1.81, 1.87, and 1.85%, respectively. Since the nanogels exhibited almost the same negative zeta potential (the zeta potential of nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) were −15.52, −15.75, −15.82 mV, respectively), there would be no marked difference in the electrostatic interaction intensity between the dye and the nanogel regardless of the kind of the nanogel. That is, the effect of the electrostatic interaction on the specific loading could be excluded. Besides the electrostatic interaction, a major factor affecting the specific loading is the cross-linking density of nanogel.11) PEI chains would be cross-linked mainly by TPPA because an excess amount of TPPA was used to cross-link the cationic polymer chains. DTPA would compete with TPPA for cross-linking PEI but the number of the ionizable group of the former cross-linker was at most 5% of the number of the ionizable group of latter one. Thus, it was thought that there would be no marked difference in the cross-linking density among three kinds of nanogels. This could account for why the specific loading in the nanogels was almost the same.
Reduction-Sensitive Release StudyFigure 6 shows the release profile of CF loaded in nanogel(2 : 1 : 0), nanogel(2 : 1 : 0.05), and nanogel(2 : 1 : 0.1) when the DTT concentration in release medium was 0, 2, 5, and 10 mM. The release degree of dye loaded in naogel(2 : 1 : 0) was less than 10% and it was almost the same regardless of DTT concentration (Fig. 6A). For example, the maximum release degree was 8.2, 8.1, 8.5, and 8.3%, respectively, when the DTT concentration was 0, 2, 5, and 10 mM. Nanogel(2 : 1 : 0) included no reducible compounds and it would remain intact in the presence of DTT. In the release profile of dye loaded in nanogel(2 : 1 : 0.05), the release was markedly promoted by DTT (Fig. 6B). A fast release took place for the first 4 h then a slow release was observed during the rest period. The release degree with DTT was much higher than the release degree without the reducing agent. For example, the maximum release degree was only 10% without DTT, and the maximum release degree was as much as 49.6 to 51.9% with DTT. DTPA (a disulfide compound) was included in nanogel(2 : 1 : 0.05) and it can be reduced to mercaptopropionic acids by DTT (a reducing agent). That is, the disulfide bond can be broken down in a reducing environment. Thus, if the nanogel containing DTPA is placed in DTT solution, the diffusivity of dye through the nanogel matrix would increase due to the breakdown of the disulfide bond, and the dye release could be promoted. The release degree was almost the same regardless of DTT concentration. For example, the maximum release degree with DTT was 49.6, 50.0, and 51.9%, respectively, when the concentration of DTT was 2, 5, and 10 mM. The concentration of 2 mM was thought to be already high enough to break down all the disulfide bond because the content of DTPA in nanogel(2 : 1 : 0.05) was as small as 7.14%. The pattern of release profile of dye loaded in nanogel(2 : 1 : 0.1) was similar to that of release profile of dye loaded in nanogel(2 : 1 : 0.05) (Figs. 6B, C). Like the release degree of dye loaded in nanogel(2 : 1 : 0.05), the release degree of dye loaded in nanogel(2 : 1 : 0.1) with DTT was much higher than the release degree without DTT. For example, the maximum release degree of dye loaded in nanogel(2 : 1 : 0.1) was only about 10% without DTT, and the maximum release degree was as much as 41.1 to 54.9% with DTT. Unlike the release degree of dye loaded in nanogel(2 : 1 : 0.05), the release degree was somewhat dependent on DTT concentration. For example, the maximum release degree was about 41.1, 49.9, and 54.9%, respectively when the DTT concentration was 2, 5, and 10 mM. The content of DTPA in nanogel(2 : 1 : 0.1) was relatively high (9.39%) thus the reduction degree of DTPA seemed to be dependent on the DTT concentration, leading to a DTT concentration-dependent release degree. The maximum release degree of dye loaded in nanogel(2 : 1 : 0.05) and in nanogel(2 : 1 : 0.1) were only 51.9 and 54.9%, respectively, even when DTT concentration was 10 mM (the highest among the concentrations tested). Since the increase in DTT concentration (from 2 to 10 mM) had no marked effect on the release degree of CF, the DTT concentration of 10 mM was thought to be enough to break down the disulfide bonds. Nevertheless, when the maximum release was reached, almost half of the loaded CF was still remained in the nanogels even under the relatively strong reducing condition (e.g. DTT concentration of 10 mM). CF is a negatively charged dye and PEI is a positively charged polymer. The electrostatic interaction would be responsible for the residual CF inside the nanogels. According to the light scattering measurement, DTPA (a disulfide compound) seemed to cross-link PEI chains in a cooperative manner with TPPA (a multi-valent anion). Even when the nanogels were put under a reducing condition and the disulfide bonds were broken down, they would maintain their integrity with aid of TPPA. The release degree of CF could increase owing to decrease in the crosslinking density. But the increase in the release degree would be limited because the dye could be trapped in the meshes of TPPA cross-linked PEI networks. This could be another reason why almost half of the loaded CF was still remained in the nanogels when the release degree reached its maximum value under a reducing condition.
Reduction-sensitive nanogel was successfully prepared by including DTPA in TPPA cross-linked nanogel. According to the light scattering study, DTPA together with TPPA seemed to act as an ionic cross-linker for PEI chains to be cross-linked into nanogel. The zeta potential of TPPA/PEI/DTPA nanogel was negative and it decreased as the amount of the acidic components (i.e., TPPA and DTPA) increased, possibly because the acidic components can be electrostatically attached to PEI chains. By FT-IR spectroscopy, Raman spectroscopy, and 1H-NMR spectroscopy, TPPA and DTPA were found to be contained in the nanogel. By colorimetric analysis, the DTPA content in the nanogel(2 : 1 : 0.05) and the nanogel(2 : 1 : 0.1) were determined to be 7.14 and 9.4%, respectively. The diameter of the nanogels was about 20–30 nm on the TEM micrographs obtained using negative staining technique, and the size and the shape were not affected markedly by DTPA possibly due to its small amount. The specific loading of CF in the nanogel (around 1.8%) was not affected by DTPA either. When DTPA was not contained in the nanogel (e.g. in case of nanogel(2 : 1 : 0)), the maximum release degree of CF was less than 10% and it was not dependent on DTT concentration. On the contrary, the release of the dye was outstandingly enhanced by DTT when DTPA was contained in the nanogel (e.g. in case of nanogel(2 : 1 : 0.05) and nanogel(2 : 1 : 0.1)). This is possibly because the diffusivity of the dye through the nanogel matrix can be increased by the DTT-induced breakdown of the disulfide bond. While the release degree of dye loaded in nanogel(2 : 1 : 0.05) was independent on DTT concentration (2, 5, 10 mM), the release degree of dye loaded in nanogel(2 : 1 : 0.1) was somewhat dependent on DTT concentration. When the DTPA content was relatively high (this was the case of nanogel(2 : 1 : 0.1)), the reduction degree of DTPA seemed to be dependent on DTT concentration, leading to the DTT concentration-dependent release degree.
This work was supported by the Technological Innovation R&D Program (S2406238) funded by the Small and Medium Business Administration (SMBA, Korea).
The authors declare no conflict of interest.
