Increased radical scavenging activity of thymoquinone and l-ascorbic acid dual encapsulated in palmitoyl-chitosan nanoparticles in a human normal lung fibroblast, MRC-5 due to synergistic antioxidative effects.

Less effective antioxidant supplementation in combating free radicals is often related to the lack of the formulation of carriers. The antioxidant may be one of the most powerful substances but is marred by poor uptake by cells when the carrier degraded and dissolved too rapidly. Nanoparticle (NP) systems are promising in overcoming the problem since they provide high surface area to enhance encapsulation and release efficiency. With the right selection of material, NP carriers could function as constructive antioxidant cargos. Generally, NPs carry only one active ingredient; this study, however, utilized chitosan nanoparticles (CNPs) and hydrophobically modified palmitoyl-chitosan nanoparticles (PCNPs) that were dual encapsulated with antioxidants of different polarities, namely, hydrophobic thymoquinone (TQ) and hydrophilic l-ascorbic acid (LAA) to evaluate their combination effects in scavenging free radicals. The antioxidants followed zero-order release kinetics with a controlled release manner for about 48 h. The interaction effects between TQ and LAA loaded in the NP systems were determined by classical isobologram (CI) values. The CI values were derived by a diphenyl picrylhydrazyl (DPPH) assay, a radical scavenging activity assay. Combined TQ and LAA had CI values of less than one, with a lower value in the PCNP system than in the CNP system. This indicates that the interaction between those antioxidants showed higher synergistic effects in PCNPs, which enhanced the DPPH radical scavenging activities. The antioxidative potential of compound(s) encapsulated in the PCNP carrier was further experimented by a reactive oxygen species (ROS) assay on a human normal lung fibroblast cell line (MRC-5) as lung is one of the organs with high accumulation of free radicals. About 48 h post treatment, the dual-loaded TQ and LAA in PCNPs showed the lowest ROS level in comparison to single-loaded antioxidants and bare antioxidant delivery. The hydrogen peroxide (H2O2) radical scavenging was influenced by both the controlled release property of the PCNP system and the synergy between TQ and LAA. In short, dual-loaded TQ and LAA in the hydrophobically modified PCNP had effectively depicted the capability of a single CS-based nanocarrier to hold more than one compound at a time to function as a potent radical scavenger.

Physicochemical characterization, cytotoxic effect and toxicity evaluation of nanostructured lipid carrier loaded with eucalyptol.

BACKGROUND: Eucalyptol is an active compound of eucalyptus essential oil and was reported to have many medical attributes including cytotoxic effect on breast cancer cells. However, it has low solubility in aqueous solutions which limits its bioavailability and cytotoxic efficiency. In this study, nanostructured lipid carrier loaded with eucalyptol (NLC-Eu) was formulated and characterized and the cytotoxic effect of NLC-Eu towards breast cancer cell lines was determined. In addition, its toxicity in animal model, BALB/c mice was also incorporated into this study to validate the safety of NLC-Eu. METHODS: Eucalyptol, a monoterpene oxide active, was used to formulate the NLC-Eu by using high pressure homogenization technique. The physicochemical characterization of NLC-Eu was performed to assess its morphology, particle size, polydispersity index, and zeta potential. The in vitro cytotoxic effects of this encapsulated eucalyptol on human (MDA MB-231) and murine (4 T1) breast cancer cell lines were determined using the MTT assay. Additionally, acridine orange/propidium iodide assay was conducted on the NLC-Eu treated MDA MB-231 cells. The in vivo sub-chronic toxicity of the prepared NLC-Eu was investigated using an in vivo BALB/c mice model. RESULTS: As a result, the light, translucent, milky-colored NLC-Eu showed particle size of 71.800 ± 2.144 nm, poly-dispersity index of 0.258 ± 0.003, and zeta potential of - 2.927 ± 0.163 mV. Furthermore, the TEM results of NLC-Eu displayed irregular round to spherical morphology with narrow size distribution and relatively uniformed particles. The drug loading capacity and entrapment efficiency of NLC-Eu were 4.99 and 90.93%, respectively. Furthermore, NLC-Eu exhibited cytotoxic effects on both, human and mice, breast cancer cells with IC50 values of 10.00 ± 4.81 µg/mL and 17.70 ± 0.57 µg/mL, respectively at 72 h. NLC-Eu also induced apoptosis on the MDA MB-231 cells. In the sub-chronic toxicity study, all of the studied mice did not show any signs of toxicity, abnormality or mortality. Besides that, no significant changes were observed in the body weight, internal organ index, hepatic and renal histopathology, serum biochemistry, nitric oxide and malondialdehyde contents. CONCLUSIONS: This study suggests that the well-characterized NLC-Eu offers a safe and promising carrier system which has cytotoxic effect on breast cancer cell lines.

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro.

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012-2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC50) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.

Excipient selection and aerodynamic characterization of nebulized lipid-based nanoemulsion loaded with docetaxel for lung cancer treatment.

Docetaxel has demonstrated extraordinary anticancer effects on lung cancer. However, lack of optimal bioavailability due to poor solubility and high toxicity at its therapeutic dose has hampered the clinical use of this anticancer drug. Development of nanoemulsion formulation along with biocompatible excipients aimed for pulmonary delivery is a potential strategy to deliver this poorly aqueous soluble drug with improved bioavailability and biocompatibility. In this work, screening and selection of pharmaceutically acceptable excipients at their minimal optimal concentration have been conducted. The selected nanoemulsion formulations were prepared using high-energy emulsification technique and subjected to physicochemical and aerodynamic characterizations. The formulated nanoemulsion had mean particle size and ζ-potential in the range of 90 to 110 nm and - 30 to - 40 mV respectively, indicating high colloidal stability. The pH, osmolality, and viscosity of the systems met the ideal requirement for pulmonary application. The DNE4 formulation exhibited slow drug release and excellent stability even under the influence of extreme environmental conditions. This was further confirmed by transmission electron microscopy as uniform spherical droplets in nanometer range were observed after storage at 45 ± 1 °C for 3 months indicating high thermal stability. The nebulized DNE4 exhibited desirable aerosolization properties for pulmonary delivery application and found to be more selective on human lung carcinoma cell (A549) than normal cell (MRC-5). Hence, these characteristics make the formulation a great candidate for the potential use as a carrier system for docetaxel in targeting lung cancer via pulmonary delivery.

Optimization, formulation, and characterization of multiflavonoids-loaded flavanosome by bulk or sequential technique.

This study involves adaptation of bulk or sequential technique to load multiple flavonoids in a single phytosome, which can be termed as "flavonosome". Three widely established and therapeutically valuable flavonoids, such as quercetin (Q), kaempferol (K), and apigenin (A), were quantified in the ethyl acetate fraction of Moringa oleifera leaves extract and were commercially obtained and incorporated in a single flavonosome (QKA-phosphatidylcholine) through four different methods of synthesis - bulk (M1) and serialized (M2) co-sonication and bulk (M3) and sequential (M4) co-loading. The study also established an optimal formulation method based on screening the synthesized flavonosomes with respect to their size, charge, polydispersity index, morphology, drug-carrier interaction, antioxidant potential through in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics, and cytotoxicity evaluation against human hepatoma cell line (HepaRG). Furthermore, entrapment and loading efficiency of flavonoids in the optimal flavonosome have been identified. Among the four synthesis methods, sequential loading technique has been optimized as the best method for the synthesis of QKA-phosphatidylcholine flavonosome, which revealed an average diameter of 375.93±33.61 nm, with a zeta potential of -39.07±3.55 mV, and the entrapment efficiency was >98% for all the flavonoids, whereas the drug-loading capacity of Q, K, and A was 31.63%±0.17%, 34.51%±2.07%, and 31.79%±0.01%, respectively. The in vitro 1,1-diphenyl-2-picrylhydrazyl kinetics of the flavonoids indirectly depicts the release kinetic behavior of the flavonoids from the carrier. The QKA-loaded flavonosome had no indication of toxicity toward human hepatoma cell line as shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide result, wherein even at the higher concentration of 200 µg/mL, the flavonosomes exert >85% of cell viability. These results suggest that sequential loading technique may be a promising nanodrug delivery system for loading multiflavonoids in a single entity with sustained activity as an antioxidant, hepatoprotective, and hepatosupplement candidate.

Successful transfer of plasmid DNA into in vitro cells transfected with an inorganic plasmid-Mg/Al-LDH nanobiocomposite material as a vector for gene expression.

The delivery of a full plasmid, encoding the green fluorescent protein gene into African monkey kidney (Vero3) cells, was successfully achieved using nanobiocomposites based on layered double hydroxides. This demonstrated the potential of using the system as an alternative DNA delivery vector. Intercalation of the circular plasmid DNA, pEGFP-N2, into Mg/Al-NO(3)(-) layered double hydroxides (LDH) was accomplished through anion exchange routes to form the nanobiocomposite material. The host was previously synthesized at the Mg(2+) to Al(3+) molar ratio R(i) = 2 and subsequently intercalated with plasmid DNA. Size expansion of the interlamellae host from 8.8 A in LDH to 42 A was observed in the resulting nanobiocomposite, indicating stable hybridization of the plasmid DNA. The powder x-ray diffraction (PXRD) results, supplemented with Fourier-transform infrared (FTIR) spectroscopy, compositional and electrophoresis studies confirmed the encapsulation episode of the biomaterial. In order to elucidate the use of this resulting nanobiocomposite as a delivery vector, an MTT assay was performed to determine any cytotoxic effects of the host towards cells. The intercalated pEGFP-N2 anion was later successfully recovered through acidification with HNO(3) after treatment with DNA-degrading enzymes, thus also showing the ability of the LDH host to protect the intercalated biomaterial from degradation. Cell transfection studies on Vero3 cells were then performed, where cells transfected with the nanobiocomposite exhibited fluorescence as early as 12 h post-treatment compared to naked delivery of the plasmid itself.