Therapeutic Efficacy of Quercetin Versus Its Encapsulated Nanoparticle on Tongue Squamous Cell Carcinoma Cell Line

Study Overview
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Brief Summary
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Squamous cell carcinoma (SCC) is the most common oral cavity carcinoma. Conventional therapeutic modalities for oral malignancy include surgery, radiotherapy and chemotherapy alone or in combinations.The major obstacle of using current anticancer drugs is; first the non-specific tissue distribution, as these drugs are unable to distinguish between normal and cancer cells.Quercetin is a bioactive flavonoid having strong antioxidant properties. .Among all the nanomaterials, polymeric nanoparticles are of significant interest for drug delivery applications due to many unique features of nanoparticle polymers.This is the first study to investigate the anticancer effects of (Quercetin) either free or encapsulated by PLGA-PEG NPs in tongue squamous cell carcinoma (TSCC) cell line.

Detailed Description
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Squamous cell carcinoma (SCC) is the most common oral cavity carcinoma. Conventional therapeutic modalities for oral malignancy include surgery, radiotherapy and chemotherapy alone or in combinations. The major obstacle of using current anticancer drugs is; first the non-specific tissue distribution, as these drugs are unable to distinguish between normal and cancer cells. Quercetin is a bioactive flavonoid having strong antioxidant properties. It is naturally present in a wide variety of fruits and vegetables. Among all the nanomaterials, polymeric nanoparticles are of significant interest for drug delivery applications due to many unique features of nanoparticle polymers. Polymeric nanocarriers have been fabricated from natural and synthetic polymers. Poly ethylene glycol-poly lactide-co-glycolic acid (PEG-PLGA) amphiphilic copolymer is an emergent system because it can be easily synthesized and possesses a lot of good qualities. Several previous in vitro and in vivo studies have evaluated the cytotoxic effects of quercetin and have revealed that it decreases cell viability and increases cell apoptotic rate in OSCC. However, the anti-cancer property of quercetin in tongue squamous cell carcinoma (TSCC) has not been studied yet. This is the first study to investigate the anticancer effects of (Quercetin) either free or encapsulated by PLGA-PEG NPs in tongue squamous cell carcinoma (TSCC) cell line.

Official Title
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Therapeutic Efficacy of Quercetin Versus Its Encapsulated Nanoparticle on Tongue Squamous Cell Carcinoma Cell Line

Conditions
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Oral Cancer

Intervention / Treatment
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* Drug: Quercetin 3,3',4',5,6-Pentahydroxyflavone, 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one
* Drug: Quercetin-encapsulated PLGA-PEG nanoparticles (Nano-QUT)
* Drug: Doxorubicin chemotherapeutic drug as a positive control


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Tongue Squamous cell carcinoma cell lines Quercetin drug. Quercetin-encapsulated PLGA-PEG nanoparticles (Nano-QUT) Application of a Quercetin drug as chemotherapeutic drug. Detection of Quercetin activity in apoptosis or cytotoxicity/cell viability. Exclusion Criteria: Any cancer cell line other than tongue Squamous cell carcinoma cell lines Any use of Quercetin other than chemotherapy. Detection of Quercetin activities other than apoptosis or cytotoxicity/cell viability
Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Non-Randomized
Intervention Model: Factorial Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Quercetin drug.<br> | Drug: Quercetin 3,3',4',5,6-Pentahydroxyflavone, 2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one<br>* Appearance (Color) Conforms Yellow Appearance (Form) Powder 1H NMR Spectrum Conforms to Structure Loss on Drying < 4 % _ Purity (HPLC) > 95 %<br>* Other names: Citrus bioflavonoid, Sophoretin; Meletin; Quercetine; Xanthaurine; Quercetol; Quercitin; Quertine; Flavin;|
| Experimental: Quercetin-encapsulated PLGA-PEG nanoparticles<br> | Drug: Quercetin-encapsulated PLGA-PEG nanoparticles (Nano-QUT)<br>* Nano QUT will prepared by PLGA-PEG nanocomposites that will be prepared by an oil-in-water (O/W) single emulsion solvent evaporation method<br>|
| Active Comparator: Doxorubicin chemotherapeutic drug<br> | Drug: Doxorubicin chemotherapeutic drug as a positive control<br>* Doxorubicin is a type of chemotherapy drug called an anthracycline. It slows or stops the growth of cancer cells by blocking an enzyme called topo isomerase 2<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Cytotoxicity/Cell viability. | MTT assay for cellular viability: The cytotoxic impacts of the tested drugs and Nano QUT-encapsulation will be measured by MTT. The (HNO-97) cells will be cultured in 96-well plates at a density of 5 × 103 cells/well. All drugs with their described concentrations will be added to the media over tongue SCC cell lines. After a day of incubation, the dissolved MTT in PBS will be added to each well at a final concentration of 5 mg/ml, and the samples will be incubated at 37 °C for 4h. Water-insoluble dark blue formazan crystals that will be formed during MTT cleavage in actively metabolizing cells will then be dissolved in dimethyl sulfoxide (DMSO). Absorbance will be measured at A455 nm, using an ELISA microplate reader | within 1 week after cell line propagation | 
| Apoptosis. | Annexin V and propidium iodide (PI) stains will be used in the determination of apoptosis after treatment with the free, nano counterpart of QUT and free Dox post 24h of their incubation over the HNO-97 cell line. The apoptotic analysis will be dedicated to differentiating between early and late apoptotic cells, as well as necrotic cells. The apoptosis of the treated and untreated HNO-97 line with the proposed free, nano counterpart of Nano-QUT and Dox will be analyzed by ﬂow cytometer apparatus | within 1 week after cell line propagation | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Gene expression of (BCL-2) . | Following treatment with the proposed free DOX, free and Nano-QUT formulations for 24 h, the cells are harvested, lysed and tested with RT-PCR using specific primers to estimate the fold change of the apoptotic signals (BCL-2 and Bax) and survival pathway (Expression of PI3K gene). GAPDH will be used as a housekeeping gene to normalize the level of target gene expression. | within 1 week after cell line propagation | 
| Gene expression of ( Bax gene) | Following treatment with the proposed free DOX, free and Nano-QUT formulations for 24 h, the cells are harvested, lysed and tested with RT-PCR using specific primers to estimate the fold change of the apoptotic Bax gene | within 1 week after cell line propagation | 
| survival pathway (Expression of pi3k gene) | Following treatment with the proposed free DOX, free and Nano-QUT formulations for 24 h, the cells are harvested, lysed and tested with RT-PCR using specific primers to estimate the fold change of survival pathway (Expression of pi3k gene) | within 1 week after cell line propagation | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Tongue, SCC, chemotherapy, Quercetin, PLGA-PEG nanoparticle