Inhibition of SARS-CoV-2-Induced NLRP3 Inflammasome-Mediated Lung Cell Inflammation by Triphala-Loaded Nanoparticle Targeting Spike Glycoprotein S1.

The COVID-19 pandemic, caused by SARS-CoV-2, poses a significant global health threat. The spike glycoprotein S1 of the SARS-CoV-2 virus is known to induce the production of pro-inflammatory mediators, contributing to hyperinflammation in COVID-19 patients. Triphala, an ancient Ayurvedic remedy composed of dried fruits from three plant species-Emblica officinalis (Family Euphorbiaceae), Terminalia bellerica (Family Combretaceae), and Terminalia chebula (Family Combretaceae)-shows promise in addressing inflammation. However, the limited water solubility of its ethanolic extract impedes its bioavailability. In this study, we aimed to develop nanoparticles loaded with Triphala extract, termed "nanotriphala", as a drug delivery system. Additionally, we investigated the in vitro anti-inflammatory properties of nanotriphala and its major compounds, namely gallic acid, chebulagic acid, and chebulinic acid, in lung epithelial cells (A549) induced by CoV2-SP. The nanotriphala formulation was prepared using the solvent displacement method. The encapsulation efficiency of Triphala in nanotriphala was determined to be 87.96 ± 2.60% based on total phenolic content. In terms of in vitro release, nanotriphala exhibited a biphasic release profile with zero-order kinetics over 0-8 h. A549 cells were treated with nanotriphala or its active compounds and then induced with 100 ng/mL of spike S1 subunit (CoV2-SP). The results demonstrate that chebulagic acid and chebulinic acid are the active compounds in nanotriphala, which significantly reduced cytokine release (IL-6, IL-1ß, and IL-18) and suppressed the expression of inflammatory genes (IL-6, IL-1ß, IL-18, and NLRP3) (p < 0.05). Mechanistically, nanotriphala and its active compounds notably attenuated the expression of inflammasome machinery proteins (NLRP3, ASC, and Caspase-1) (p < 0.05). In conclusion, the nanoparticle formulation of Triphala enhances its stability and exhibits anti-inflammatory properties against CoV2-SP-induction. This was achieved by suppressing inflammatory mediators and the NLRP3 inflammasome machinery. Thus, nanotriphala holds promise as a supportive preventive anti-inflammatory therapy for COVID-19-related chronic inflammation.

The in vitro effects of black soldier fly larvae (Hermitia illucens) oil as a high-functional active ingredient for inhibiting hyaluronidase, anti-oxidation benefits, whitening, and UVB protection.

Objective: Larvae of Hermitia illucens, or black soldier fly larvae (BSFL), have been recognized for their high lipid yield with a remarkable fatty acid profile. BSFL oil (SFO) offers the added value of a low environmental footprint and a sustainable product. In this study, the characteristics and cosmetic-related activities of SFO were investigated and compared with rice bran oil, olive oil and krill oil which are commonly used in cosmetics and supplements. Methods: The physicochemical characteristics were determined including acid value, saponification value, unsaponifiable matter and water content of SFO. The fatty acid composition was determined using GC-MS equipped with TR-FAME. The in vitro antioxidant properties were determined using DPPH, FRAP and lipid peroxidation inhibition assays. Antihyaluronidase (anti-HAase) activity was measured by detecting enzyme activity and molecular docking of candidate compounds toward the HAase enzyme. The safety assessment towards normal human cells was determined using the MTT assay and the UVB protection upon UVB-irradiated fibroblasts was determined using the DCF-DA assay. The whitening effect of SFO was determined using melanin content inhibition. Results: SFO contains more than 60% polyunsaturated fatty acids followed by saturated fatty acids (up to 37%). The most abundant component found in SFO was linoleic acid (C18:2 n-6 cis). Multiple anti-oxidant mechanisms of SFO were discovered. In addition, SFO and krill oil prevented hyaluronic acid (HA) degradation via strong HAase inhibition comparable with the positive control, oleanolic acid. The molecular docking confirmed the binding interactions and molecular recognition of major free fatty acids toward HAase. Furthermore, SFO exhibited no cytotoxicity on primary human skin fibroblasts, HaCaT keratinocytes and PBMCs (IC50 values > 200 µg/mL). SFO possessed significant in-situ anti-oxidant activity in UVB-irradiated fibroblasts and the melanin inhibition activity as effective as well-known anti-pigmenting compounds (kojic acid and arbutin, p < 0.05). Conclusion: This study provides scientific support for various aspects of SFO. SFO can be considered an alternative oil ingredient in cosmetic products with potential implications for anti-skin aging, whitening and UVB protection properties, making it a potential candidate oil in the cosmetic industry.

KLVFF Conjugated Curcumin Microemulsion-Based Hydrogel for Transnasal Route: Formulation Development, Optimization, Physicochemical Characterization, and Ex Vivo Evaluation.

Curcumin is a potent natural compound used to treat Alzheimer's disease (AD). However, the clinical usefulness of curcumin to treat AD is restricted by its low oral bioavailability and difficulty permeating the blood-brain barrier. To overcome such drawbacks, various alternative strategies have been explored, including the transnasal route. However, rapid mucociliary clearance in the nasal cavity is a major hindrance to drug delivery. Thus, designing a delivery system for curcumin to lengthen the contact period between the drug and nasal mucosa must be employed. This study describes the optimization of KLVFF conjugated curcumin microemulsion-base hydrogel (KCMEG) to formulate a prototype transnasal preparation using the response surface method to improve a mucoadhesive property. A central composite design was employed to optimize and evaluate two influencing factors: the concentration of carbopol 940 and the percentage of KLVFF conjugated curcumin microemulsion (KCME). The physicochemical properties, anti-cholinesterase activity, and anti-aggregation activities of KCME were investigated in this study. The studied factors, in terms of main and interaction effects, significantly (p < 0.05) influenced hardness and adhesiveness. The optimized KCMEG was evaluated for pH, spreadability, and mucoadhesive properties. Ex vivo nasal ciliotoxicity to optimize KCMEG was performed through the porcine nasal mucosa. KCME was transparent, with a mean globule size of 70.8 ± 3.4 nm and a pH of 5.80 ± 0.02. The optimized KCMEG containing 2% carbopol 940 showed higher in vitro mucoadhesive potential (9.67 ± 0.13 min) compared with microemulsion and was also found to be free from nasal ciliotoxicity during histopathologic evaluation of the porcine nasal mucosa. The result revealed that both the concentration of carbopol 940 and the percentage of KCME play a crucial role in mucoadhesive properties. In conclusion, incorporating a mucoadhesive agent in a microemulsion can increase the retention time of the formulation, leading to enhanced brain delivery of the drug. Findings from the investigation revealed that KCMEG has the potential to constitute a promising approach to treating AD via transnasal administration.

Targeting Spike Glycoprotein S1 Mediated by NLRP3 Inflammasome Machinery and the Cytokine Releases in A549 Lung Epithelial Cells by Nanocurcumin.

Chronic inflammation and tissue damage can result from uncontrolled inflammation during SARS-CoV-2 or COVID-19 infections, leading to post-acute COVID conditions or long COVID. Curcumin, found in turmeric, has potent anti-inflammatory properties but limited effectiveness. This study developed nanocurcumin, a curcumin nanoparticle, to enhance its physical and chemical stability and investigate its in vitro anti-inflammatory properties upon CoV2-SP induction in lung epithelial cells. Nanocurcumin was prepared by encapsulating curcumin extract in phospholipids. The particle size, polydispersity index, and zeta potential of nanocurcumin were measured using dynamic light scattering. The encapsulated curcumin content was determined using HPLC analysis. The encapsulation efficiency of curcumin was 90.74 ± 5.35% as determined by HPLC. Regarding the in vitro release of curcumin, nanocurcumin displayed a higher release content than non-nanoparticle curcumin. Nanocurcumin was further investigated for its anti-inflammatory properties using A549 lung epithelial cell line. As determined by ELISA, nanocurcumin showed inhibitory effects on inflammatory cytokine releases in CoV2-SP-stimulated conditions, as evidenced by a significant decrease in IL-6, IL-1ß and IL-18 cytokine secretions compared with the spike-stimulated control group (p < 0.05). Additionally, as determined by RT-PCR, nanocurcumin significantly inhibited the CoV2-SP-stimulated expression of inflammatory genes (IL-6, IL-1ß, IL-18, and NLRP3) compared with the spike-stimulated control group (p < 0.05). Regarding the inhibition of NLRP3 inflammasome machinery proteins by Western blot, nanocurcumin decreased the expressions of inflammasome machinery proteins including NLRP3, ASC, pro-caspase-1, and the active form of caspase-1 in CoV2-SP-stimulated A549 cells compared with the spike-stimulated control group (p < 0.05). Overall, the nanoparticle formulation of curcumin improved its solubility and bioavailability, demonstrating anti-inflammatory effects in a CoV2-SP-induced scenario by inhibiting inflammatory mediators and the NLRP3 inflammasome machinery. Nanocurcumin shows promise as an anti-inflammatory product for preventing COVID-19-related airway inflammation.

Surface Modification of Curcumin Microemulsions by Coupling of KLVFF Peptide: A Prototype for Targeted Bifunctional Microemulsions.

Curcumin is one of the most promising natural therapeutics for use against Alzheimer's disease. The major limitations of curcumin are its low oral bioavailability and difficulty in permeating the blood-brain barrier. Therefore, designing a delivery system of curcumin to overcome its limitations must be employed. KLVFF, a peptide known as an amyloid blocker, was used in this study as a targeting moiety to develop a targeted drug delivery system. A prototype of transnasal KLVFF conjugated microemulsions containing curcumin (KLVFF-Cur-ME) for the nose-to-brain delivery was fabricated. The KLVFF-Cur-ME was developed by a titration method. A conjugation of KLVFF was performed through a carbodiimide reaction, and the conjugation efficiency was confirmed by FTIR and DSC technique. KLVFD-Cur-ME was characterized for the drug content, globule size, zeta potential, and pH. A transparent and homogeneous KLVFF-Cur-ME is achieved with a drug content of 80.25% and a globule size of 76.1 ± 2.5 nm. The pH of KLVFF-Cur-ME is 5.33 ± 0.02, indicating non-irritation to nasal tissues. KLVFD-Cur-ME does not show nasal ciliotoxicity. An ex vivo diffusion study revealed that KLVFF-Cur-ME partitions the porcine nasal mucosa through diffusion, following the Higuchi model. This investigation demonstrates the successful synthesis of a bifunctional KLVFF-Cur-ME as a novel prototype to deliver anti-Aß aggregation via an intranasal administration.

Validated HPTLC and antioxidant activities for quality control of catechin in a fermented tea (Camellia sinensis var. assamica).

Miang, a Thai traditional fermented tea (Camellia sinensis var. assamica), is exploited as nutraceutical and cosmeceutical ingredients despite limited standardization studies. Thus, this research aimed to develop a simple and rapid method for miang quality control using catechin and high-performance thin-layer chromatography (HPTLC) validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the Association of Official Analytical Collaboration (AOAC). The developing solvent consisting of toluene: ethyl acetate: acetone: formic acid (6:6:6:1 v/v/v/v) showed acceptable specificity with R f value of 0.54 ± 0.02 and linearity with correlation coefficient of 0.9951. The recovery was 98.84%-103.53%, and the RSD of intra- and inter-day precision was 0.70%-3.00% and 1.93%-4.94%, respectively. Miang ethyl acetate fraction is suggested to be attractive ingredient due to rich catechin (25.78 ± 0.53%), prolonged stability at 40 ◦C, and strong antioxidants determined by the assays of ABTS (IC50 = 3.32 ± 0.74 mg/ml), FRAP (89.05 ± 15.49 mg equivalent of FeSO4/g), and inhibition of lipid peroxidation (IC50 = 4.36 ± 0.67 mg/ml).

Dermal targeting of Centella asiatica extract using hyaluronic acid surface modified niosomes.

The work aimed to develop Centella asiatica extract-loaded niosomes (CAE-Nio) and surface modified niosomes by hyaluronic acid (CAE-Nio-HA) to enhance transdermal penetration. Niosome formulations were prepared by film hydration method using Tween 60 and Span 60 as nonionic surfactants, cholesterol and various CAE contents. Various HA concentrations were investigated to obtain optimized CAE-Nio enhancing further skin penetration. Results showed that niosomes prepared from Tween 60 yielded suitable CAE encapsulated niosomes with mean particle size and zeta-potential of 155 nm and -15 mV, respectively. The niosomes exhibited high encapsulation efficiency (%EE) and drug loading capacity (%DL) of 71-77% and 3-7%, respectively. Incorporating HA to niosome decreased %DL and caused larger particle size and increased zeta-potential in a dose dependent manner while %EE remained unaffected. The sustained-release behaviour of CAE from all niosomes was under a diffusion controlled mechanism. Asiaticoside, a relatively polar compound from CAE-Nio-HA could penetrate through the stratum corneum and dermis in a larger amount than from CAE-Nio and CAE solution. CAE-Nio-HA formulations showed good stability under low temperature (4 °C and 25 °C) for periods longer than 4 months. In conclusion, the developed Nio-HA is a promising delivery system for asiaticoside to enhanced dermal absorption, permeation and accumulation in viable epidermis and dermis layers. This system can also be applied to other hydrophilic natural active compounds.

FLT3, a prognostic biomarker for acute myeloid leukemia (AML): Quantitative monitoring with a simple anti-FLT3 interaction and flow cytometric method.

BACKGROUND: Overexpression of fms-like tyrosine kinase 3 (FLT3) protein in leukemia is highly related to poor prognosis and reduced survival rate in acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients. Simple but efficient quantification of FLT3 protein levels on the leukemic cell surface using flow cytometry had been developed for rapid determination of FLT3 on intact cell surface. METHODS: Quantitation protocol for FLT3 biomarker in clinical samples was developed and validated. Cell model selection for calibration curve construction was identified and evaluated. Selected antibody concentrations, cell density, and incubation time were evaluated for most appropriate conditions. Comparison of the developed FLT3 determination protocol with the conventional Western blot analysis was performed. RESULTS: EoL-1 cell line was selected for using as positive control cells. Calibration curve (20%-120% of FLT3 positive cells) and quality control (QC) levels were constructed and evaluated. The results demonstrated good linearity (r2  > 0.99). The intra- and inter-day precision and accuracy, expressed as the coefficient of variation (%CV) and % recovery, were <20% and fell in 80%-120% in all cases. When compared with Western blotting results, FLT3 protein expression levels in leukemia patient's bone marrow samples were demonstrated in the same trend. CONCLUSIONS: The effective, reliable, rapid, and economical analytical technique using the developed flow cytometric method was demonstrated for FLT3 protein determination on leukemic cell surface. This method provided a practical analysis of FLT-3 biomarker levels which is valuable for physician decision in acute leukemia treatment.

Enhancement of anti-inflammatory activity of polyphenolic flavonoid rutin by encapsulation.

The cellular mechanisms underlying the anti-inflammatory activity of rutin which has been found to have in vivo inhibitory effects merit more evaluation. The effects of rutin and encapsulated-rutin on lipopolysaccharide (LPS)-induced IL-6 secretion, NF-κB expression, as well as protein denaturation were investigated. The secretion of IL-6 was not found to have significantly reduced upon incubation with either rutin or encapsulated-rutin at all concentrations. At 100 µg/mL, the cells treated with encapsulated-rutin brought about slightly reduced IL-6 secretion but significantly inhibited NF-kB protein expression and protein denaturation in comparison with rutin. Inflammation can be resolved through many mechanisms. The inhibition of IL-6 and NF-kB can serve not only to terminate inflammation but also to inhibit other cytokines or mechanisms. Further investigations are necessary to clarify, verify and establish the anti-inflammatory mechanisms of rutin. Additionally, the encapsulation is an interesting technique for enhancing rutin activity.

Development of Microemulsion Delivery System of Essential Oil from Zingiber cassumunar Roxb. Rhizome for Improvement of Stability and Anti-Inflammatory Activity.

The present study aims to investigate the major constituents of the essential oil from Zingiber cassumunar rhizome (EO) and to develop microemulsions with enhanced chemical stability and anti-inflammatory activity of EO. The major constituents of EO were terpinen-4-ol (40.5 ± 6.6%) and sabinene (17.4 ± 1.4%) as determined by gas chromatography-mass spectrometry. These compounds were responsible for the anti-inflammatory activities of EO. Sabinene and terpinen-4-ol significantly reduced nuclear factor-kappa B (NF-kB) expression by 47 ± 5 and 78 ± 8%, respectively (p < 0.001) and significantly reduced the interleukin-6 (IL-6) secretion levels to 64 ± 4% (p < 0.05) and 50 ± 1% (p < 0.001), respectively. EO microemulsions, developed using the system of EO/Tween 20 and propylene glycol (2:1)/water, showed the internal droplet size in the range of 211.5 ± 63.3 to 366.7 ± 77.8 nm. Both EO and EO microemulsions were shown to be safe for human use since there was no apparent toxic effect on human peripheral blood mononuclear cells. Interestingly, EO microemulsion could significantly protect sabinene from the evaporation after heating-cooling stability test, which leads to a good stability and high efficacy. Moreover, EO microemulsions significantly enhanced the anti-inflammatory effect comparing to the native EO. Therefore, microemulsions were attractive delivery system for natural anti-inflammatory compounds since they could enhance both efficacy and stability of EO.

LFA-1 on leukemic cells as a target for therapy or drug delivery.

Leukemia therapeutics are aiming for improved efficacy by targeting molecular markers differentially expressed on cancerous cells. Lymphocyte function-associated antigen-1 (LFA-1) expression on various types of leukemia has been well studied. Here, the role and expression of LFA-1 on leukemic cells and the possibility of using this integrin as a target for drug delivery is reviewed. To support this rationale, experimental results were also included where cIBR, a cyclic peptide derived from a binding site of LFA-1, was conjugated to the surface of polymeric nanoparticles and used as a targeting ligand. These studies revealed a correlation of LFA-1 expression level on leukemic cell lines and binding and internalization of cIBR-NPs suggesting a differential binding and internalization of cIBR-NPs to leukemic cells overexpressing LFA-1. Nanoparticles conjugated with a cyclic peptide against an accessible molecular marker of disease hold promise as a selective drug delivery system for leukemia treatment.