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.

Increased Cytotoxic Efficacy of Protocatechuic Acid in A549 Human Lung Cancer Delivered via Hydrophobically Modified-Chitosan Nanoparticles As an Anticancer Modality

“…The smallest nanoparticle size of CNP obtained from this study was 82.24 ± 2.67 nm which by using 250 uL TPP while PCNP was 90.23 ± 2.67 nm by using 200 uL TPP. This result was congruent with the findings of Kavi Rajan et al where the optimum chitosan to TPP ratio of about 3:1 [ 46 ]. Thereafter, particle size increased exponentially, indicating the formation of aggregates and nanoparticle clusters after this threshold resulting in the existence of excess TPP in the aqueous system, which may promote further interaction between the CNPs/pCNPs, thus initiating agglomerated nanoparticles with larger sizes [ 47 , 48 ].…”

“…The particle size of pCNP-PCA increased from 90.2 to 196.3 nm, a 117.6% surge from empty pCNP. As this expansion correlated with previous study [ 46 ], the inclusion of a hydrophobic moiety within pCNP-PCA has affected its expansion compared to CNP-PCA at similar PCA concentrations used for encapsulation. This was ascribed to an increased amount of PCA encapsulated in pCNP-PCA, due in part towards a tighter hydrophobic-hydrophobic interaction forming between the palmitoyl groups in pCS with PCA prior to nanoparticle formation.…”

Chlorogenic Acid Enhances Doxorubicin-Mediated Cytotoxic Effect in Osteosarcoma Cells

“…Surely, the restricted solubility and permeability have always hindered the CGA efficacy as a nutraceutical compound. More recently, CGA encapsulation in a nano-sized colloidal delivery vector has been proposed as a possible approach to enhance its antioxidant properties and increase chemopreventive efficacy at an even lower concentration [64]. Moreover, even though an initial CGA Phase I clinical trial has recently been concluded, demonstrating that its injection is safe and well-tolerated in recurrent high grade glioma patients, collecting safety data in a larger statistical sample is absolutely necessary to define the tolerance and pharmacokinetics of this compound (NCT02245204).…”

Hesperidin and Chlorogenic Acid Synergistically Inhibit the Growth of Breast Cancer Cells via Estrogen Receptor/Mitochondrial Pathway

“…Among numerous attempts to develop such a capacity, nanoencapsulation has been extensively exploited as a novel strategy to deliver anti-cancer drugs particularly for flavonoid phytochemicals due to their poor bioavailability [43,44]. The use of chitosan, rebaudioside A, and directly loading onto gold nanoparticles all have been shown to be feasible approaches and profoundly enhance the anti-cancer activity of chlorogenic or hesperidin [45][46][47][48]. With this concept in mind, our next endeavor is to encapsulate our herbal compounds with chitosan or nanolize chlorogenic acid and hesperidin via dry-heating treatment [49] to assess the delivery efficacy and therapeutic improvement in a murine model.…”