Research On Doxorubicin-loaded Mesoporous Magnetic Fe₃O₄ Nanoparticles To Induce Apoptosis In MCF-7 Breast Cancer Cells

Breast cancer is the second most common type of malignancy worldwide with approximately 1.4 million new cases every year. Breast cancer accounts for nearly 23% of total cancer cases and 15% of overall cancer related deaths among females, making it one of the deadliest diseases. At present, conventional treatment modalities such as chemotherapy, surgical intervention, radiotherapy, and targeted therapies are the treatment of choice. However, multidrug resistance, lack of selectivity, and rapid clearance of drugs from the systemic circulation result in non-specific toxicity to normal cells and reduce the chemotherapeutic efficacy in cancer cells.Doxorubicin (DOX), an important anthracycline derivative, is a broad spectrum anticancer drug with wide clinical applications against multiple cancers including breast cancer. Intracellularly, DOX acts by the intercalation of DNA which disrupts the replication and transcription process in cancer cells and finally cell death. The anticancer effect of DOX is hampered by rapid clearance from body, poor target selectivity, and serious side effects (such as cardiotoxicity). Therefore, it have many limitations in clinical applications.Selective targeting of chemotherapeutic drugs toward the cancer cells overcomes the limitations involved in chemotherapy. Magnetic nanoparticles (MNP) which can be selectively targeted to the cancer tissues in the presence of magnetic field are gaining increasing importance. Mainly polymer coated MNP in which polymer is coated on the nanoparticles offer many advantages including lack of toxicity, biocompatibility, biodegradability and cell adhesion. In addition, polymer coating prevents the drug from premature release, renders biocompatibility and prevents their agglomeration.The present study is aimed at developing DOX-loaded chitosan coated mesop-orous magnetic nanoparticles (DOX-CMMN) and to study its anticancer activity against breast cancer cells (MCF-7). The protective layer of chitosan will protect the release of drugs and improve the biocompatibility of magnetic systems. For this purpose, Fe3O4 based magnetic nanoparticles were formed which were then made mesoporous by chemical and heat treatment. DOX was loaded on to the pores of the nanoparticles followed by chitosan coating on the surface. The selective anticancer potential of DOX-CMMN was evaluated in the presence of applied alternating current magnetic field (ACMF) that facilitates drug release due to heating and enhances cancer cell death due to thermal therapy.1. Develop DOX-loaded chitosan coated mesoporous Fe3O4 magnetic nanoparticles material&method:1g of FeCl3·6H2O and FeCl2·4H2O (2:1) was mixed in 25ml of ethylene glycol (EG). This mixture was stirred at 80℃ for 1h. Following which 2g of sodium acetate and 5ml of ethylenediamine (EDA) was added to the homogenous solution. The temperature of the solution was gradually increased to 160℃ under constant stirring. The temperature was gradually raised to 180℃ within 6h and allowed to cool to room temperature. The resulting magnetic nanoparticles were separated from the solution mixture with the aid of a magnet and then it was washed with ethanol and water. DOX was loaded into MMN by incubation method. 100mg of MMN was incubated with DOX in 5ml of water and was left aside for 1h. The mixture was sonicated for 20 min and unentrapped drug was separated. 100mg of DOX-MMN was dispersed in a 10ml aqueous solution containing 1% span-80 to this solution,30ml of chitosan solution (1%) was added under constant magnetic stirring for 3h. Next, the mixture was mixed in ultrasonication bath for 60min. The resulting CMMN were separated from solution mixture with the aid of a magnetic field. The drug loading efficiency of CMMN was evaluated by spectrometer method. DOX-CMMN were centrifuged at 15,000 rpm for 15 min. The supernatant was collected to estimate the amount of free drug. The amount of drug in the supernatant was determined by UV-VIS spectrometer at 480nm. The drug loading and enbedding ratio of CMMN was calculated through the formula. The mean diameter and size distribution were analyzed using dynamic light scattering technique by Zetasizer. The samples were measured at 25℃. Each sample was measured in triplicate. Scanning electron microscopy (SEM) was used to determine the morphology and surface texture. The magnetic properties of MNP and CMNP were evaluated by vibrating the sample using magn etometer. spectrophotometer method is used to measure the Doxorubicin (DOX) solution in 200～500 nm range of ultraviolet-visible absorption spectrum. We can observe that 480 nm is the absorption peak, so determine 480 nm as measurement wavelength. Configuration standard concentration of 0.2 mg/ml,0.4 mg/ml,0.6 mg/ml,0.8 mg/ml,1 mg/ml DOX solution, measured at 480 nm wavelength absorption value, draw standard absorption curve of DOX. The DOX release from CMMN nanoparticles was investigated in phosphate buffered saline (PBS, pH 7.4) and acetate buffered saline (ABS, pH 5.5). The DOX-CMMN were dispersed in lml of PBS and sealed in a dialysis bag. The dialysis bag was in turn kept in a 50ml Falcon tube containing 25ml of the respective release media. The whole assembly was placed in a shaker bath maintained at 37℃. In 1 h,2 h,4 h,8 h,12 h,16 h,24 h and 36 h,48 h interval time,1 ml dialysate was suctioned, and measured 480 nm wavelength absorbance by a spectrophotometer method, calculate the content of DOX. Samples were poured back into the dialysate after testing. The amount or percentage of drug released was plotted against time intervals.RESULT:The preparation of DOX-loaded mesoporous magnetic nanoparticles (DOX-MMN) is successful by solvothermal method and the temperature is increased to 180℃ to introduce pores in the magnetic particles. The DOX was incubated with porous MMN at room temperature where the drug was loaded into the nanoparticles. Finally, MMN were coated with chitosan to form a thin-polymer coated MMN. Owing to its porous nature and appreciable aque-ous dispersibility, CMMN showed a high loading capacity for DOX. The CMMN exhibited >90% of entrapment efficiency with an active drug loading of 19%. The high drug loading might be due to the hydrophilic nature of DOX and presence of NH3+ group. The positive charge of drug was attracted toward the negative charge of MMN. In addition, porous nature of MMN further facilitated the drug loading of DOX. The mean particle size of DOX-CMMN was found to be 120 nm. The polydispersity index of nanoparticles was around 0.2 indicating the success of formulation strategy. The morphology and size of DOX-CMMN were further confirmed by SEM imaging. The particles were spherical aggregates with smooth surface on the particle border. The particles were aggregated due to strong magnetic dipole-dipole interactions. The particle size measured by DLS was consistent with SEM although the former was measured in aqueous state and dried state. Polymer coated magnetic particles have showed superparamagnetic behavior as that of uncoated or original MMN at room temperature. The saturation magnetization of the CMMN was slightly less compared to MMN confirming its potential magnetic property. The slight reduction in magnetization was due to the presence of polymer layer. This polymer molecule could potentially reduce the magnetic dead layer on the surface of MMN.480 nm wavelength absorption value on different concentrations of DOX solution at were 3.7069,7.1951,10.6403,14.5028, 17.1009 (1/trans). Concentration and absorbance had good linear correlation. The regression equation Y=17.0618+0.4611 X, correlation coefficient R2=0.9981. In vitro DOX-CMMN particle release experiment, cumulative release of drugs in two different pH solution were tested within interval of 1h,2h,4h,8h,12h,16h,24h,36h, and 48h. Use the single factor analysis of variance, the data were expressed as mean± sd, P<0.001 were accepted as statistically. The result showed significant difference between two groups. The release rate in PBS solution (pH7.7) were 2.38±0.21; 3.67±0.37; 6.24±1.37; 10.09±1.74; 16.35±2.02; 24.95±3.02; 38.16±3.94; 51.74±3.49; 58.16±4.86. The release rate in ABS solution (pH5.5) were 4.04±0.34; 5.41±0.42; 10.82±1.28; 16.33±2.01; 27.70±3.39; 38.53±4.95; 55.17±4.90; 80.73±3.31; 94.67±3.39. CMMN exhibited a sustained release pattern in both the pH conditions. Especially, higher drug release was observed in acidic media (pH 5.5). At the end of 24h,38.16% of DOX released in pH 7.4 conditions while 55.17% of drug release was in pH 5.5 conditions during the same time period. At the end of the study (48h), about 94.67% of drug was released in acidic media on comparing to that of only 58.16% in physiological media..2. Research on DOX-CMMN inducing apoptosis in MCF-7 breast cancer cells The MCF-7 breast cancer cell was cultured in RPMI 1640 medium. The cells were maintained in a humidified incubator at 37℃ and 5% CO2. MCF-7 cells were seeded at a density of 1 X 104 per 100ml RPMI 1640 into 96-well microtiter plates. Cells were allowed to attach for 24 h.0.001,0.01,0.1,0.5,1,5,10,50,100,200μg/ml concentrations of blank CMMN to evaluate its biocompatibility. Cytotoxic potential was evaluated by MTT assay. Cells with 0.001,0.01,0.1,0.5,1,5,10μg/ml concentrations of free DOX and DOX-CMMN were treated and incubated for 24h. Untreated cells were considered as control. The following day, plates were washed treated with 100ml of MTT solution. The absorbance of each plate was read at 570nm using a microplate reader. All experiments were repeated 6 times. Cells were incubated with respective formulations, washed with PBS, fixed with 4% paraformaldehyde, and viewed under optical microscope. The synergistic activity of formulations in the presence and absence of applied magnetic field was evaluated in MCF-7 cancer cells. Cells were seeded at a density of 1 X 104 and were treated with free DOX and DOX-CMMN and incubated for 6h. Plates were divided into two sections and one section of the plates was completely enclosed in a parafilm and exposed to ACMF (250 kHz) for 5 min and 10 min under sterile conditions. The cells were incubated for 24h after that. Cells were subjected to cell viability assay by MTT method. All experiments were repeated 6 times.Result:In vitro after treated with blank nanoparticles, the MCF-7 cells viability were 99.81±7.14; 99.70±4.33; 99.83±3.62; 99.23±3.31; 99.03±4.30; 99.83±6.51; 96.53±7.20; 94.39±3.47; 87.22±5.59; 85.01±4.80. Blank nanoparticles did not exhibit any appreciable cytotoxicity up to a maximum tested concentration of 200 mg/ml. Throughout all the concentrations, cell viability remained>85% indicating its excellent biocompatible nature. The cytotoxic potential of DOX and DOX-CMMN showed that both the formulations exhibited a concentration-dependent cytotoxicity. It can be seen that DOX-CMMN exhibited a superior cytotoxic effect than when compared to that of free DOX. The IC50 value of DOX was around 2.76 mg/ml while it was 1.25 mg/ml for DOX-CMMN. The morphological imaging was carried out on MCF-7 cells. Control group presented a normal morphology pertaining to healthy untreated cells, while DOX-CMMN treated cells showed completely different and spherical cells. Both free DOX and DOX-CMMN exposed group were subject to hyperthermic conditions under an applied ACMF. DOX killed 30% of cancer cells in the absence of ACMF, while a marginal increase in cell killing efficiency was observed when exposed to ACMF. Interestingly, DOX-CMMN showed a killing efficiency of 40% in the absence of ACMF, however killing efficiency significantly increased to 65% for 5 min exposure of ACMF. The cell killing efficiency further increased (90%) when the ACMF expose time prolonged to 10 min.Conclusions:In conclusion, DOX-loaded chitosan coated mesoporous magnetic nanoparticles were (DOX-CMMN) prepared and evaluated its anticancer efficiency against MCF-7 breast cancer cell line. DOX-CMMN exhibited a controlled and sustained release of drug without any burst release phenomenon. DOX-DMMN showed a concentration-dependent cell proliferation inhibitory action against breast cancer cells. The blank nanoparticles showed excellent biocompatibility with cell viability>85% at the maximum tested concentration. Our results showed that chitosan coated magnetic system has high potential for breast cancer targeting. The present study showed that magnetic nanoparticles can be targeted to tumor cells under the presence of oscillating magnetic field. The combined effect of chemotherapy and thermotherapy remarkably enhanced the therapeutic efficacy. However, further in vivo studies are needed to be carried out to show the therapeutic potential of the present delivery systems.