INTRODUCTION:

BC is a life-threatening disease that is nearly 95% of the cases originates from epithelial cells of glandular milk ducts or breast lobules (carcinomas)¹. The latest Jordan Cancer Registry (JCR) reports show that breast cancer accounts for 20.6% of all cancer cases in the country, increasing to 39.4% among females². It is hence essential to utilize the medicinal potential of natural substances in addition to existing chemotherapies and to develop “nano-based drug delivery systems” (NDDSs) in order to reduce the adverse effects and improve therapy efficiency.

Gemcitabine produces 14 % to 37 % response rates in chemotherapy-naive breast cancer patients and 12 % to 30% in patients previously treated with anthracyclines and/or taxanes when used in isolation.

It also results in a significantly greater response rate (41.4% vs. 26.2%), a significantly longer time to progression (6.1 vs. 4 months), and a significantly higher overall survival (18.6 vs. 15.8 months) when used in combination with Paclitaxel against breast cancer³^,⁴. NDDS use in cancer therapy should be explored, as they have shown a considerable potential for improving the efficacy of anticancer medications, lowering their toxicity to normal cells, and overcoming drug resistance^5,6. Anti-BC compounds are already being delivered using many major NDDS classes⁷. It is also noteworthy that, for treating pancreatic cancer in vitro, gemcitabine and tocotrienols were loaded into niosomes made from cholesterol, span 60, and D-tocopherylpolyethylene glycol 1000 for increased efficacy⁸. Natural products also play an important role in the detection of new anticancer agents and many of these natural anticancer drugs are used clinical settings⁹.

Date Seeds contain a considerable amount of oil, which is high in phenolic compounds, tocols (tocopherols and tocotrienols), and phytosterols¹⁰. Because of their ability to scavenge lipoperoxyl radicals, tocopherols and tocotrienols are known to be powerful natural antioxidants that protect the components of cellular membranes¹¹. The biological effects of tocopherols include neuroprotective, anticancer, anti-inflammatory, cardioprotective, immune-stimulatory, diabetic, hepatoprotective, and nephroprotective properties¹².

Although the anticancer effects of both DSO and GEM, have been separately confirmed, the anticancer effects of DSO-niosomes and GEM-niosomes against MCF-7 human breast cancer cells have not been previously examined. The purpose of this study was to prepare DSO-loaded niosomes and explore their anti-breast cancer activity as well as their effects when combined with GEM using MCF-7 human breast cancer cells.

MATERIALS AND METHODS:

Materials and Chemical Reagents:

Cholesterol, chloroform, sodium hydroxide, span 60 (pharmaceutical grade), phosphate-buffered saline (PBS, pH 7.4), accutase solution, sodium nitrite, aluminum chloride, sodium carbonate and Folin-ciocalteu phenol reagent were purcased from Sigma Aldrich, USA. Ethanol, trypan blue dye and DMSO were obtained from GCC – UK, while methanol and phosphoric acid were provided by Tedia-America. Deionized water was purchased from AAU lab and GEM HCl (as pour perfusion I.V. ampoule) was procured from THYMOORGAN^®.Date seeds were purchased from a shop in Aghwar, Jordan. Solubilization solution / stop mix and yellow dye were procured from Promega – USA, while quercetin, and gallic acid were acquired from GenoChem World –Valencia.

Date Seed Oil Extraction:

Soxhlet extraction method was adopted to extract oil from the date seeds according to the procedure described by Disher et al with some modifications¹⁰. Briefly, 50g of ground seeds were transferred into a cellulose thimble (of 30mm x 200mm dimensions) which was inserted in the extraction chamber of a 500-mL distilled flask that contained 250mL of n-hexane-containing solvents and a condenser-equipped 250mL Soxhlet apparatus. The oil was subsequently extracted from the date seeds using n-hexane under reflux (at 69ºC) for 1, 2, 4, and 6hours (10–12cycles/h). Finally, rotating evaporater was used to evaporate hexane under reduced pressure.

Determination of Total Flavonoids in Date Seed Oil:

The total flavonoids content (TFC) of date seeds was determined by the method described by Kim¹³. First, 4 ml of distilled water was added to 100µL of date seed extract. Next, 0.3ml of sodium nitrite solution (5%) and 0.3mL of aluminum chloride solution (10%) were added. The resulting sample was incubated in test tubes for 5 minutes at room temperature before 2mL of sodium hydroxide (1M) and 10mL of distilled water were added to achieve the final volume. After completely vortexing the solution, the absorbance at 510 nm was calculated using quercetin (50–750mg/L) as standard. For the quercetin stock solution preparation, 0.5g of quercetin was dissolved in 100mL of methanol, followed by serial dilutions to obtain the required concentrations. Absorbance was determined in triplicate for all standards and samples.

Determination of Total Phenol in Date Seed Oil:

The total phenolic content (TPC) was estimated using the Folin-Ciocalteu assay¹⁴with minor modifications. Briefly, 100µL of date seed oil was thoroughly mixed with 500µL of Folin-Ciocalteu phenol reagent (previously diluted in distilled water at the 1:10 ratio), after which 400µL of sodium carbonate (7.5% w/v in water) was added. After allowing the mixture to stand for 60 minutes at room temperature, the absorbance was measured at 765 nm using a UV-Vis spectrophotometer.

TPCs were calculated by calibration curve achieved from determining the absorbance of an identified concentration of gallic acid standard (1–100mg ⁄100mL of 50% ethanol). The concentrations are stated as milligrams of gallic acid equivalents per 100g of fresh weight.

Niosomes Formulations:

For niosomes formation, 50mg of date seed oil, 50mg of span 60 and 50mg cholesterol was dissolved in 10mL chloroform in the 50mL round-bottom flask of the rotary evaporator at 100rpm under 600 mbar partial vacuum, after which the pressure was decreased gradually to 400 mbar using a 45-50°C water bath. As a result, a thin niosomal film was formed on the inner flask wall which was then hydrated with 10ml PBS (pH 6.8) by vortexing for 30 minutes, thus ensuring complete hydration of the niosomal film. The final sample was stored at -20℃ until required for further analyses.

Preparation of Gemcitabine Niosomes (Niosomes Hydration):

The gemcitabine stock solution was prepared by adding 25mL of normal saline to the ampoule of Gemcitabine HCl (Pour Perfusion I.V. THYMOORGAN®, 1000 mg) to achieve 38mg/mL standard solution concentration. For GEM-niosomes loading, 87µL of the gemcitabine stock solution was diluted with 9.913µL normal saline before adding it to the niosomes prepared by hydration under constant stirring for 60 minutes at room temperature using vortex device¹⁵. DL = 0.2%. The water soluble drug was found on the hydrophilic part of the niosomes.

Determination of Particle Size and Zeta-potential of GEM-niosomes:

Malvern Zetasizer Nano Z^® device (Malvern, Model ZEN3600) was utilized to define the average size, charge, and polydispersity index (PDI) at 25⁰C. For this purpose, 100µL of the sample was diluted with 2900µl of deionized water followed by sonication for 20 minutes using Probe Sonicator (SON-003) with the following settings: pulsating ultrasound waves, 3 s on /2 s off, for 60 rounds at 45 MHz to obtain niosomes of progressively smaller size. Next, the sample was added to transparent disposable cuvettes to allow the size and the zeta potential to be determined using Zeta Sizer Instrument. All measurements were performed in triplicate¹⁶. For the preparation of the date seed oil niosomes (drug-Free Niosomes), 50µL of the sample was diluted with 950µl deionized water followed by 10 minutes sonication.

GEM Quantification via High-performance Liquid Chromatography (HPLC):

Shimadzu Liquid Chromatography was used to analyze GEM at room temperature within the λ = 200 - 300nm spectral range using an SPD-M20A diode array detector. As a stationary phase, a Luna C-18 column of 250 mm x 4.6mm dimensions (i.d. 5µm) was utilized. As an isocratic mobile phase, phosphate buffer was mixed with methanol at a 50:50% v/v ratio (pH 7.4, adjusted with HCl). The injection volume and the flow rate were set at 2µm and 1.0ml/minutes, respectively. The 266nm detection wavelength was found to be sufficient for chromatographic analysis with excellent response, and the analysis was completed in under six minutes. All these chromatographic conditions were carried out at room temperature¹⁷.

Sample Preparation for HPLC (Calibration Curve):

GEM stock solution was prepared by adding 25mL of normal saline to the ampoule of GEM HCl as pour perfusion I.V. (THYMOORGAN^®, 1000 mg), with 38 mg/mL standard solution concentration, after which 10 µL of the stock solution was added to 990µL of the mobile phase. Next, to prepare 3.8, 7.6, 11.4, 15 and 19 µg/mL working standard concentrations, serial dilutions using; 10, 20, 30, 40 and 50µL were performed using the mobile phase (pH=7.4) to complete the volume to 1mL. The resulting samples were then placed in glass vials to generate the HPLC calibration curve by plotting the area under the curve (AUC) on the Y-axis against the concentration (on the X-axis). Each sample was evaluated in triplicate. Finally, the calibration curve was used to ascertain the linearity degree and formulate the mathematical equation.

Determination of GEM-niosomes Encapsulation Efficiency (EE%):

For determining the EE%, 30 µL of GEM-niosomes was diluted with 970 µl of the mobile phase and was vortexed for 2 minutes, after which it was filtered through a syringe filter and injected. The entrapment drug concentration was determined using the equation obtained from the calibration curve, whereby the peak area offset was established from the Y-axis intercept, and the acquired concentration was used to the following equation:

Amount of drug encapsulated in niosomes

EE (%) =----------- ---------- --------------------------- x 100

Amount of total drug added in the preparation

In vitro Cell Viability Assay:

Prior to use, as the breast cancerous cell line (MCF-7) was kept in a long-storage freezer at -80 °C, it was warmed to 37°C using a water bath before transferring it to 10mL Dulbecco's Modified Eagle Medium (DMEM) comprising of 1mM sodium pyruvate, 2mM L-glutamine and 10% fetal bovine serum. After 5 minutes’ centrifugation at 5000rpm, the medium was carefully removed, leaving cancerous cells adsorbed to the wall of the test tube. These cells were re-suspended in 5mL of medium and were placed into a T25 flask which was placed overnight in a humidified incubator at 37°C. The next day, cells were placed in sterile T75 cell culture flasks and their growth was monitored until approximately 80% growth had been achieved. Prior to further use, to remove supernatant, cancerous cells were rinsed twice with PBS, after which 2mL of trypsin/EDTA was added to ensure their detachment from the flask wall. Once a single cell suspension had been achieved, 10ml of fresh medium was added to neutralize trypsin. Cell suspension was then centrifuged for 10 minutes at 2000rpm and the adsorbed cells were re-suspended in 10ml of complete DMEM medium (stock). The prepared stock was mixed with 100mL of trypan blue dye in an Eppendorf tube, which was shaken thoroughly before applying to the content to a slip-covered slide, allowing cells to be counted under a microscope.

Determination of Viable Cell Number:

Trypan blue is usually used as a stain in dye exclusion procedures for practical cell counting.as trypan blue does not enter living cells (which thus remain transparent), whereas it enters and stains the dead cells’ cytoplasm (which therefore appear blue under the microscope)¹⁸. Thus, the same approach was adopted in this study, whereby for determining the viable cell number, 100µl of cell suspension was added into a microcentrifuge tube, followed by 100µl of 0.4% trypan blue solution (w/v), and the content were mixed well. Next, four identical sections were viewed under inverted microscope, whereby each section was separated into equal-sized segments, allowing colorless dots representing viable malignant cells to be counted. As 111 cells were identified in the four sections, each one contained 27.75 cells on average. This value was multiplied by two (to account for 100µL cells and 100 µL dye), ten (media volume), and 10 ̂ 4 (four sections). The resulting figure represents the total cell count in millions per 10mL of medium, and was approximately 2,600,000.

In the next step, 4mL of the stock solution was removed and the volume was increased to 20mL by adding 6mL of DMEM medium to obtain 1 000 000 cells. These cells were then placed on three 96-well culture plates and were allowed to develop in a CO₂ incubator for 24 hours. Finally, 100μl was added to each well to obtain about 1 000 000 cells.

Measurement of Cytotoxicity on Cultivated MCF-7 Cells:

For assessing their cytotoxicity, MCF-7 was seeded in a 96-well plate at the optimal density of 5000cell/well and left to adhere overnight in humidified incubator at 37°C. After incubation, cells were treated with a suitable concentration of GEM as a free drug, GEM-niosomes, and drug-free niosomes. For this purpose, 250µL of the required sample was diluted with 750µL of medium, and 250µL of this dilution was added to 750µL of medium. Six further serial dilutions were performed to obtain 0.04, 0.160, 0.645, 2.58, 10.33, 41.32, 165.31mg/L final concentrations. The resulting cells were incubated in humidified incubator at 37°C for 24 hours before the MTT Assay.

MTT Cytotoxicity Assay:

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test was used to detect cell growth. In live cells, the yellow color of tetrazolium salt transforms into purple formazan, and the color intensifies as the number of living cells increases. As a part of this study, cell viability test was performed using MTT following a protocol described by Mosmann¹⁹. Briefly, 100μl dye solution was placed in a CO₂ incubator for three hours. The solubilization solution/stop mix was then applied and the sample was left at room temperature for further 60 minutes. Absorbance was measured immediately at 590nm and 630 nm in multi-well plate spectrophotometer (Biotech, USA). The average absorbance in the control wells was taken as 100% survival, and the IC₅₀ values were defined as the drug concentrations that inhibited the cell growth by 50%.

In vitro Scratch Assay:

The in-vitro scratch or wound healing assay was used to study cancer cell migration, in line with the procedure described by Grimmig et al²⁰. Briefly, scratches were created on a confluent cell monolayer, causing the cells on the scratch edge to move toward the center, closing the scratch and establishing new cell–cell connections.

Previously, 12 wells MCF-7 cells were seeded (200,000 cell/well) in sterile 12-well cell culture plates and were incubated at 37°C in 5% CO₂ for 24 h. The next day, vertical scratch was created in the middle of the MCF-7 cells using a 200µL sterile micropipette tip, after which all wells were washed twice with sterile PBS and fresh pre-warmed media were added. The medium was used as negative control and all treatments were performed in triplicate.

For the free niosomes, only IC₅₀ was measured. Finally, scratch images were captured directly after treatment and after 72 hours using Phase Contrast Microscope P. MICRO-001 (Nikon) with a 4X magnification objective piece, and the wound closure rate (%) was monitored by calculating the closure area using Motic Images plus 2.0 software.

The rate of wound closure was calculated using the following formula:

[Area day 2]

Rate of wound opening (%) = -------------- x 100

[Area day 1]

Statistical Analysis:

All statistical analysis was performed in Graph Pad Prism 8.0 and MS. Excel. P values ≤ 0.05 were considered significant.

RESULTS AND DISCUSSION:

Total Flavonoids:

As quercetin²¹ was chosen as a standard, five working standards (in 50, 250, 350, 500 and 750mg/L concentrations) were prepared and the absorbance was measured using 1cm quartz cuvette, with distilled methanol serving as blank. The calibration curve was drawn (Figure 1.). The concentration of Total Flavonoids content (TFC) in the date seed oil was found to be 413.66mg/L.

Figure 1- Quercetin Calibration Curve

Total Phenol Results:

Gallic Acid was chosen as a standard, and five working standards were prepared in 50, 100, 250, 350 and 500 mg/L concentrations, whereby the absorbance was measured using 1cm quartz cuvette, with distilled methanol as blank. The Gallic Acid Calibration Curve. shown in (Figure 2). Based on the resulting calibration curve, TPC concentration in the DSO was found to be 231mg/L. Total flavonoids and phenolic acid act as primary antioxidants with anti-inflammatory and antimicrobial properties, and are also known to protect DNA from oxidative damage and inhibit tumor cells growth.

Figure 2-Gallic Acid Calibration Curve

Niosomal Characterization of GEM-niosomes Particle Size and Stability:

The ideal nano-size range can be determined using a particle size analyser, laser diffraction, dynamic light scattering, and direct imaging techniques. In the present study, laser diffraction was adopted, given that when a light beam (laser) is dispersed by a series of particles, the depression angle is inversely proportional to particle size. The particle size, zeta potential and PDI for niosomes, as well as GEM-niosomes, were thus determined (Table 1).

Table I: The particle size, zeta potential and polydispersity Index (PDI)

	Sonication time	Size, Z-Ave	Zeta Potential, ZP	PDI
	(min)	(d.nm)	(mV)
Noisomes	No	576-590	-8.20 – -9.12	0.271-0.289
Noisomes	10	90-102	-6.8 – -7.9	0.236-0.241
Niosomes with GEM	No	1200-1400	------	0.90-1.00
Niosomes with GEM	10	275-286	-31 – -32	0.29-0.39
Niosomes with GEM	20	124-126	-26 – -28	0.27-0.29

The results demonstrate that niosomes formed without sonication had about 5 times greater diameter than the niosomes formed using sonication, which was in the 90-102 nm range, concurring with the results reported by Gharbavi²². The particle diameter and PDI for GEM-niosomes without sonication was in the 1200-1400 nm range, declining to 275-286 nm after 10 minutes’ sonication. Further sonication resulted in even smaller particle size (diameter = 124-126 nm), confirming the importance of sonication time²³, which also affected the PDI, given that the values for GEM-niosomes were nearly 50% lower than those obtained without sonication.

According to the pertinent literature²⁴, the optimal niosomal particle diameter should be around 100 nm, and the zeta potential should be in the +30.0 - -30 mV range. The optimal drug loaded niosomal diameter should be around 200 nm (Figure 3).

Figure 3-Particle size of DSO-niosomes with GEM (sonication time = 20 min)

When this optimal size is achieved, the niosomal preparation has a beneficial effect on tumor tissue accumulation, as smaller niosomes are more likely to interfere with leaky tumor capillaries and extravagate into tumor tissue. As sonication duration was shown in this study to be inversely related to vesicle size, these findings concur with those reported by Sezgin-Bayindir²⁵, who showed that 60 minutes sonicated was needed to attain the required particle size reduction. Moreover, PDI was decreased by sonication, which improved dispersion homogeneity. Again, these results are consistent with those obtained by Nowroozi²⁶ and Yeo²⁷.

Storage Stability:

The particle size of GEM-niosomes remained relatively stable after four 4 weeks of storage, as the average diameter was still below 200nm, the PDI was close to 0.2, and the mean zeta potential of GEM-niosomes (measured in a charge cuvette in triplicate) was 24mV (Table 2). These results are in accordance with the findings reported for rifampicin-loaded niosomes²⁸ and niosomal encapsulating fosinopril²⁹.

Table II. GEM- niosomes stability for four weeks.

Loaded-Niosomal Drug	(Weeks)	Average Particle Size (nm)	Charge (mV)	PDI
GEM-Niosomes	First	125 ± 2.04	-27 ± 1.0	0.201 ± 0.038
	Second	128 ± 3.01	-22± 1.47	0.206 ± 0.044
	Third	146 ± 3.89	-20 ± 1.5	0.24 ± 0.08
	Fourth	170 ± 4.60	-19 ± 1.5	0.26 ± 0.09

High Performance Liquid Chromatography (HPLC) of GEM-niosomes:

GEM calibration curve for different drug concentrations were obtained using HPLC aiming to determine the mean peak area (Figure 4). The encapsulation efficiency (EE%) of GEM-niosomes was determined after performing thin film hydration followed by probe sonication by determining the concentration of encapsulated drug (Figure 5). EE% was calculated using the following equation as shown the Actual (45069) and Theoretical (54526)

Amount of drug encapsulated in niosomes

EE (%) =----------- ---------- --------------------------- x 100

Amount of total drug added in the preparation

EE% = 82%

Figure 4- GEM Calibration Curve

Figure 5- Peak area of entrapped GEM-niosomes

The encapsulation efficiency is an important parameter for the characterization of niosomal vesicles which was found in our formulation to be 82%, concurring with the percentages (37% to 96%) reported by other authors^30,31,32 The results revealed the effect of lipid concentration and type of surfactant used on the drug encapsulation efficiency of niosomal vesicles. For determining in vitro cell viability at two wavelengths (590, 630 nm), after seeding and treating the MCF-7 cancerous cells in a 96-well, ELx800 absorbance microplate reader (Biotech) was used (Figure 6).

Figure 6- The half inhibitory concentration (IC₅₀) values of different treatments on MCF-7 cell line

These results (Figure 7A-C) were in good agreement with those obtained in previous studies performed on different drugs using MCF-7^33,
34. Moreover, the findings regarding DSO are consistent with the previously reported results, which indicated that date seed extracts are effective in different types of cancers, including breast, liver, lung³⁵, and colorectal cancer cells³⁶. Therefore, our study marks the first attempt to use DSO- loaded niosomes against breast cancer, and can thus serve as a confirmation of the antitumor activity of DSO.

Figure 7- Cell viability of different treatments against MCF-7 cell line after 72 hrs. for, A: GEM-niosomes, B: GEM, C: DSO-niosomes

In vitro Scratch Assay:

The treatment concentrations for the migration study were calculated from the IC₅₀ value (4.041nM), resulting in 2 IC₅₀= 8.082 nM, IC₅₀=4.041 nM, and 0.5 IC₅₀= 2.020 (12-well plates were treated) (Table 3). The free niosomes showed 55.74% % opening while the media showed 20.57%. After 3-day incubation period, the degree of wound closure was assessed under florescent microscope, allowing the effect of the free drug, niosomes and the GEM with niosomes at different concentrations to be assessed and compared (Figure 8). The obtained results were consistent with those obtained by other authors for other antitumor agents, such as paclitaxel, vinblastine, colchicine and podophyllotoxin^37,38,39.

Table III- Treatment concentrations for Scratch Assay on 12-well plates

% of Opening	Niosomes with GEM	Free Drug
IC₅₀	79.37%	67.52%
0.5 IC₅₀	68.73%	63.45%
2IC₅₀	_53.22%	53.79%

CONCLUSIONS:

This study revealed for the first time that “GEM-DSO-loaded niosomes” showed about 10-fold stronger activity against MCF-7 cells compared to the free drug (GEM). We prepared anionic date seed oil niosomes utilizing thin film hydration method followed by probe sonication. The prepared final formula was optimized and fully characterized, allowing the optimum particle size, PDI and zeta potential to be determined. The targeted niosomes had 100 ±10 nm diameter, while those loaded with GEM had an average diameter of 125±15. GEM was almost fully loaded to DSO-niosomes with the EE% of 82%. The prepared GEM-niosomes showed good stability after four weeks of storage at -7 °C.

These promising results need to be confirmed through further in vivo studies, and can potentially lead to new treatment opportunities. However, further investigations regarding; the in vitro GEM release and GEM-niosome diffusion are needed, as well as additional research into the in vitro and in-vivo GEM-niosomes cytotoxicity, focusing on more aggressive breast cancer cell lines.

ACKNOWLEDGEMENTS:

The authors are Al-Ahliyya Amman University, Amman, Jordan for the financial support granted to cover the publication fee of this research article.

DECLARATIONS:

Funding: This research was funded by Al-Ahliyya Amman University, Amman, Jordan.

CONFLICT OF INTEREST:

The authors hve no conflicts of interest to declare.

Ethical approval: The study was approved by the Institutional Ethics Committee.

AUTHOR CONTRIBUTIONS:

N. S and MO.A. designed and supervised the project. M. A. performed and wrote the original draft. MO.A. edited and revised the manuscript.

REFERENCES:

1. V. N. Dange, S. J. Shid, C.S. Magdum, S.K. Mohite. A Review on Breast cancer: An Overview. Asian J. Pharm. Res. 2017; 7(1): 49-51.

2. Scholl AR, Flanagan MB. Educational Case: Invasive Ductal Carcinoma of the Breast. Academic Pathology 2020; 7: 2374289519897390. https://doi.org/10.1177/2374289519897390

3. Silvestris N, Cinieri S, La Torre I, Pezzella G, Numico G, Orlando L, Lorusso V. Role of gemcitabine in metastatic breast cancer patients: A short review. Breast 2008; 17(3): 220–226. https://doi.org/10.1016/j.breast.2007.10.009.

4. Majed Jary Mohammed, Zeyad Kadhim Oleiwi, Muntadher Abdulabbas Hasan Al-Hilo, Ahmed Kareem Hussein Mubarak, Ehab Kareem Obaid, Ali Jabbar Radhi. Synthesis and Study Biological Activity of Gemcitabine Linked Heterocyclic Hybrids. Research J. Pharm. and Tech. 2020; 13(7): 3257-3261.

5. Markman JL, Rekechenetskiy A, Holler E, Ljubimova JY. Nanomedicine therapeutic approaches to overcome cancer drug resistance. Advanced Drug Delivery Reviews 2013; 65(13–14): 1866–1879. https://doi.org/10.1016/j.addr.2013.09.019

6. Kshitij B. Makeshwar, Suraj R. Wasankar. Niosome: a Novel Drug Delivery System. Asian J. Pharm. Res. 3(1): Jan.-Mar. 2013; Page 15-19..

7. Yap KM, Sekar M, Fuloria S, Wu YS, Gan SH, Mat Rani NNI, Subramaniyan V, Kokare C, Teng Lum P, Begum MY, Mani S, Meenakshi DU, Sathasivam KV, Fuloria NK. Drug Delivery of Natural Products Through Nanocarriers for Effective Breast Cancer Therapy: A Comprehensive Review of Literature. International Journal of Nanomedicine 2021; 16(November): 7891–7941. https://doi.org/10.2147/ijn.s328135

8. Taranjit Kaur, Charanjit Kaur, Iqbaljit Kaur, Parminderjit Kaur. Preparation and characterization of In Situ Gel of Gemcitabine Hydrochloride loaded Nanoparticles used for the treatment of Pancreatic Cancer. Research Journal of Pharmacy and Technology. 2021; 14(12):6609-6.

9. M Lavanya, Asish Bhaumik, A Gopi Reddy, Ch Manasa, B Kalyani, S Sushmitha. Evaluation of Anticancer Activity of Ethanolic and Ethylacetoacetate Extracts of Sweet Cherry Against Human Breast Cancer Cell Line MCF-7. Research J. Pharmacology and Pharmacodynamics.2016; 8(2): 65-70

10. Disher, I., Ali, M., & Alhattab, T. Extraction of Date Palm Seed Oil (Phoenix Dactylifera) by Soxhlet Apparatus. International Journal of Advances in Engineering & Technology (IJAET) 2015; 8, 261–271.

11. Ahsan H, Ahad A, Iqbal J, Siddiqui WA. Pharmacological potential of tocotrienols: a review. Nutrition & Metabolism 2014; 11(1): 1-22. https://doi.org/10.1186/1743-7075-11-52.

12. Abdeldjabbar Messaoudi, Messaouda Dekmouche, Zehour Rahmani, Cheyma Bensaci. Phenolic profile, Antioxidant potential of date (Phoenix dactylifera Var. Degla Baidha and Deglet-Nour) seeds from Debila region (Oued Souf, Algeria). Asian J. Research Chem. 2021; 14(1):37-41.

13. Kim DO, Jeong SW, Lee CY (2003) Antioxidant capacity of phenolic phytochemicals from various cultivars of plums. Food Chemistry 2003; 81(3): 321–326. https://doi.org/https://doi.org/10.1016/S0308-8146(02)00423-5

14. Saafi, E, Amira EA, Issaoui M, Hammami M, Achour L (2009) Phenolic content and antioxidant activity of four date palm (Phoenix dactylifera L.) fruit varieties grown in Tunisia. International Journal of Food Science & Technology 2009; 44(11): 2314–2319. https://doi.org/10.1111/j.1365-2621.2009.02075.x

15. Kushnazarova RA, Mirgorodskaya AB, Zakharova LY. Niosomes modified with cationic surfactants to increase the bioavailability and stability of indomethacin. Russian Chemical Bulletin 2021; 70(3): 585-591. https://doi.org/10.1007/s11172-021-3129-z

16. De Silva L, Fu JY, Htar TT, Muniyandy S, Kasbollah A, Wan Kamal WHB, Chuah LH. Characterization, optimization, and in vitro evaluation of Technetium-99m-labeled niosomes. International Journal of Nanomedicine 2019; 14: 1101–1117. https://doi.org/10.2147/IJN.S184912.

17. Charde M, Shinde M, Welankiwar A, Jitendra K. Development of analytical and stability testing method for vitamin A palmitate formulation. International Journal of Pharmaceutical Chemistry 2015; 5(4): 104–114. https://doi.org/10.7439/ijpc

18. Stepanenko AA, Dmitrenko VV. HEK293 in cell biology and cancer research: phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene 2015; 569(2): 182-190. https://doi.org/10.1016/j.gene.2015.05.065

19. Mosmann T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. Journal Of lmmunological Methods 1983; 65(1-2): 55–63. https://doi.org/10.1039/c6ra17788c

20. Grimmig R, Babczyk P, Gillemot P, Schmitz KP, Schulze M, Tobiasch E. Development and evaluation of a prototype scratch apparatus for wound assays adjustable to different forces and substrates. Applied Sciences 2019; 9(20): 4414. https://doi.org/10.3390/app9204414

21. Roma Mathew, Joyamma Varkey. Development of Solid Self Nanoemulsifying Drug Delivery System of Quercetin. Asian Journal of Research in Pharmaceutical Sciences. 2022; 12(3):183-8.

22. Gharbavi M, Amani J, Kheiri-Manjili H, Danafar H, Sharafi A. Niosome: A Promising Nanocarrier for Natural Drug Delivery through Blood Brain Barrier. Advances in Pharmacological Sciences 2018; 2018. https://doi.org/10.1155/2018/6847971

23. Alireza Doroudi, Ehsan Rezaee, Seyyed Mostafa Saadati, Ali Kiasat, Faramarz Ahmadi, Behrooz Etesami, Mostafa Erfani. The Role of Sonication for the Preparation of Infection-Seeking Radiotracer Sample Versus the Standard Method. Research J. Pharm. and Tech. 2018; 11(3): 987-995.

24. Clogston JD, Patri AK. Zeta potential measurement. Characterization of nanoparticles intended for drug delivery. Humana Press 2011; 697: 63–70. https://doi.org/10.1007/978-1-60327-198-1_6

25. Sezgin-Bayindir Z, Yuksel N. Investigation of Formulation Variables and Excipient Interaction on the Production of Niosomes. AAPS PharmSciTech 2012; 13(3): 826–835. https://doi.org/10.1208/s12249-012-9805-4

26. Nowroozi F, Almasi A, Javidi J, Haeri A, Dadashzadeh S. Effect of Surfactant Type, Cholesterol Content and Various Downsizing Methods on the Particle Size of Niosomes. Iranian Journal of Pharmaceutical Research 2018; 17(Suppl2): 1–11. PMID: 31011337; PMCID: PMC6447874.

27. Yeo LK, Chaw CS, Elkordy AA. The Effects of Hydration Parameters and Co-Surfactants on Methylene Blue-Loaded Niosomes Prepared by the Thin Film Hydration Method. Pharmaceuticals 2019; 12(2): 46. https://doi.org/10.3390/ph12020046

28. Khan DH, Bashir S, Figueiredo P, Santos HA, Khan MI, Peltonen L Process optimization of ecological probe sonication technique for production of rifampicin loaded niosomes. Journal of Drug Delivery Science and Technology 2019; 50: 27–33. https://doi.org/https://doi.org/10.1016/j.jddst.2019.01.012

29. Hnin HM, Stefánsson E, Loftsson T, Asasutjarit R, Charnvanich D, Jansook P. Physicochemical and Stability Evaluation of Topical Niosomal Encapsulating Fosinopril/γ-Cyclodextrin Complex for Ocular Delivery. Pharmaceutics 2022; 14(6) :1147. https://doi.org/10.3390/pharmaceutics14061147

30. Ahad A, Raish M, Al-Jenoobi FI, Al-Mohizea AM. Sorbitane Monostearate and Cholesterol based Niosomes for Oral Delivery of Telmisartan. Current Drug Delivery 2018; 15(2): 260–266. https://doi.org/10.2174/1567201814666170518131934

31. Rasul A, Imran Khan M, Ur Rehman M, Abbas G, Aslam N, Ahmad S, Abbas K, Akhtar Shah P, Iqbal M, Al Subari AMA, Shaheer T, Shah S. In vitro Characterization and Release Studies of Combined Nonionic Surfactant-Based Vesicles for the Prolonged Delivery of an Immunosuppressant Model Drug. International Journal of Nanomedicine 2020; 15: 7937–7949. https://doi.org/10.2147/IJN.S268846

32. Mahawar Sheetal. Development and Characterization of Docetaxel Encapsulated pH-Sensitive Liposomes for Cancer Therapy. Research J. Pharma. Dosage Forms and Tech. 2013; 5(3): 151-160 .

33. Dabbagh Moghaddam F, Akbarzadeh I, Marzbankia E, Farid M, khaledi L, Reihani AH, Javidfar M, Mortazavi P. Delivery of melittin-loaded niosomes for breast cancer treatment: an in vitro and in vivo evaluation of anti-cancer effect. Cancer Nanotechnology 2021; 12(1): 1-35. https://doi.org/10.1186/s12645-021-00085-9

34. Zarrabi Ahrabi N, Tabaie SM, Jahanshiri M. Study of Cytotoxic Effects of Caffeine- Loaded Niosomes on Human Breast Cancer Cells MCF 7. Journal of Sabzevar University of Medical Sciences 2021; 28(5): 663–674. http://jsums.sinaweb.net/article_1447.html

35. Al-Sheddi, Ebtesam S. Anticancer potential of seed extract and pure compound from Phoenix dactylifera on human cancer cell lines. Pharmacognosy Magazine 2019; 15(63): 494-9. http://www.phcog.com/text.asp?2019/15/63/494/258401

36. Rezaei M, Khodaei F, Hooshmand N. Date Seed Extract Diminished Apoptosis Event in Human Colorectal Carcinoma Cell Line. MOJ Toxicol 2015; 1(4): 00017. DOI: 10.15406/mojt.2015.01.00017

37. L. Krishnasamy, M. Masilamani Selvam, Bharathi Ravikrishnan. Anticancer property of Colchicine isolated from Indigofera aspalathoids. Research J. Pharm. and Tech. 2016; 9(4): 386-390.

38. Kim O, Hwangbo C, Kim J, Li DH, Min BS, Lee JH (2015) Chelidonine suppresses migration and invasion of MDA-MB-231 cells by inhibiting formation of the integrin-linked kinase/PINCH/α-parvin complex. Molecular Medicine Reports 12(2): 2161–2168. https://doi.org/10.3892/mmr.2015.3621

39. Lü S, Wang J. Homoharringtonine and omacetaxine for myeloid hematological malignancies. Journal of Hematology & Oncology 2014; 7(1): https://doi.org/10.1186/1756-8722-7-2

Received on 22.12.2022 Modified on 13.03.2023

Research J. Pharm. and Tech 2023; 16(9):4179-4187.

DOI: 10.52711/0974-360X.2023.00684

	Day 1	Day 3
Media
IC₅₀Nio+GEM
0.5 IC₅₀Nio+GEM
2IC₅₀Nio+GEM
IC₅₀Free Drug
0.5 IC₅₀Free Drug
2IC₅₀Free Drug
IC₅₀Free Niosomes