Synthesis, characterization and molecular dynamics simulation of the polyacrylates membranes

2.1 Characterization of the polyacrylates membrane

The Fourier transform infrared (FTIR) spectra of the membranes 4, 5 and 6 are recorded in Figure 1. The three membranes have similar characteristic peaks in the FTIR spectra owing to 80% of A and B in the monomer units. Here the characteristic peaks of the membrane 4 are assigned in detail: a wide peak from 3600 to 3200 cm^-1 is the stretching vibration of O-H, 2954 cm^-1 is the stretching vibration of C-H; 1598, 1494 and 1454 cm^-1 are the aromatic ring skeleton vibration; 758 and 694 cm^-1 are the bending vibration of C-H in the aromatic ring; 1732 cm^-1 is the stretching vibration of C=O in ester group; 1174 cm^-1 is the stretching vibration of C-O-C in ester group; 1245 cm^-1 is the stretching vibration of C-O in O-C₆H₅; 1045 cm^-1 is the stretching vibration of C-O in CH₂OH. The absence of absorption peaks at ~1620 cm^-1 and ~810 cm^-1 implies the successful reaction of radical polymerization with the C=C bonds in the monomer units (32, 33).

Figure 1:

The FTIR spectra of the membranes: (A) membrane 4, (B) membrane 5 and (C) membrane 6.

The assignment of absorption peaks in the FTIR spectra of membrane 5 are 3600–3200 cm^-1 (ν_O–H), 2952 cm^-1 (ν_C–H), 1598, 1496 and 1456 cm^-1 (ν_C–C, aromatic ring), 752 and 692 cm^-1 (δ_C–H, aromatic ring), 1732 cm^-1 (ν_C=O), 1172 and 1244 cm^-1 (ν_C–O–C), 1045 cm^-1(ν_C–O(H)). The assignment of absorption peaks in the FTIR spectra of the membrane 6 are 3600–3200 cm^-1 (ν_O–H), 2956 cm^-1 (ν_C–H), 1598, 1496 and 1456 cm^-1 (ν_C–C, aromatic ring), 756 and 692 cm^-1 (δ_C–H, aromatic ring), 1733 cm^-1 (ν_C=O), 1172 and 1247 cm^-1 (ν_C–O–C), 1045 cm^-1 (ν_C–O(H)).

The differential scanning calorimetry (DSC) thermograms of membranes 4, 5 and 6 are recorded in Figure 2. The values of the T_g of the membranes 4, 5 and 6 were -10.776, 20.354 and 8.148°C, respectively.

Figure 2:

The DSC thermograms of the membranes: (A) membrane 4, (B) membrane 5 and (C) membrane 6.

2.2 In vitro cytotoxicity studies

Cytotoxicity is one of the important properties for biomedical material. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay is widely used to evaluate the in vitro cytotoxicity. It is based on the reaction between MTT and mitochondrial succinate dehydrogenases in living cells to form a purple formazan which is not soluble in water but is soluble in dimethyl sulfoxide (DMSO). Cytotoxicity of the liquid extracts of the polyacrylates membrane was tested by the MTT assay in L929 cells.

Usually, the cytotoxic effects in cell cultures are mainly caused by released monomers. When the curing of a polyacrylate is not complete, unconverted monomers can be released from the polymer into the medium phase and cause cytotoxicity. Table 1 shows the relative growth ratio (RGR) data of the membranes 4, 5 and 6. During the 3-day cell culture period, all the RGR values were above 90%. This indicated that all polymers presented very low cytotoxicity.

2.3 In vitro release of clonidine HCl

Table 2 depicts the cumulative amount of clonidine transporting through the membranes 4, 5 and 6 with 14 μm thickness. Compared with the previous reported membrane 3 (17), the permeation rates follow the order of membrane 4>membrane 3>membrane 6>membrane 5.

2.4 Effect of the structure of the polymers on the release behaviors

It is well known that the release molecules firstly are absorbed onto the membrane and then diffuse through it. Usually, the release rate of drug molecules in the membrane is considerably affected by the mobility of polymer chains (29, 31) and free volume of the polymer membrane (27, 28).

The mobility of polymer chains can be analyzed by the MSD of the polymer chains. The larger slope of the MSD curve reflects the higher chains’ mobility. The MSD of the polymer chains are shown in Figure 3. It was found that the chains’ mobility follows the order of membrane 4>membrane 3>membrane 6>membrane 5. As we know the glass transition temperature (T_g ) is another parameter to reflect the mobility of polymer chains by experimental measurement. Lower T_g value reflects the higher chains’ mobility. The T_g s of membranes 4, 5 and 6 were -10.776, 20.354 and 8.148°C, respectively, and the T_g of membrane 3 as recorded in (17) was 4.179°C. The chains mobility obtained from T_g s agreed well with the MSD results.

Figure 3:

The MSD curves of the polymer chains: (A) membrane 3, (B) membrane 4, (C) membrane 5 and (D) membrane 6.

There are two phases in the polymer membrane: an occupied volume by polymer chains, and a free volume. The larger FFV reflects the higher amount and larger size of the voids in the polymer bulk, and contributes to the release of the drug molecules inside the polymers. The overall FFVs (the FFV probed by a probe with the radius 0.00 Å) of four different polymer membranes are listed in Table 3. The order of the FFV results is: membrane 4>membrane 3>membrane 6>membrane 5.

The simulation results of MSD and FFV shows the same tendency with the permeation rates of drug molecules transporting through the polymer membranes. By comparing the structures of the third monomer, we can acquire the following structure-performance relationship: the polymer chains’ mobility decreases, and permeation rate correspondingly decreases with the increase in the amount of side groups, such as membranes 3 and 4; the fractional free volume of the polymer chains decreases, and permeation rate correspondingly decreases with the increase in the volume of the side group, such as membranes 3, 5 and 6.

4.1 Materials

2-Hydroxy-3-phenoxypropyl acrylate, 4-hydroxybutyl acrylate, 2-butoxyethyl acrylate, 2-phenoxyethyl methacrylate and isodecyl methacrylate (Sigma-Aldrich Corporation, WI, USA) were purified by vacuum distillation under reduced pressure and stored at 4°C for further use. Benzoyl peroxide and clonidine HCl (National Medicine Corporation, Shanghai, China) were used as received.

The L929 murine fibroblast cell was purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). High glucose DMEM, fetal bovine serum (FBS), penicillin/streptomycin, trypsin and phosphate-buffered saline (PBS) were purchased from Thermo-Fisher HyClone (Logan, UT, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Aladdin (Shanghai, China).

All the other chemicals (National Medicine Corporation, Shanghai, China) received were of the highest purity and used without further purification.

4.2 Synthesis of the polyacrylates membranes

The synthesis of the polyacrylates membranes was as previously published (17), in brief, three monomers with 3% (w/w) benzoyl peroxide were dissolved completely and then were treated under the UV light (wavelength: 200–400 nm; power: 3 kW) for about 5 min. Three monomers in membrane 4 were 2-hydroxy-3-phenoxypropyl acrylate, 4-hydroxybutyl acrylate and 2-butoxyethyl acrylate with the weight ratio of 4:4:2. Three monomers in membrane 5 were 2-hydroxy-3-phenoxypropyl acrylate, 4-hydroxybutyl acrylate and 2-phenoxy ethyl methacrylate with the weight ratio of 4:4:2. Three monomers in membrane 6 were 2-hydroxy-3-phenoxypropyl acrylate, 4-hydroxybutyl acrylate and isodecyl methacrylate with the weight ratio of 4:4:2.

4.3 Characterization of the polyacrylates membranes

FTIR spectra of the thin freeze-dry membranes were recorded on a Nicolet iS10 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA USA) at a resolution of 4 cm^-1 with 16 times of scanning over a wavenumber range of 450–4000 cm^-1.

The values of the glass transition temperature (T_g ) of the membranes were collected on a DSC 204F1 differential scanning calorimeter (Netzsch, Selb, Germany). The samples weighing approximately 5 mg were first heated from 20°C to 200°C at a heating rate of 10°C/min and then held for 5 min to eliminate the thermal history. Subsequently, the samples were cooled to -60°C under a N₂ atmosphere and then heated again from -60°C to 200°C at a rate of 10°C/min.

The film thickness of dried membranes was measured using a digital meter (Shanghai Measuring and Cutting Tools Factory, Shanghai, China) with 0.001 mm accuracy. Five measurements were taken for each sample.

4.4 In vitro cytotoxicity studies

The L929 cell with high sensitivity to toxin, was selected for study of cytotoxicity (34). L929 cells were cultured in DMEM supplied with 10% FBS, 1% penicillin/streptomycin and incubated at a standard culture condition (37°C, 5% CO₂ in air) (Thermo Fisher Scientific, Waltham, MA, USA). The culture medium was refreshed every 2 days.

The cytotoxic effects of the membranes were evaluated by an indirect cytotoxicity test using the MTT assay (35). The cytotoxicity tests of samples were done according to ISO 10993-5 (Third Edition, 2009). L929 cells were seeded into 96-well plates at a seeding density of 5000 cells/well and incubated overnight. The sample was cut to 30 mg per disk, and then two sides of the sample were sterilized with UV for 24 h, respectively. Each sample was incubated in 5 ml DMEM medium for 5 days at 37°C, followed by filtration and mixing with 10% (v/v) FBS. One hundred micro liters of the sample extra was transferred into each well. The blank control groups were prepared by the same procedure without sample treatment. Five replicates of each sample were performed in each plate. After 1, 2 and 3 days treatment, respectively, the treatment medium was removed, 150 μl of cultur medium and 20 μl of MTT solution (5 mg/ml in PBS) were added to each well. After 4 h incubation the medium was discarded, 150 μl of DMSO was added to each well, and then the plate was shaken for 10 min to dissolve the formazan crystals. The optical density (OD) was measured at 492 nm with a reference wavelength 630 nm using a Multiskan MK3 microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). All samples were repeated three times to ensure reproducibility.

The relative growth ratio (RGR) was calculated using the following equation, and the cytotoxicity was evaluated according to Table 4. The lower the RGR% value, the higher the cytotoxic potential of the test sample. According to ISO 10993-5:2009, if RGR% is reduced to <75% of the blank, it has a cytotoxic potential.

RGR(%)=OD490(sample)-OD630(sample)OD490(blank)-OD630(blank)×100

4.5 In vitro release study

The release of the drug from the membrane was carried out using a modified Franz cell with 0.785 cm² of diffusion area and a receptor chamber of 9 ml volume. The prepared membrane was used as the diffusion membrane. An aqueous solution of 3.0 mg/ml clonidine HCl was used as the tested drug. PBS (pH 7.4) was used as a diffusion medium in the receptor chamber, stirred at 200 rpm at 37°C. At predetermined time intervals, 200 μl solution in the receptor chamber was withdrawn and then replaced an equal volume of fresh PBS. The samples withdrawn were analyzed for clonidine content by HPLC. Each test was carried out in triplicate (36).

The cumulative amount of clonidine HCl was calculated as follows:

Q=CnV+∑i=1i=n-1CiViA

where Q is the cumulative amount of the drug (μg/cm²), V is the volume of receptor solution (ml), V_i is the volume of sample withdrawn (ml), C_n and C_i are the drug concentrations of the receptor solution and of the sample withdrawn (μg/ml), respectively, and A is the diffusion area (cm²).

When the cumulative drug amount (Q_t , μg/cm²) was plotted vs. time (T, h), the slope of the linear portion of the plot was presented as the permeation rate [J, μg/(cm²·h)]. The intercept on the x-axis was presented as the lag time (T_L , h).

4.6 HPLC analysis of clonidine HCl

The HPLC system (Waters, Milford, USA) consisted of a 1525 binary pump, a 717 plus auto-sampler and a 2487 dual-wavelength UV absorbance detector. Data acquisition and processing were dealt with by Waters Empower professional software. The mobile phase was a mixture of a buffer solution (1.16 g of d-10-camphorsulfonic acid dissolved in 1000 ml of 0.1 m sodium acetate): acetonitrile: methanol in the volume ratio of 6:1:1, and then adjusted to pH 3.0 with phosphate acid. The mobile phase was filtered through a 0.45 μm porosity filter and degassed. The liquid chromatograph was equipped with a 5 μm, 4.6 mm×150 mm C8 column (Agilent Eclipse XDB) with a flow rate of 1 ml/min. The volume of each injected sample was 20 μl, the wavelength of the UV detector was set at 220 nm, and the run time was 10 min (36).

4.7 Molecular dynamics simulation

As a computer simulation method, molecular dynamics (MD) simulation can calculate the physical movements of atoms and molecules properties of the membrane at the molecular level (37, 38). Polymer chain mobility was analyzed by mean-square displacement (MSD) and free volume characteristics of membrane were investigated by a Connolly surface.

MD simulations were carried out using “Amorphous cell”, “Discover” and “Forcite” modules of Materials Studio 7.0 (Accelrys Software Inc., San Diego, USA). The energy minimization process was conducted using the smart minimizer method. Non-bond cutoff distance was defined as 12.5 Å (with a spline width of 1.0 Å and a buffer width of 0.5 Å).

First, an amorphous cell containing a polymer chain consisting of 100 repeat unit was built. Then, geometry optimization was performed by setting a maximum number of iterations to 50,000 or convergence tolerance energy to 0.001 kcal/mol. And then, a 200 ps dynamics equilibration process on the system was run with 1 fs time step in the NVT (T=298 K) ensemble to obtain the equilibrium molecular structures and the atomic trajectory was recorded for the analysis of mean square displacement (MSD) of the polymer chain.

To estimate free volume inside each polymer membrane, fractional free volume (FFV) was calculated as follows (38, 39):

FFV=VfreeVfree+Voccupy

where V_free and V_occupy are the free volume and occupied volume.