Breast cancer is the most prevalent type of cancer that is found and is the main cause of cancer-related deaths in women worldwide [1,2]. Breast cancer mortality rates have remained largely stable over the past three decades, despite significant improvements in chemotherapy treatment [3]. Increasing age, active and passive smoking, microbial infections, and genetic predisposition are just a few of the numerous risk factors connected to breast cancer. These multifaceted risk factors have a significant impact on how cancer develops and spreads, which ultimately affects the efficiency and results of cancer therapy [4-7]. It has been acknowledged that the main difficulties or restrictions of chemotherapy treatment are the initial response to the drugs and the ensuing development of resistance, which causes relapse and the metastatic spread to vital organs like the lungs, liver, and bones [8]. A promising approach for delivering chemotherapeutics is to use nanotechnology-based materials as they improve tumor targeting, increase drug penetration into tumors, and decrease side effects [9,10]. Breast cancer is one of many malignancies for which cisplatin is frequently used as a chemotherapeutic agent [11]. The drug significantly affects cancer cells by attaching to the DNA molecule, causing apoptosis and necrosis to occur [12]. Notwithstanding, nephrotoxicity and neurotoxicity are among the side effects of cisplatin administration [13]. The creation of drug delivery systems with the intention of influencing and managing drug distribution within the body has received a lot of attention over the last few decades. One method uses nanoliposomes, which can easily pass through the interendothelial cell gap of newly formed tumor capillaries and collect inside the tumor. These nanoliposomes are packed with anticancer agents. These innate characteristics aid in boosting the concentration of anticancer medications specifically within the tumor, thereby lowering their toxicity in healthy tissues [14,15]. Researchers have had difficulty creating the ideal nanoliposome formulation for the drug cisplatin, despite prior attempts to deliver it using a variety of carriers. Researchers optimized cisplatin nanoliposomes in this study and assessed the toxicity of the nanoliposomes on breast cancer cell lines.

Cisplatin, cholesterol, and polyethylene glycol 3350 were obtained from Sigma-Aldrich Co., UK. Lecithin was obtained from Acros Co., Belgium. RPMI 1640 cell culture medium was obtained from Gibco Co., Germany. Additionally, the T-47D cell line was purchased from the cell bank of the Pasteur Institute in Iran.

The reverse phase evaporation method was used for producing liposomal nanoparticles. Briefly, 100 ml of 96% ethanol was used to dissolve 10 mg of cisplatin, 150 mg of lecithin, 60 mg of cholesterol, and 72 mg of polyethylene glycol 3350 (with a molar ratio of 55: 40: 5%). For two hours, the mixture was stirred at 140 rpm while being heated to 37 C. A rotary evaporator was used to separate the solvent following complete dissolution. In two separate additions, the resulting thin film was dissolved in 15 cc of phosphate buffer (pH 7.4). The formulations were then placed in an ultrasonic bath (Bandelin Sonorex Digitec, Germany) and sonicated for 5 minutes.

Four milliliters of phosphate-buffered saline (PBS) were used to dissolve one milligram of the formulation. Then, a Zetasizer (Nano ZS3600, Malvern Instruments, UK) was used to measure the zeta potential and mean diameter of the nanoliposomes as well as their absorption at 633 nm.

40 mg of the formulation were centrifuged for an hour at 4 degrees Celsius and 13,200 rpm to ascertain the rate of drug entrapment. Using a spectrophotometer (UV1800, Shimadzu Co.), the optical absorbance of the supernatant from each formulation was then measured at 220 nm. Then, using Formulas 1 and 2, the rate of drug loading and encapsulation efficiency were calculated.

(1) Encapsulation percent=(PC-CS )/(PC ) x 100

(2) Drug loading percent=(C )/(W ) x 100

In Formula 1, PC: primary cisplatin and CS: cisplatin in supernatant in mg/ml

In Formula 2, C: cisplatin content in the nanoliposomes and W: weight of nanoliposomes in mg/ml

Nanoliposomal drugstability study

For a month, the nanoliposomes were kept in a refrigerator at 4°C. Following this, the above-described procedure was used to determine the nanoliposome properties once more.

A dialysis bag with a cut-off of 10,000 Daltons was filled with 1 ml of the liposomal drug suspension and its control in order to assess the release pattern of nanoliposomal cisplatin. The bag was then submerged in 100 ml of pH 7.4 PBS buffer and stirred for 35 hours at 140 rpm at room temperature. Then, 1 ml of the used PBS buffer was taken out and swapped out for 1 ml of brand-new PBS buffer. The samples’ optical absorbance was determined at 227 nm, and a standard curve was used to determine the concentration of the free drug. The drug release profile was finally plotted versus time.

In RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 g/ml streptomycin, and 100 U/ml penicillin, the T-47D cell line was cultured. At 37°C, 5% CO2, and 90% humidity, the cell culture was kept alive. A 96-well plate was filled with 100 of a cell suspension containing 10,000 cells for each cell culture. The plate was incubated with 5% CO2 at 37°C. The cells’ supernatant was removed after 24 hours. The cells were then treated with various concentrations of the standard cisplatin drug, control, and the nanoliposomal cisplatin formulation. After another 24 hours, this process was repeated.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to test for cytotoxicity. The cells were exposed for 48 hours before the supernatant was taken out and a 100 µl solution of MTT (0.5 mg/ml) was added. After the cells had been incubated for 3 hours, the culture medium was then added with a 100 µl solution of isopropanol. After 30 minutes, a plate reader (Synergy Multi-Mode Elisa Reader, Bio-Tek, USA) was used to measure the formazan product’s optical absorbance at 570 nm. Finally, the pharm program was used to determine the IC50 value.

The SPSS software, version 11, was used to analyze the results. The results of three independent tests, each with two samples, are shown as Mean±SD. The statistical test was performed using the unpaired-samples t-test, and the significance level was set at 0.05.

Chemotherapy is still the mainstay treatment for various cancers. However, it has run into problems like low tumor selectivity and MDR (multidrug resistance). One creative solution to these problems is the use of nanotechnology materials for targeted drug delivery [16,17]. This study’s goals included improving cisplatin nanoliposomes and determining how toxic they were to breast cancer cell lines. The results of the study showed that cisplatin’s cytotoxic effects were increased in liposomes as opposed to in its free form. In conclusion, nanoliposome synthesis methods have proven to be an invaluable asset in improving therapeutic drugs, particularly in the area of chemotherapy [18]. Nevertheless, it is essential to carefully assess the toxicity and fate of nanoliposomes before using them for pharmaceutical applications [19]. Numerous studies have investigated the effects of different medications formulated in nanoliposomes in various cell lines. In the case of the breast cancer cell line MCF-7, Koohi Moftakhari Esfahani et al. [20] evaluated the cytotoxicity of liposomal paclitaxel. Their analysis of the drug release showed that the maximum amount of paclitaxel, or about 5.53%, was released from liposomes in just 28 hours. Additionally, their research showed that nanoliposomal paclitaxel had more cytotoxic effects than free paclitaxel. Pegylated nanoliposomal artemisinin was investigated by Dadgar et al. [21] as a potential treatment for breast cancer cell lines. They found that over a 48-hour period, the drug release amounted to roughly 5.17%. According to their research, artemisinin’s cytotoxicity displayed a greater effect when it was packaged in pegylated nanoliposomes than when it was used alone. The dialysis tubing method was used to examine the drug release profile of nanoparticles containing cisplatin. The release process first showed rapid diffusion, which was followed by a phase of slower diffusion. According to the results, the majority of drug release happened in the first four hours. It is clear that nanoliposomal cisplatin is more effective at killing breast cancer cells when compared to free cisplatin when looking at their respective cytotoxic effects. This is explained by the fact that it has a phospholipid structure similar to the bilayer membrane found in breast cell lines. Because of their structural similarity, these cells can be penetrated more easily by nanoliposomal cisplatin, which then releases the drug directly into the target cells, killing them [18]. Using the MTT method, we successfully loaded cisplatin onto liposomal nanoparticles in this study and assessed the cytotoxicity of both the free form of cisplatin and its liposomal formulation on a cell line from a human breast carcinoma. We also looked at the produced nanoparticles’ physicochemical characteristics. The ability of the liposomal nanocarrier to lengthen the drug’s half-life in the bloodstream and play a protective role can be credited for the liposomal formulation’s improved performance. The presence of polyethylene glycol during nanoparticle preparation enables them to remain hidden and stay in circulation for longer periods despite the low loading seen in this study. As a result, this prolonged time in circulation increases the effectiveness of the drug [22]. As a result, our research shows that free cisplatin does not have the same cytotoxic effects on breast cell lines as nanoliposomal cisplatin. This suggests that this formulation has the potential to be a potent substitute for upcoming chemotherapeutic strategies for the treatment of breast cancer.

Co-Author Contributions

Authors’ contribution Fateme Mohammadinezhad, Armin Talebi, Elham Saberian, Somayeh Eslami, Parizad Ghanbarikondori, and Zeinab Jabbari Velisdeh performed the experimental tests. Reza Nahavandi, and Maryam Vesal designed the nanoparticle. Ali Habiba did the drug release test. Cell culture was carried out by Azim Akbarzadeh Khiyavi and Paniz mirmoghaddam. Armin Sedighi set up and performed the work with the laboratory devices. Mohammadreza Allahyartorkaman wrote the article.

Statement of Transparency and Principals:

· Author declares no conflict of interest

· Study was approved by Research Ethic Committee of author affiliated Institute.

· Study’s data is available upon a reasonable request.

31 10 350-354 11 2014 10.1007/s13770-014-0024-9 Babaei M Ardjmand M Akbarzadeh A Seyfkordi A Tissue Engineering and Regenerative Medicine Efficacy Comparison of Nanoniosomal and Pegylated Nanoniosomal Cisplatin on A172 Cell Line 4 1671-1685 12 2022 Jabbarzadeh Kaboli P Shabani S Sharma S Partovi Nasr M Yamaguchi H Hung M American Journal of Cancer Research Shedding light on triple-negative breast cancer with Trop2-targeted antibody-drug conjugates 6 1403-1412 45 2015 Bayindir ZS Be AB Yüksel N Turkish Journal of Medical Sciences Paclitaxel-loaded niosomes for intravenous administration: pharmacokinetics and tissue distribution in rats 9 09 DC05-08 8 2014 10.7860/JCDR/2014/8886.4864 Torkaman MRA Nasiri MJ Farnia P Shahhosseiny MH Mozafari M Velayati AA Journal of clinical and diagnostic research: JCDR Estimation of Recent Transmission of Mycobacterium Tuberculosis Strains among Iranian and Afghan Immigrants: A Cluster-Based Study 06 1 12 18515 9 2019 10.1038/s41598-019-55112-y Allahyartorkaman M Mirsaeidi M Hamzehloo G Amini S Zakiloo M Nasiri MJ Scientific Reports Low diagnostic accuracy of Xpert MTB/RIF assay for extrapulmonary tuberculosis: A multicenter surveillance 06 242-244 17 2019 10.1016/j.jgar.2018.12.022 Amini S Hoffner S Allahyar Torkaman MR Hamzehloo G Nasiri MJ Salehi M Sami Kashkooli G et al Journal of Global Antimicrobial Resistance Direct drug susceptibility testing of Mycobacterium tuberculosis using the proportional method: A multicenter study Pt 4 04 463-465 64 2015 10.1099/jmm.0.000021 Torkaman MRA Kamachi K Nikbin VS Lotfi MN Shahcheraghi F Journal of Medical Microbiology Comparison of loop-mediated isothermal amplification and real-time PCR for detecting Bordetella pertussis 1 02 10-18 23 2014 10.1016/j.breast.2013.10.006 Blanco E Ferrari M Breast (Edinburgh, Scotland) Emerging nanotherapeutic strategies in breast cancer 1 44-54 6 2016 Hosseinian S Khajavi Rad A Hadjzadeh MAR Mohamadian Roshan N Havakhah S Shafiee S Avicenna Journal of Phytomedicine The protective effect of Nigella sativa against cisplatin-induced nephrotoxicity in rats 14 9 08 115 38 2021 10.1007/s12032-021-01561-3 Shabani S Moghadam MF Gargari SLM Medical Oncology (Northwood, London, England) Isolation and characterization of a novel GRP78-specific single-chain variable fragment (scFv) using ribosome display method 04 152-167 85 2016 10.1016/j.biomaterials.2016.01.061 Kanamala M Wilson WR Yang M Palmer BD Wu Z Biomaterials Mechanisms and biomaterials in pH-responsive tumour targeted drug delivery: A review 29 2 09 359-366 47 2012 10.1016/j.ejps.2012.06.020 Kowapradit J Apirakaramwong A Ngawhirunpat T Rojanarata T Sajomsang W Opanasopit P European Journal of Pharmaceutical Sciences: Official Journal of the European Federation for Pharmaceutical Sciences Methylated N-(4-N,N-dimethylaminobenzyl) chitosan coated liposomes for oral protein drug delivery 05 132-143 18 2015 10.1016/j.actbio.2015.02.022 Li M Tang Z Zhang Y Lv S Li Q Chen X Acta Biomaterialia Targeted delivery of cisplatin by LHRH-peptide conjugated dextran nanoparticles suppresses breast cancer growth and metastasis 437-444 6 2011 10.2147/IJN.S15997 Zhang X Yang H Gu K Chen J Rui M Jiang GL International Journal of Nanomedicine In vitro and in vivo study of a nanoliposomal cisplatin as a radiosensitizer 07 154528 247 2023 10.1016/j.prp.2023.154528 Mehralizadeh H Nazari A Oruji F Roostaie M Hosseininozari G Yazdani O Esbati R Roudini K Pathology, Research and Practice Cytokine sustained delivery for cancer therapy; special focus on stem cell- and biomaterial- based delivery methods Mujoriya R Babu Bodla R available at http://hdl.net/123456789/97,2012 Design and development of niosomal delivery system for Ketoprofen. 2012 3 609-617 20 2021 10.22037/ijpr.2020.114260.14757 Hashemi A Bigdeli R Shahnazari M Oruji F Fattahi S Panahnejad E Ghadri A et al Iranian journal of pharmaceutical research: IJPR Evaluation of Inflammasome Activation in Peripheral Blood Mononuclear Cells of Hemodialysis Treated Patients with Glomerulonephritis 4 10 568-573 88 2016 10.1111/cbdd.12786 Poy D Akbarzadeh A Ebrahimi Shahmabadi H Ebrahimifar M Farhangi A Farahnak Zarabi M Akbari A Saffari Z Siami F Chemical Biology & Drug Design Preparation, characterization, and cytotoxic effects of liposomal nanoparticles containing cisplatin: an in vitro study 2 02 167-177 86 2014 10.1016/j.ejpb.2013.04.001 Bulcão RP Freitas FA Dallegrave E Venturini CG Baierle M Durgante J Sauer E et al European Journal of Pharmaceutics and Biopharmaceutics: Official Journal of Arbeitsgemeinschaft Fur Pharmazeutische Verfahrenstechnik e.V In vivo toxicological evaluation of polymeric nanocapsules after intradermal administration 4 10 358-360 28 2013 10.1007/s12291-013-0296-1 Esfahani MKM Alavi SE Movahedi F Alavi F Akbarzadeh A Indian journal of clinical biochemistry: IJCB Cytotoxicity of liposomal Paclitaxel in breast cancer cell line mcf-7 4 10 410-412 28 2013 10.1007/s12291-013-0306-3 Dadgar N Alavi SE Esfahani MKM Akbarzadeh A Indian journal of clinical biochemistry: IJCB Study of toxicity effect of pegylated nanoliposomal artemisinin on breast cancer cell line 7 07 1355-1359 37 2015 10.1007/s10529-015-1813-5 Shahbazian S Akbarzadeh A Torabi S Omidi M Biotechnology Letters Anti-cancer activity of pegylated liposomal trans-anethole on breast cancer cell lines MCF-7 and T47D