Increased ROS Scavenging and Antioxidant Efficiency of Chlorogenic Acid Compound Delivered via a Chitosan Nanoparticulate System for Efficient In Vitro Visualization and Accumulation in Human Renal Adenocarcinoma Cells

Abstract

Naturally existing Chlorogenic acid (CGA) is an antioxidant-rich compound reported to act a chemopreventive agent by scavenging free radicals and suppressing cancer-causing mechanisms. Conversely, the compound's poor thermal and pH (neutral and basic) stability, poor solubility, and low cellular permeability have been a huge hindrance for it to exhibit its efficacy as a nutraceutical compound. Supposedly, encapsulation of CGA in chitosan nanoparticles (CNP), nano-sized colloidal delivery vector, could possibly assist in enhancing its antioxidant properties, in vitro cellular accumulation, and increase chemopreventive efficacy at a lower concentration. Hence, in this study, a stable, monodispersed, non-toxic CNP synthesized via ionic gelation method at an optimum parameter (600 µL of 0.5 mg/mL of chitosan and 200 µL of 0.7 mg/mL of tripolyphosphate), denoted as CNP°, was used to encapsulate CGA. Sequence of physicochemical analyses and morphological studies were performed to discern the successful formation of the CNP°-CGA hybrid. Antioxidant property (studied via DPPH (1,1-diphenyl-2-picrylhydrazyl) assay), in vitro antiproliferative activity of CNP°-CGA, and in vitro accumulation of fluorescently labeled (FITC) CNP°-CGA in cancer cells were evaluated. Findings revealed that successful formation of CNP°-CGA hybrid was reveled through an increase in particle size 134.44 ± 18.29 nm (polydispersity index (PDI) 0.29 ± 0.03) as compared to empty CNP°, 80.89 ± 5.16 nm (PDI 0.26 ± 0.01) with a maximal of 12.04 μM CGA loaded per unit weight of CNP° using 20 µM of CGA. This result correlated with Fourier-Transform Infrared (FTIR) spectroscopic analysis, transmission Electron Microscopy (TEM) and field emission scanning (FESEM) electron microscopy, and ImageJ evaluation. The scavenging activity of CNP°-CGA (IC₅₀ 5.2 ± 0.10 µM) were conserved and slightly higher than CNP° (IC₅₀ 6.4±0.78 µM). An enhanced cellular accumulation of fluorescently labeled CNP°-CGA in the human renal cancer cells (786-O) as early as 30 min and increased time-dependently were observed through fluorescent microscopic visualization and flow cytometric assessment. A significant concentration-dependent antiproliferation activity of encapsulated CGA was achieved at IC₅₀ of 16.20 µM as compared to CGA itself (unable to determine from the cell proliferative assay), implying that the competent delivery vector, chitosan nanoparticle, is able to enhance the intracellular accumulation, antiproliferative activity, and antioxidant properties of CGA at lower concentration as compared to CGA alone.

Keywords: 1,1-Diphenyl-2-picrylhydrazine assay; MTT assay; chemopreventive; chitosan nanoparticles; chlorogenic acid; drug encapsulated chitosan nanoparticles; in vitro accumulation of encapsulated chlorogenic acid; nanobiotechnology.

Figure 1

Influence of TPP volume on percentage of utilized amine group in combined graph bar CNP-F1, CNP-F2, and CNP-F3. The percentage of utilized primary amine (-NH₃⁺) groups increases with TPP addition. The figure suggests that with increasing TPP volume, more phosphate ions, -PO₄³⁻ groups were available to readily interact with -NH₃⁺ groups to form CNP, hence reducing the availability of free -NH₃⁺ groups. Error bars represent the standard deviation (SD) of three independent replicates of the experiment. * shows degree of significant difference, p < 0.0001 compared to control, 0 µL of TPP volume.

Figure 2

The influence of TPP volume of (A) CNP-F1, (B) CNP-F2, and (C) CNP-F3 on average particle size distribution (diameter in nm, nm), and polydispersity index (PDI). The average particle size distribution was illustrated in graph bar and PDI was illustrated in line graph. CNP° represents the optimal parameter required to synthesize stable and evenly distributed nanoparticle sizes. Error bars represent the SD of three independent replicates of the experiment. * shows degree of significant difference, p < 0.0015 compared to 20 µL of TPP volume.

Figure 3

Reproducibility of DLS analyses for the optimal parameter, CNP° using 600 µL CS (0.5 mg/mL) and 200 µL TPP (0.7 mg/mL). Consistency in synthesizing nanoparticle with average 80.89 ± 5.16 nm with PDI value of 0.26 ± 0.01 were observed in all three replicates; showing that at this parameter, stable and monodispersed CNP° were consistently able to be synthesized.

Figure 4

Effect of different final concentrations of CGA on average particle size distribution (nm) and polydispersity index, (PDI) of CNP°. The average particle size distribution is illustrated in bar graph and PDI is illustrated in line graph. Error bars represent the SD of three independent replicates of the experiment. * shows degree of significant difference, p < 0.0086 compared to control, CNP°.

Figure 5

Images of Transmission Electron Microscopy (TEM) morphological analysis of (A) CNP° and (B) CNP°-CGA. Particles appeared as irregular spherical-shape for all parameters used.

Figure 6

Images of FESEM morphological analysis of (A) CNP° and (B) CNP°-CGA. Particles appeared as irregular, dispersed spherical-shape for all parameters.

Figure 7

(A) Dynamic Light Scattering (DLS) result of CNP° and (B) normal distribution and frequency graphs of CNP° particle sizes (nm) obtained by measuring particle size in FESEM image in (Figure 6A) using ImageJ software. Comparable particle size range of CNP° was observed between DLS (80.89 ± 5.16 nm) and FESEM (50 nm and 70 nm). The slight difference between the two analysis is because DLS measures the particle size in hydrodynamic condition, while FESEM measures in a dry condition.

Figure 8

(A) DLS result of CNP°-CGA and (B) normal distribution and frequency graphs of CNP°-CGA particle sizes (nm) obtained by measuring particle size in FESEM image in (Figure 6B) using ImageJ software. Comparable particle size range of CNP° was observed between DLS (134.44 ± 18.29 nm) and FESEM (80 nm to 160 nm). The slight difference between the two analysis is because DLS measures the particle size in hydrodynamic condition, while FESEM measures in a dry condition.

Figure 9

FTIR spectra of CS, TPP, CNP°, CGA, and CNP°-GCA. The successful formation of CNP° and CNP°-CGA were discerned through the presence of functional group peaks. Freeze dried samples were used to ensure sensitivity of analysis, also to remove non-specific background peaks. Alphabet labeling denotes important functional groups of samples, and is further elaborated in Table 3.

Figure 10

Antioxidant capacity of CGA, CNP°-GCA and ascorbic acid as standard at different concentrations. Error bars represent the SD of three independent replicates of the experiment. Dotted line indicates the IC₅₀.

Figure 11

Flow cytometry study of in vitro accumulation of fCNP°-CGA in 786-O cells at (C) 30 min, (D) 6 h, (E) 24 h, and (F) 48 h. Overlap peaks of particular time point on (A) control and (B) FITC only peaks were shown in each figure to show the accumulation of fCNP°-CGA in 786-O cells. The accumulation of fCNP°-CGA in 786-O cells showed a time-dependent trend, indicated through the increase of fluorescent intensity over time. Accumulation of fCNP°-CGA was observed as early as 30 min (C) and subsequently increased 6 h, 24 h, and 24 h post treatment (D–F).

Figure 11

Flow cytometry study of in vitro accumulation of fCNP°-CGA in 786-O cells at (C) 30 min, (D) 6 h, (E) 24 h, and (F) 48 h. Overlap peaks of particular time point on (A) control and (B) FITC only peaks were shown in each figure to show the accumulation of fCNP°-CGA in 786-O cells. The accumulation of fCNP°-CGA in 786-O cells showed a time-dependent trend, indicated through the increase of fluorescent intensity over time. Accumulation of fCNP°-CGA was observed as early as 30 min (C) and subsequently increased 6 h, 24 h, and 24 h post treatment (D–F).

Figure 12

In vitro cytotoxicity test of CNP° at different concentrations. Error bars represent the SD of three independent replicates of the experiment. At the highest dosage, CNP° appeared to be non-toxic to 786-O cells upon 24 h post-treatment. * shows degree of significant difference, p < 0.0305 compared to control.

Figure 13

In vitro cytotoxicity test of CGA and CNP°-CGA at different concentrations. Error bars represent the SD of three independent replicates of the experiment. CGA alone was found to be non-toxic even at its highest dosage of 12 µM (24 h post-treatment). Conversely, a noticeable dose-dependent decrease in 786-O cell viability were observed upon encapsulation of CGA in CNP°. * shows degree of significant difference, P < 0.0001 compared to control.