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ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 3  |  Page : 480-490

Development of α-tocopherol surface-modified targeted delivery of 5-fluorouracil-loaded poly-D, L-lactic-co-glycolic acid nanoparticles against oral squamous cell carcinoma


1 Department of Oral and Maxillofacial Surgery, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Oral Pathology and Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India
3 Department of Clinical Laboratory Science, College of Applied Medical Sciences, King Khalid University; Research Centre for Advanced Materials Science, King Khalid University, Abha, Kingdom of Saudi Arabia

Date of Web Publication29-May-2019

Correspondence Address:
Dr. Saurabh Srivastava
Department of Oral and Maxillofacial Surgery, King George's Medical University, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_263_18

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 > Abstract 


Objective: The aim of the study to develop surface modified targeted moiety α-tocopherol (α-t) encapsulated with 5-fluorouracil (5-FU)-poly-D, L-lactic-co-glycolic acid nanoparticles (PLGA NPs) toward the anticancer activity against oral squamous cell carcinoma (OSCC).
Materials and Methods: 5-FU was conjugated with the polymer, PLGA by ionic cross-linking and α-tocopherol use as a functionalized surface moiety. Characterization, drug entrapment efficiency, and in-vitro drug release system were optimized at different pH 7.4 and pH 4.5. The in-vitro cell was performed to optimize the anticancer activity through MTT assay and apoptotic staining assay was also performed by flow cytometry to evaluate the cellular apoptotic activity and cellular uptake.
Results: The particle size was distributed within an average range of 145–162 nm, the polydispersity index values lie 0.16–0.30, and the surface charge was at the negative side, –17mV to –23mV. The in vitro drug release system showed more sympathetic situation at pH 7.4 as compared to pH 4.5, for targeted NPs, approximately 86% and 69%, respectively. The non-targeted 5-FU-PLGA NPs showed drug release of 83% and 64% at pH 7.4 and 4.5 subsequently. In vitro anticancer activity confirmed the intense inhibition by α-t-FU-PLGA NPs of 79.98% after 96 h treatment of SCC15 cells and confirmed the steady-state inhibition of 83.74% after 160 h incubation in comparison to 5-FU-PLGA NPs. Subsequently, the early apoptosis, 27.98%, and 16.45%, and late apoptosis, 47.29%, and 32.57%, suggested the higher apoptosis rate in targeted NPs against OSCC.
Conclusions: The surface modified α-t-FU-PLGA NP was treated over SCC15 cells, and the oral cancer cells have shown the high intensity of cellular uptake, which confirmed that the target moiety has successfully invaded over the surface of cancer cells and shown advanced targeted delivery against OSCC.

Keywords: Anticancer, fluorouracil, nanoformulation, oral squamous cell carcinoma, poly-D,L-lactic-co-glycolic acid, squamous cell carcinoma-15 cells, targeted moiety


How to cite this article:
Srivastava S, Gupta S, Mohammad S, Ahmad I. Development of α-tocopherol surface-modified targeted delivery of 5-fluorouracil-loaded poly-D, L-lactic-co-glycolic acid nanoparticles against oral squamous cell carcinoma. J Can Res Ther 2019;15:480-90

How to cite this URL:
Srivastava S, Gupta S, Mohammad S, Ahmad I. Development of α-tocopherol surface-modified targeted delivery of 5-fluorouracil-loaded poly-D, L-lactic-co-glycolic acid nanoparticles against oral squamous cell carcinoma. J Can Res Ther [serial online] 2019 [cited 2019 Jun 24];15:480-90. Available from: http://www.cancerjournal.net/text.asp?2019/15/3/480/251400




 > Introduction Top


Cancer is the uncontrolled expansion of flawed cells to build into a pulpy mass called tumor, which affects surrounding healthy tissues to made collusion.[1] It is the topmost reason for the loss of life all around the world, the responsibility of new patients was increased around 14.5 million till 2012,[2] and it is rising at a high rate of approximately 50% up to the next upcoming decades.[3],[4] Specifically, in India, approximately 1.2 million malignant subjects were reported,[5] and it was estimated that the approximately 1 million new cancer patients will be raised by 2026.[6]

Oral cancer is the most frequent type of solid tumor globally [7] and comes under the top three most occurred types of cancer in India only.[8] Oral cancer could be seen in the inner part of mouths, such as tongue, lips,[9] inside part of the cheek, tooth gum parts, and all the related part.[10] Annually, 25 billion fresh subjects have been reported, and from them, 0.55 million cases were excluded from India till 2014,[11] with maximum cases within an average age of 45–55 years.[12] The rate of survival of oral cancer is 5 years.[13] The prime risk factor for oral cancer is tobacco, about 75%, including any type of tobacco use.[14],[15]

Squamous cell carcinoma (SCC),[16] established within the squamous cells, flat cells, underline the entire human body. The dissimilar forms of SCC were determined, and the most traditional forms are verrucous and basaloid.[17],[18] SCC over the oral cavity calls oral SCC (OSCC) and about 80% of oral cancer is SCC.[19],[20] OSCC may also the outcome of one of the continual irritations, such as dental cavity, much use of mouthwash, betel nuts, smoking and smokeless,[21],[22] and alcoholism, including other aspects, immunosuppression, catastrophic metabolism, deficiency in the enzyme, which regulates DNA, human genetic perceptivity,[23] human papillomavirus,[24],[25] and diet.[26] OSCC evoked on mucosa and premalignant condition, mostly leukoplakia, erythroplakia, dysplasia, lichen planus, and oral submucous fibrosis.[27],[28],[29],[30] Approximately 45% of OSCC proposed from the tongue surface.[31] In vitro cells studies with drug treatment quantify and explain biological characteristics of the destructive infirmity and provide a provisional mechanism to understand oral cancer.[32],[33]

OSCC was primarily treated by surgical incision additionally chemo-radio-therapy,[34] and the distinctive class of chemodrugs may be introduced against OSCC, which may be alone or in combination. There are different classes of chemotherapeutic drugs, which are used to treat oral cancer such as alkylating, nucleotide, anthracylines, alkaloids, and others.[35],[36] These agents have the competence to kill the tumor tissue through distraction in cell proliferation.[37]

5-Fluorouracil (5-FU),[38] nucleotide analog, has the consequential efficacy against OSCC through anticipating chromosomal cloning to suppress thymidylate synthesis exertion and ultimately block the transfiguration of deoxyuridine monophosphate to deoxythymidine monophosphate, resulting in inhibition of DNA replication through deoxythymidine triphosphate and further cell proliferation.[39],[40],[41] The conventional chemotherapy of 5-FU has utmost restriction over nonspecificity, limited half-life, less bioavailability, and limited therapeutic index, besides less unyielding to OSCC.[42] It was suggested that the drawback of 5-FU conventional dosage may be reduced through the nanoparticles (NPs)-targeted therapy specifically against OSCC.[43] The targeted drug delivery has the ability to directly inflate over the affected region.[44] It could interim cancer cells by inhibiting the protein, which is susceptible to tumor proliferation. The efficiency of 5-FU as a chemotherapeutic agent could be enhanced through a chambered drug–polymer nanoparticulate system that targeted over a specific region.[45] The 5-FU being designed to engulf in a bucket of polymer and surface would be protected through the target moiety, which has the capability to bind with a surface protein of the OSCC cells and made a way to transfer the drug directly on specific site and antiproliferate OSCC cells exclusively and may not affect the normal cells.[46],[47] In addition, the nanoformulation drug delivery has the competence of enhanced permeability and retention (EPR) effect,[48],[49] which assists aggregation of the drug-loaded-nanoparticulate system on leaky tumor tissues as compared to normal tissues.[50],[51] Poly-D, L-lactic-co-glycolic acid (PLGA) polymer extensively used to prepare the nanoparticulate control-release system for the drug, which is enclosed within the PLGA complex and specifically exaggerates the bioavailability of poor absorbed low lipophilic drug like 5-FU.[52],[53],[54] The targeted drug delivery of 5-FU NPs formulation was specifically design to attach over OSCC cells, according to the biochemical behavior of tumor site [55],[56] and thus provide an advanced drug delivery platform to minimize the side effects, toxicity,[57] and nonspecific inhibition over surrounding normal cells.[58] This advancement may improve the drug efficacy, bioavailability, prolonged release, and targeted over affected region.[59],[60]

α-Tocopherol (α-t), a fellow of Vitamin E group,[61] acquires phenol hydroxyl group having a maximal biological activity along with nontoxic characteristics and vigorously absorbed by humans.[62] The concussion of α-t in the inhibition of chronic diseases affirmed to be concord with oxidative stress and influences apoptosis in OSCC cells and tempers the accessibility of cancer cell surface that enhances the penetration of 5-FU.[63],[64] The targeting moiety, α-t, has the ability to bind with a specific class of receptors over the surface of cancer cells and fabricate the path to deliver the anticancer drug 5-FU. Infrequent target moieties such as peptides, folic acid, vitamins, and antibodies were composite within a network for targeted drug delivery system.[65],[66]

The goal of the study to formulate α-t surface-modified targeted PLGA NPs engulfed 5-FU and their evaluation on the basis of control-release system and OSCC cell treatment. The targeted α-t-FU-PLGA NPs were compared with nontargeted 5-FU nanoformulation, 5-FU-PLGA NPs on SCC15 cells, followed by in vitro assays, which were performed to assess the result through inhibitory cytotoxicity study and the affinity of in vitro cellular uptake and cell targeting and cell apoptosis rate [Figure 1].
Figure 1: Schematic representation for formulation of targeted α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and nontargeted 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and its squamous cell carcinoma-15 cell internalization

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 > Materials and Methods Top


Chemicals

5-FU 99% was purchased from (Cas No. 51.21.9) Sigma-Aldrich, India, and α-t was purchased from TCI Chemicals Pvt. Ltd. (India) (Cas No. 59-02-8). PLGA was purchased from Sigma-Aldrich, India (Product No. 808482-5G), SCC15, cancer cells were obtained from National Centre for Cell Sciences, Pune, India, and the rest chemicals used for experiments, standard analytical grade, were acquired from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India.

Preparation of targeted α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

5-FU was conjugated to the polymer PLGA by ionic cross-linking and α-t used as a functionalized surface moiety,[67] for the preparation of α-T-FU-PLGA NPs. PLGA 34.50 mg was dissolved in acetic acid 1% w/v (10 ml), and the pH was maintained at 4.8. The drug 5-FU was added to that solution. The solution was added in 0.5% polyvinyl alcohol solution and allowed for magnetic stirring for 1 h. The 5-FU-PLGA solution was allowed to ultrasonicated for 12 min at 20% amplitude to facilitate the solubility and retrieved a homogeneous amalgamation followed by washing with deionized water, then lyophilized, and stored at 4°C.

Surface functionalized of α-tocopherol as targeted moiety on 5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

30.25 mg of α-t was added in pH 7.4 phosphate-buffered saline (PBS). Subsequently, 17.5 ml 0.1% (w/v) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide aqueous solution was added drop-wise, under low-velocity magnetic stirring condition about 2 h to form cross-links. The formations of NPs have been formed impulsively under the carbodiimide reaction. The prepared 5-FU-PLGA NPs were activated by adding in 7.4 PBS and 250 μl of N-hydroxysuccinimide (1 mg/ml) under magnetic stirring for 3 h after that unreacted chemical has been washed with PBS. Both the solutions were added under magnetic stirring further for 3 h followed by overnight incubation and ultrasonicated [68] for 15 min, and the pellets were collected after washed with PBS. Eventually, the targeted α-T-FU-PLGA NPS were obtained and used for furthermore experiments.

Characterization of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

The particle size, zeta potential, and polydispersity index (PDI) of targeted α-T-FU-PLGA and nontargeted 5-FU-PLGA NPs were optimized through the instrument Malvern Zetasizer ver. 7.12 (Malvern Instrument, UK.), and morphology of the NPs was obtained through scanning electron microscope (SEM) (Quanta 450 FEG, the Netherlands).

In vitro drug-release system and drug entrapment efficiency

To study the release system and entrapment efficiency (EE) of targeted NPS and nontargeted NPs, a standard curve has been plotted between 5-FU concentration (μg/ml) and absorbance (nm) [Figure 2]. The absorbance of the solution of 5-FU was established by the ultraviolet (UV) spectrophotometer (PerkinElmer, Lambda 25, USA) at absorption maxima 267 nm. The α-t-FU-PLGA/5-FU-PLGA NPs were accommodated into a dialysis bag and engrossed at different pH 7.4 and pH 4.5 in PBS. The dialysis bag (Sigma-Aldrich, India) procured as per the protocol. The USP dissolution appliance grade 1 basket type was used to perform the analysis, and the speed of the apparatus was 100 rpm, 37°C. The dialysis bag was poured into the dissolution solution (gastric fluid maintained pH 7.4 and 4.5), and the 5 ml supernatant at definite time interval in h (0, 20, 40, 60, 80, 120, 160) was withdrawn and further analyzed for drug content through established standard calibration curve of the 5-FU solution using UV visible spectrophotometer and the in vitro drug release was calculated by the given formula. The EE value was monitored through high-speed centrifugation of 5-FU-PLGA/α-t-FU-PLGA NPs at 12,000 rpm for 45 min. The supernatant was further filtered with 0.2 μm membrane filter. The collected sample was further allowed to evaluate the absorbance value through UV spectroscopy and compare with earlier plotted graph of 5-FU solution, and percentage of EE was calculated by given formula.
Figure 2: Standard calibration curve of the 5-fluorouracil solution in between concentration (μg/ml) versus absorbance (nm)

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  • In vitro drug release (%): Concentration of drug at different time interval/absolute bulk of the drug × 100
  • EE (%): The absolute bulk of the drug − The unbound drug particles in supernatant/the absolute bulk of the drug × 100.


Cell culture

Human tongue SCC15, as oral cancer cell lines, cultured (DMEM/F12) and enriched with 10% heat-inactivated fetal bovine serum followed by addition of 1% concentration ratio of penicillin and streptomycin, and further, SCC15 cells were preserved in typical humidified incubator supplied with 5% CO2, 95% air at 37°C ± 0.5°C.

Cytotoxicity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles by methyl thiazolyl tetrazolium assay

SCC15 cells were fixed with a frequency of 10,000 cells into 96-well plates and acquiesced to abide by 24 h. The SCC15 cells were exhibited to 10 μL of α-t-FU-PLGA/5-FU-PLGA NPs at predetermined time intervals (24, 48, and 72 h) in a different dose; methyl thiazolyl tetrazolium (MTT) (0.2 mg/ml) was composite to all the well plate and sustenance for 4–6 h. 250 μL DMSO was mixed after the removal of medium and further vibrated for 12 min.

Cytotoxicity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles in drug-resistant squamous cell carcinoma 15 cells

The drug-resistant SCC15 cell lines were placed in into 96-well plates with a density of 10,000 cells per plates for 24 h, centrifuged, and then procured; the drug-resistant cells were again incubated different drug concentration of α-t-FU-PLGA/5-FU-PLGA NPs at dose 0, 0.25, 1.50, 3.0, 4.5, 6.0, and 7.5 μg/ml.

Therapeutic productivity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5- fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles against squamous cell carcinoma 15 cells

The therapeutic productivity of targeted α-t-FU-PLGA and nontargeted 5-FU-PLGA NPs as antiproliferating agent established against SCC15 cell lines through MTT method. The malignant cells, SCC15, were fixed with the frequency of 5000 cells into 96-well plates for the overnight incubation, the cells were treated with different concentrations 1.0, 0.5, and 0.1 mg/ml, and the antiproliferating effect of the α-t-FU-PLGA/5-FU-PLGA NPs was examined. The cell viability of the NPs was quantified in 96 h and indirectly the cytotoxicity effects of the targeted and nontargeted NPs.

In vitro cellular uptake of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

SCC15 cell lines were seeded into six-well plates and allowed to treat with for 1 day; after that, the cells were exhibited to formulated α-t-FU-PLGA and nontargeted 5-FU-PLGA NPa labeled with distinct concentration of fluorescein isothiocyanate (FITC) for 4–6 h, and the cellular uptake and targeting were observed by fluorescent microscopy (Carl Zeiss, Germany) using 485 nm excitation for FITC and fluorescence intensity within the treated SCC15 cells being quantified by a microplate reader.

Cell apoptosis by α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

AV-FITC/PI (Invitrogen, Thermo Fisher Scientific, India) apoptotic staining assay was performed by flow cytometry analysis to obtain the programmed cell death.[69] The SCC15 cells were incubated with α-t-FU-PLGA/5-FU-PLGA NPs formulation to attain the rate of cell apoptosis.[70] The SCC15 cells treated with α-t-FU-PLGA/5-FU-PLGA NPs were matured in six-well plates at the density 5 × 103 cells. The treated cells were washed with PBS, and 10 μL AV-FITC conjugate and PI staining solution were poured to each cells solution and incubated at 25°c for 15 min and defended from light, and the fluorescence of the SCC15 cells was examined through flow cytometry (BD Influx Model, USA).

Data analysis

All the scientific data were expressed as in the standard deviation and mean and one-way ANOVA analysis and least significance difference test applied for comparison of the groups. Prism 7.0 (GraphPad, Fay Avenue, Suite 230, La Jalla, CA 92037, USA) statistical software, was used for data analysis (P > 0.05).


 > Results Top


Characterization of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

The surface conjugation of α-t as a targeted moiety on 5-FU-PLGA NPs eventually increased in average particle size ranges from 145 nm to 160 nm as the nontargeted NPs formulation having the negative surface charge about −17 mV and increased in α-t functionalized α-t-FU-PLGA nanoformulation as shown in [Figure 3]a and [Figure 3]b. The PDI, particle size, and zeta potential are summarized in [Table 1]. The SEM of the α-t-FU-PLGA/5-FU-PLGA nanoformulation confirmed that as a spherical shape of the nanoparticles with the easy surface as shown in [Figure 4].
Figure 3: Zetasizer peak report for particle size, zeta potential, and polydispersity index of α-tocopherol-5-fluorouracil-poly-D,L-lactic- co-glycolic acid/5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles. (a) Particle size and polydispersity index distribution of the nanoparticles; (b) zeta potential distribution of nanoparticles

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Table 1: Particle size, polydispersity index, and zeta potential of targeted a-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles/nontargeted 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles (n=3; mean±standard deviation)

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Figure 4: Scanning electron microscope showing spherical nanoparticles, easy surface, no adherence between the particles in α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles/5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles

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In vitro drug-release system and entrapment efficiency

The in vitro release study and EE of α-t-FU-PLGA/5-FU-PLGA NPs were standardized through a standard calibration curve of the 5-FU solution, which was plotted a linear regression ranging from 0.1 to 10 μg/ml; a straight line was found between 5-FU concentration (μg/ml) and absorbance (nm) through the UV spectrophotometer at absorption maxima 267 nm. In vitro drug release of NPs also depends upon particle size, when size is less, the surface area of NPs increases, and more surface area comes in contact with the medium, resulting in a faster release. The cumulative in vitro drug release at pH 7.4 and pH 4.5 to identify the pH-susceptible difference in a 5-FU release from the nanoparticulate system and the EE of 5-FU loaded in α-t-FU-PLGA/5-FU-PLGA NPs were 68% and 63%, respectively, for both the nanoformulations. The total amount of commutative in vitro drug release at the pH 4.5 was increased 70% at the time range of up to 160 h; the results of 5-FU-PLGA NPs showed steady-state release approximately at the time 140–160 h and 63% in α-t-FU-PLGA NPs which had targeted moiety. In vitro drug-release data for α-t-FU-PLGA and 5-FU-PLGA NPs in pH 7.4 showed rapid releases at time range 40–60 h about 35%–60% and 25%–50%, respectively, which was followed by the cumulative drug release up to 160 h about 85% and 82%, respectively. The slope of the graph for in vitro release system for the α-t-FU-PLGA and 5-FU-PLGA NPs at different pH (7.4 and 4.5) ranges confirm the optimum drug release at different time intervals up to 160 h, as shown in [Figure 5], and the release kinetic at the pH 7.4 and 4.5 confirms that cumulative drug release strongly influences the pH of the dissolution solution. The data for drug release are summarized at a different time interval in [Table 2].
Figure 5: In vitro releases of α-tocopherol-5- fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles in phosphate-buffered saline at pH 7.4 and 4.5

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Table 2: In vitro drug release of targeted a-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and nontargeted 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles

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Cytotoxicity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles in dose-time dependent on squamous cell carcinoma 15 cells

The cytotoxicity study was performed in reference to time-dose dependent manner. 5-FU-PLGA/α-t-FU-PLGA NPs have inhibited the growth of SCC15 cells at different time interval and α-t-FU-PLGA NPs have shown high inhibition rate as compare to 5-FU-PLGA NPs. The targeted α-t-FU-PLGA NPs (8.0 μg/ml) have confirmed the high percentage of inhibition rate 79.39%, 56.93%, and 46.63 at different time interval (24, 48 and 72 h respectively). In other hand non-targeted 5-FU-PLGA NPs (8.0 μg/ml) have shown comparatively low inhibition rate 69.45%, 48.96% and 36.28 at different time interval (24, 48 and 72 h respectively) in SCC15 cells the results confirmed the intense inhibition of OSCC by α-t-FU-PLGA NPs as shown in the graph of [Figure 6]a and [Figure 6]b. The inhibition rate for α-t-FU-PLGA NPs was increased as nontargeted 5-FU-PLGA NPs approximately in lesser intense 45.29% and 39.58%, respectively, for both at time 24 h in SCC15, as the time increases the cytotoxicity effects of α-t-FU-PLGA NPs in SCC15 showed higher percentage of inhibition of 79.98% at time 96 h and confirmed approximately steady-state inhibition of 83.74% up to 160 h, as 5-FU-PLGA NPs showed lower inhibition rate up to 59.25% at the time 160 h as plotted graph in [Figure 7].
Figure 6: Cytotoxicity study, inhibition rate of α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles in squamous cell carcinoma-15 cells detected by methyl thiazolyl tetrazolium assay. (a) Inhibition rate of different dose-concentration-time (24, 48, 72 h) of 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles on squamous cell carcinoma-15 cells. (b) Inhibition rate of different dose-concentration-time (24, 48, 72 h) of α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles on squamous cell carcinoma-15 cells

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Figure 7: Cytotoxicity study, inhibition rate of α-tocopherol-5- fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles on squamous cell carcinoma-15 cells up to time 160 h

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Cytotoxicity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolicacid nanoparticles in drug-resistant squamous cell carcinoma 15 cells

The drug-resistant cell line was fixed to optimize the inhibitory activity of α-t-FU-PLGA/5-FU-PLGA NPs in drug-resistant SCC15 cells. IC50 values of the inhibitory activity of α-t-FU-PLGA/5-FU-PLGA NPs on drug-resistant SCC-15 cell were 13.19 and 23.25 μg/ml. The drug resistance for the cytotoxicity of α-t-FU-PLGA/5-FU-PLGA NPs in respect to dose-dependent manner, inhibition rate was remarkably high in α-t-FU-PLGA approximately 80% at the concentration 8.0 μg/ml with a comparison to inhibition rate for 5-FU-PLGA NPs, 65% at the concentration of 8.0 μg/ml and inhibition rate with respect to time-dependent [Table 3], cytotoxicity in α-t-FU-PLGA nanoparticles was higher (58%) in comparison to 5-FU-PLGA nanoparticles (45%), shown in [Figure 8]a and [Figure 8]b.
Table 3: Inhibitory effects of targeted a-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and nontargeted 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles on the proliferation of the squamous cell carcinoma-15 cells, 5-fluorouracil resistance cell line (n=3; mean±standard deviation)

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Figure 8: Inhibition of α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles on drug-resistant squamous cell carcinoma-15 cells detected by methyl thiazolyl tetrazolium assay. (a) Dose-concentration-dependent inhibition rate; (b) time-dependent inhibition rate

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Therapeutic productivity of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles in squamous cell carcinoma 15 cells

The therapeutic productivity of α-t-FU-PLGA/5-FU-PLGA NPs was examined on the basis of the cellular update, cytotoxicity investigation of targeted nanoformulation, α-t-FU-PLGA NPs and nontargeted 5-FU-PLGA NPs on the SCC15 cell line; the nanoformulations produced expected level of toxicity to the cell at different concentration (0.1, 0.5, and 1.0 mg/ml). The α-t-FU-PLGA NPs produced higher percentage of cell viability, 83.59%, 67.82%, and 41.25% in all concentration levels 1, 0.5, and 0.1 mg/ml, respectively, confirmed the therapeutic productivity, and the internalization of 5-FU and nontargeted 5-FU-PLGA NPs produced less cell viability 65.23%, 39.82%, 29% at the concentration of 1.0, 0.5, and 0.1 mg/ml, respectively, which confirmed the comparatively less therapeutic productivity of 5-FU, as shown in [Figure 9].
Figure 9: Cell viability assays of α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles in squamous cell carcinoma-15 cells at the concentration of 1.0 mg/ml, 0.05 mg/ml, and 0.1 mg/ml

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In vitro cellular uptake and targeting of α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

In vitro cellular uptake analysis of targeted drug-loaded α-t-FU-PLGA NPs and nontargeted 5-FU-PLGA NPs was examined in SCC15 cancer cells by fluorescent microscopy. FITC-labeled α-t-FU-PLGA NPs crucially inflate the acquisition of drug into SCC15 as compared nontargeted NPs. In [Figure 10], FITC labeled α-t-FU-PLGA/5-FU-PLGA NPs were assembled in the cytoplasm when treated with SCC15. The robust color fluorescence was distinguished in SCC15 cells, designed the endocytosis of a vast number of α-t-FU-PLGA NPs, and ascribed to extent of the targeting moiety. The exposure of SCC15-5-FU-PLGA NPs showed the modest strength fluorescence. The higher intensity of cellular uptake for α-t-FU-PLGA NPs described that target moiety of NPs successfully invaded the cancer cells via receptor-mediated endocytosis in OSCC.
Figure: 10: Cellular uptake of fluorescein isothiocyanate labeled α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles. (a) α-Tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles, (b) 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles in squamous cell carcinoma-15 cells by fluorescent microscopy analysis on the scale bar is 50 μm

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Cell apoptosis by α-tocopherol-5-fluorouracil-poly-D, L-lactic-co-glycolic acid/5-fluorouracil-poly-D, L-lactic-co-glycolic acid nanoparticles

Cell apoptosis assay was performed to investigate the cell death rate in SCC15, which was investigated by nontargeted NPs (5-FU-PLGA NPs) and targeted NPs (α-t-FU-PLGA NPs). The NPs have incubated with SCC15 cells approximately 96 h, with the exposure of AV-FITC/PI staining protocol by flow cytometry. The data confirm the apoptosis for early and late phase as shown in [Figure 11], and flow cytometry investigation for the ratio of AV/PI-positive cells was treated with α-t-FU-PLGA/5-FU-PLGA NPs. The observation for early apoptosis, 27.98% and 16.45%, and late apoptosis, 74.29% and 61.13%, of SCC15 cells for α-t-FU-PLGA/5-FU-PLGA NPs, respectively.
Figure 11: Flow cytometry analysis of quantitative apoptosis and apoptosis of squamous cell carcinoma-15 cells and the corresponding cell (%) in early and late apoptosis for α-tocopherol-5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles and 5-fluorouracil-poly-D,L-lactic-co-glycolic acid nanoparticles

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 > Discussion Top


In current scenario, advancement in the treatment system against cancer takes multiple levels up to increase the survival rate of affected population.[71] Cancer is a massive health problem which effects almost every part of the world. Mostly, Asian countries are reported higher rate cancer death, approximately 3 million.[2] In that, oral tumor is highly reported and underlined tumor because of ignorance and consumption of tobacco.[72] The prime treatment of oral cancer is surgery with chemotherapy and radiotherapy.[73] Chemotherapy is the treatment which is used to treat oral cancer by killing or inhibiting the growth of tumor cells.[74] The anticancer drug, 5-FU, was used as a chemotherapeutic agent against oral cancer treatment. Although 5-FU has high efficacy, it may be also restricted with limited therapeutic index and reduced bioavailability because of its conventional dosage formulation.[75] 5-FU may also cause some side effects and toxicity because of overdose and nonspecific treatment. Hence, these drawbacks were always demands to overcome nonspecific activity of 5-FU along with minimum toxicity.[43] Nanotechnology may enhanced the effectiveness and biological activity of 5-FU with targeted delivery as surface attachment of functional moiety, α-t along with PLGA NPs engulfed 5-FU drug molecules. The earlier report showed very less effective release of drug as this study enhances the release up to 80% with control release of delivery up to 5 days over the specific region.[76],[77] The polymeric NPs, as PLGA nanoparticulate system,[78] loaded 5-FU was prepared through ultrasonication method and α-t, tagged upon the surface of PLGA-FU NPs to bind with the cancer cell surface and release the drug. The targeted α-t-FU-PLGA NPs showed higher cytotoxicity in comparison to nontargeted 5-FU-PLGA NPs because of surface modification with the tagging of α-t. The targeted moiety α-t acted to increase the endocytosis toward SCC15 cells. α-t-modified PLGA-FU NPs showed enhanced cellular permeation and enhanced cancer cell accumulation.[79] The extracellular fluid may activate the α-t tagging and influx the accumulation within the cytosol and adhere over SCC15 cells and accurately followed the EPR effects over tumor site. On the other hand, nontargeted 5-FU-PLGA NPs showed limited cell uptake due to nonmodified surface. The over-treated damaged cells may produce drug resistance. The results of cytotoxicity of drug-resistant SCC15 cells may produce high inhibition with concentration of NPs. The IC50 values of α-t-FU-PLGA NPs showed high regulation of growth inhibition and reduced level of cell survival via growth inhibition in G1 phase. The PLGA polymer nanoparticulate system provided a parallel advancement as surface modification by α-t to the drug delivery of 5-FU.[48] This system may sort the issue which limited the dissolution, solubility, therapeutic efficacy, and biological activity against OSCC. The in vitro encapsulation of cytotoxicity may enhance the 5-FU efficacy about 5-fold and cellular uptake was increased as time-dependent manner. The apoptosis study confirmed the cell internalization and produced thymineless cell death affecting the cellular pathways. Flow cytometry revealed the early apoptosis through targeted drug delivery of 5-FU. The in vitro drug assessment plots a significant result data for formulated α-t-modified PLGA-FU NPs against OSCC, SCC15 cells.


 > Conclusion Top


Farthermost, the objectives of the current work were to formulate the targeted NPs specifically on oral cancer region and uncollision with beside healthy cells. In this study, targeted α-t-functionalized surface moiety successfully attached within 5-FU-loaded PLGA nanoformulation and comparatively nontargeted 5-FU-PLGA nanoformulation was also synthesized. The characterization studies of blended NPs in both formulations were confirmed the uniformed dispersion, particle size, and negative charges. The cumulative in vitro drug-release pattern of the α-t-FU-PLGA/5-FU-PLGA NPs gradually increased with concentration-time-dependent manner. The assessment of cytotoxicity in dose-time-dependent and drug-resistant in SCC15 cells confirmed the ability of α-t-FU-PLGA/5-FU-PLGA NPs to inhibit the growth of dividing cancer cells. The physiology of SCC15 cells and on the basis of result data of increased cellular uptake, cellular augmentation, specific cellular targeting, cell viability, and cellular toxicity confirmed the successful formulation of targeted α-t-FU-PLGA NPs. It is concluded that the attractive-targeted drug within the polymeric nanoformulation provides a possible platform for delivering the drug to the specific site for trigged therapeutic action toward oral cancer.

Acknowledgment

Authors are thankful to Indian Council of Medical Research (ICMR), New Delhi, India for providing financial support.

Financial support and sponsorship

Indian Council of Medical Research (ICMR), New Delhi, India (Project id-45/21/2013-NAN-BMS).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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