Study on characteristics, properties, and morphology of poly(lactic acid)/chitosan/hydroquinine green nanoparticles

2.1 Materials

Poly(lactic acid) (density 1.25 g/cm³, molecular weight 1.42×10⁴ Da), CS (in powder, DD >77%, viscosity 1220 cPs), Hq (in white powder, purity≥98%) were purchased from Sigma Aldrich (USA). Dichloromethane (DCM) and acetic acid were of analytical reagent grade and used without further purification were provided by Guangdong Guanghua Chemical Factory Co. (China).

2.2 Preparation

An aqueous solution of the Hq drug, Hq dissolved in ethanol was poured into the PLA solution using solvent DCM to form an emulsion of water/oil. Next, the emulsion mixture of water/oil was added into 1% acetic acid solution dissolved CS and polyethylene oxide (PEO) calculated by weight. The emulsion process was carried out for 15 min in a MAS-II microwave machine (Sineo Microwave, China). The PCHq nanoparticles were collected by centrifugation and then washed several times with distilled water in order to remove excessive PEO emulsifier before lyophilizing using a FreeZone 2.5 equipment (Labconco, USA). In this study, the PCHq nanoparticles samples were prepared at 10, 20, 30, and 50 wt% Hq (in comparison with PLA weight) and abbreviated as PCHq10, PCHq20, PCHq30, and PCHq50, respectively.

2.3 Characterization

The FTIR spectra of the PCHq nanoparticles were analyzed at room temperature by using the Nicolet/Nexus 670 spectrometer (USA). Each sample was recorded with 16 scans at a resolution of 4 cm⁻¹.

The size distribution of the PCHq nanoparticles was measured using a Zetasizer particle size analyzer (Malvern, England).

Thermal property of the PCHq nanoparticles was analyzed by using a differential scanning calorimetry (DSC-60) thermogravimetric analyzer (Shimadzu, Japan) from room temperature to 400°C at a heating rate of 10°C/min under argon atmosphere.

The morphology of the nanoparticles was observed on the FESEM images conducted using the S-4800 FESEM instrument (Hitachi, Japan). FESEM images were taken of sputtered samples with platinum coating.

The Hq released content from the nanoparticles in different pH solutions was calculated by UV-Vis spectroscopy method using a CINTRA 40, GBC spectrometer (USA).

3.1 FTIR spectra

The FTIR spectra of Hq, PC, and PCHq nanoparticles are shown in Figure 1. In the FTIR spectrum of Hq, the characteristic band at 3178 cm⁻¹ can be assigned to –OH bending vibration. The peaks appeared at 2929, 1622, 1510, 1238, and 1033 cm⁻¹ corresponding to CH₃ group, aryl C=C and C=N– conjugated group, C–N amine group and C–O–C stretching vibration group, respectively. The FTIR spectra of PCHq nanoparticles indicated the characteristic peaks of the stretching vibrations of C=N, C–N, and C=C groups in Hq. In addition, the shift of wavenumbers of the groups such as C=O, C=N, C–N, C=C, C–O–C, –OH, –NH₂, and COOH in CS, PLA and Hq could be observed for the PCHq nanoparticles in comparison with the original PLA, CS, and Hq (Table 1). This could be explained by formation of hydrogen-bonding and dipole-dipole interactions between C=O group in PLA with OH and NH₂ groups in CS, and OH, C=N, and C–O groups in Hq.

Figure 1:

Fourier transform infrared spectra of hydroquinine (Hq), poly(lactic acid)/chitosan (PLA/CS) (PC), and PLA/CS/hydroquinine (PCHq) nanoparticles.

3.2 The particle size distribution

The particle size distribution diagrams of PCHq nanoparticles using different Hq content (10, 20, 30, and 50 wt%) are presented in Figure 2.

Figure 2:

Particle size distribution diagrams of the PCHq nanoparticles using different Hq content.

It is clear that the particle size of PCHq nanoparticles ranged from 115 to 200 nm. The average particle size of PCHq nanoparticles was smaller than that of the PC nanoparticles (without Hq). The change in the particle size distribution can be clarified by the hydrogen-bonding and dipole-dipole between NH₂ and OH groups in CS with C=O group in PLA, and C=N, C–O, and OH groups in Hq. This confirmed that Hq was incorporated into polymeric nanoparticles [4, 7]. The average particle size of the PCHq20 nanoparticle was smaller than that of other nanoparticles (Figure 2). This value of PCHq nanoparticles was smaller than that of PC nanoparticles loaded other drugs such as rifamicine, anthraquinone, and lamivudine [7, 9, 10]. The particle sizes of PC/rifamicine, PC/anthraquinone, and PC/lamivudine nanoparticles were 180–220, 100–200, and 300–350 nm, respectively.

3.3 DSC analysis

The thermal property of PCHq nanoparticles could be remarkably affected by the crystallization characteristics of PLA and CS. The data of DSC analysis of PLA, CS, and PCHq nanoparticles using different Hq content are shown in Table 2.

From the DSC diagrams (Figure 3) and Table 2, it can be seen that neat PLA has a glass transition temperature (T_g) of 79.7°C, and a melting temperature (T_m) of 189°C. The T_g of CS is 110.7°C. The PCHq nanoparticles had T_g values between the T_gs of PLA and CS. The shift of T_g of PCHq nanoparticles in comparison with T_g of PLA and CS can be explained by the hydrogen-bonding and dipole-dipole interaction between OH, NH₂, C=O, and C=N groups in CS, PLA, and Hq as a rearrangement of the crystal structure of PLA. This displayed the simultaneous crystallization in PCHq and nanoparticles occurred due to the interactions as aforementioned. Thus, the degree of crystallinity (χ_c) of the PCHq nanoparticles was higher than that of neat PLA.

Figure 3:

Differential scanning calorimetry diagrams of PLA, CS, and PCHq nanoparticles using different Hq content.

3.4 Morphology

The FESEM images of Hq and PCHq nanoparticles using different Hq content were expressed in Figure 4. It can be seen that Hq had an amorphous form and was irregularly sized, ranging between 1 and 5 μm (Figure 4A).

Figure 4:

Field emission scanning electron microscopy images of Hq (A), PCHq nanoparticles using 20 wt% Hq (B), 30 wt% Hq (C), and 50 wt% Hq (D).

Figure 4(B–D) showed that the PCHq nanoparticles having a spherical shape with basic particle size was in the range 70–250 nm. The PCHq nanoparticle using 20 wt% Hq (PCHq20) had regular particle size and single dispersion. Its particle size was smaller than that of the nanoparticles using other Hq content (about 60–200 nm). However, all PCHq nanoparticles were agglomerated to form the particles with bigger size. The PCHq20 nanoparticle was less agglomerated than the other samples.

3.5 In vitro drug release

3.5.2 Setting up calibration equation of Hq in different pH solutions

The calibration equation of Hq in pH 2.0 solution and pH 7.4 solution were set up by using the UV-Vis spectroscopy method. Their linear regression coefficients were calculated according to the Excel software from the obtained data. The maximum wavelength of Hq in pH 2.0 and pH 7.4 solutions were 250.67 and 234.73 nm, respectively.

The calibration equation of Hq in pH 2.0 solution was y=37357x+0.022 with R²=0.997 (approximate 1) showed a linear dependence of absorbance on the Hq content at λ_max=250.67 nm in the range of 3–12 g/ml (Figure 5A). Therefore, this wavelength was used to investigate the Hq content released from the PCHq nanoparticles according to testing time (30 h).

Figure 5:

The absorbance versus different Hq content in pH 2.0 and pH 7.4 solutions.

Similarly, the calibration equation and the regression coefficient of Hq in pH 7.4 solution were displayed in Figure 5B. The calibration equation y=30556x+0.059 with R²=0.998 indicated a linear dependence of absorbance on the Hq content at λ_max=234.73 nm in the range of 3–12 g/ml.

3.5.4 Effect of initial Hq content

The Hq released content from the PCHq nanoparticles using 10–50 wt% (comparison with the PLA weight) in pH 2.0 and pH 7.4 solutions was shown in Figure 6.

Figure 6:

In vitro Hq released content from PCHq nanoparticles according to testing time.

The Hq content released from the PCHq nanoparticles included fast released period for the first testing time and then a controlled released period (slower release). The first fast released period occurred on the surface of the samples. The slower Hq release for the second testing period was started after eight testing hours because it took time for Hq to diffuse through the polymer matrix. It can be seen that the Hq released content from the PCHq nanoparticles in pH 7.4 solution was higher than that in pH 2.0 solution. This can be explained by: in pH 2.0 solution, the Hq released partially from the PCHq nanoparticles reacted with the acid solution to reduce the amount of Hq in the solution. This is consistent with the view in biomedical: Hq is poorly absorbed in the stomach, where its pH is small.

3.5.5 Release kinetic modeling

The HQ released kinetic study from the PCHq nanoparticles using 10–50 wt% of initial HQ content in pH 2.0 and pH 7.4 solutions was determind by different models such as zero order model (ZO), first order model (FO), Higuchi model (HG), Hixson-Crowell model (HCW), and Korsmeyer-Peppas model (KMP) [11].

The Hq released process from the PCHq nanoparticles using 10–50 wt% of initial Hq content in pH 7.4 solution was carried out for testing 30 h according to various kinetic models as performed in Figure 7. Figure 7(A–D) indicated that the R² values of Hq released process from the nanoparticles according to FO, HG, and HCW models were 0.941, 0.901, 0.966, and 0.922, respectively. The highest R² value (0.979, Table 3) and all R² values in Table 4 belonged to the KMP model which was most suitable for reflecting Hq released process from the PCHq nanoparticles in pH 7.4 solution (Figure 7E).

Figure 7:

The Hq released kinetic from the PCHq20 nanoparticles in pH 7.4 solution [zero order model (A), first order model (B), Higuchi model (C), Hixson-Crowell model, and (D) Korsmeyer-Peppas model (E)].

Table 3:

Regression equations and the regression coefficient (R²) of Hq released from the PCHq20 nanoparticles in pH 7.4 solution according to different kinetic models.

Model	Regression equation	R2
Zero order	y=0.557x+28.58	0.941
First order	y=0.010x+1.584	0.901
Higuchi	y=9.526+23.87	0.966
Hixson-Crowell	y=−0.031x+1.282	0.922
Korsmeyer-Peppas	y=0.269x−1.184	0.979

Table 4:

Parameters of regression equation reflected Hq released process from the PCHq nanoparticles in pH 7.4 solution according to different kinetic models.

Model	PCHq10	PCHq20	PCHq30	PCHq50
ZO
R2	0.895	0.929	0.948	0.839
k	1.213	1.585	0.935	0.77
FO
R2	0.819	0.901	0.904	0.784
k	0.021	0.010	0.008	0.009
HG
R2	0.964	0.948	0.979	0.913
k	8.171	11.93	6.583	5.562
HCW
R2	0.847	0.922	0.921	0.803
k	−0.027	−0.031	−0.024	−0.023
KMP
R2	0.984	0.979	0.974	0.929
k	0.24	0.269	0.215	0.233

The linear regression equations and the linear regression coefficient (R²) of Hq released from the PCHq20 nanoparticles in pH 7.4 solution according to different kinetic models were presented in Table 3.

Similarly, the Hq released process from the PCHq nanoparticles using 10–50 wt% of initial Hq content in pH 2.0 solution was carried out in 30 h according to various kinetic models as shown in Table 5. The parameters of regression equations (R² and k) were calculated by using the different models (ZO, FO, HG, HCW, and KMP). The highest R² values (0.948–0.995) corresponding to the Korsmeyer-Peppas model also expressed this model was suitable for Hq released process from the PCHq nanoparticles in pH 2.0 solution.

Table 5:

Parameters of regression equation reflected Hq released process from the PCHq nanoparticles in pH 2 solution according to different kinetic models.

Model	PCHq10	PCHq20	PCHq30	PCHq50
ZO
R2	0.852	0.967	0.925	0.938
k	0.52	0.375	0.409	0.345
FO
R2	0.896	0.968	0.891	0.905
k	0.016	0.005	0.007	0.007
HG
R2	0.987	0.971	0.968	0.974
k	14.0	10.91	2.902	2.436
HCW
R2	0.912	0.969	0.903	0.917
k	−0.018	−0.013	−0.016	−0.015
KMP
R2	0.995	0.986	0.948	0.968
k	0.177	0.337	0.173	0.169

Table 4 shows that the parameters of regression equation such as regression coefficient (R²) and constant (k) that displayed the release process of Hq from PCHq nanoparticles with different contents of Hq in pH=7.4 are calculated based on different models (ZO, FO, HG, HCW, and KMP).