Synthesis and application of magnetite dextran-spermine nanoparticles in breast cancer hyperthermia
Cancer treatment has been very challenging in recent decades. One of the most promising cancer treatment methods is hyperthermia, which increases the tumor temperature (41–45 °C). Magnetic nanoparticles have been widely used for selective targeting of cancer cells. In the present study, magnetic dextran-spermine nanoparticles, conjugated with Anti-HER2 antibody to target breast cancer cells were developed. The magnetic dextran-spermine nanoparticles (DMNPs) were prepared by ionic gelation, followed by conjugation of antibody to them using EDC-NHS method. Then the Prussian blue method was used to estimate the targeting ability and cellular uptake. Cytotoxicity assay by MTT showed that antibody-conjugated MNPs (ADMNPs) have no toxic effect on SKBR3 and human fibroblast cells. Finally, the hyperthermia was applied to show that synthesized ADMNPs, could increase the cancer cells temperature up to 45 °C and kill most of them without affecting normal cells. These observations proved that Anti-HER2 conjugated magnetic dextran-spermine nanoparticles can target and destroy cancer cells and are potentially suitable for cancer treatment.
KeywordsCancer hyperthermia Anti-HER2 Magnetic nanoparticles Dextran-spermine
Breast cancer is a malignant tumor that originates from healthy mammary gland cells which is most frequent among women aged between 50 and 70. It is the most common type of cancer among women which affects one in eight women on average (Alphandery 2014; Matsen and Neumayer 2013; Tinoco et al. 2013). Surgery, chemotherapy and radiotherapy are the most common strategies to treat breast cancer. These conventional cancer therapies, having limitations such as toxic side effects and drug resistance, have often failed to completely eliminate the tumor (Chalkidou et al. 2011; Chen et al. 2014; Debnath et al. 2016; Sahu et al. 2013).
Hyperthermia is the application of heat to destroy the tumor cells or sensitize them to drugs and radiation by increasing blood flow and inducing immune responses. The tumor cells, by having poorly developed vessels and nervous system, so insufficient oxygen supply and inability to dissipate heat, are sensitive to temperatures in the range of 41–45 °C, which damages tumor cells irreversibly, while normal cells can tolerate even higher temperatures (Chen et al. 2009; Kawashita et al. 2005; Lin et al. 2012; Meenach et al. 2010; Purushotham and Ramanujan 2010; Rao et al. 2013; Sen et al. 2011). An adequate amount of heat should be delivered to the tumor so that it reaches the desired temperatures, or it may induce resistance. The magnetic fluid hyperthermia is a non-invasive method which can deliver the desired heat to deep-seated tumors without damaging healthy tissues. It predominantly uses superparamagnetic iron oxide nanoparticles (SPIONs) which are biocompatible, non-toxic and easy to synthesize. They have raised great attention in biomedical applications such as magnetic resonance imaging, drug delivery and magnetic hyperthermia where SPIONs produce heat in an alternating magnetic field (AFM) thorough either Néel or Brownian relaxation (Lin et al. 2012; Ma et al. 2004; Sadhukha et al. 2013; Stocke et al. 2016; Yallapu et al. 2011).
The magnetic particles can be introduced into the body via the systemic circulation and guided to the target site under the influence of magnetic field or via direct injection into the tumor. However, in case of intravenous injection, the particle size should be controlled as well as surface modification in a way that inhibits protein adsorption and phagocytosis. SPIONs, having a hydrophobic nature, tend to aggregate, so have a short half-life in blood circulation, leading to poor bioavailability. To resolve this challenge, polymeric and inorganic nanoparticles, liposomes, micelles and phospholipid complexes have been used to encapsulate them for delivery. Among them, polymeric nanoparticles are emerging as one of the best options due to their higher stability (Cole et al. 2011a; Lin et al. 2012; Liu et al. 2011; Rao et al. 2013). The particles can be delivered to the tumor either passively thorough vascularization and the enhanced permeation and retention effect (EPR) or actively thorough receptor-mediated endocytosis. However, active targeting results in higher local concentrations of nanoparticles and lower systemic concentrations, which is essential for more effective treatment (Cole et al. 2011; Kruse et al. 2014; Lin et al. 2012; Ling et al. 2017; Ruoslahti et al. 2010; Wuang et al. 2008).
In this study an atni-HER2 conjugated dextran-spermine magnetic nanoparticle was developed for breast cancer magnetic hyperthermia. Atni-HER2 is a humanized IgG monoclonal antibody directed against the extracellular domain of the human epidermal growth factor receptor 2 (HER-2), which is overexpressed in some types of breast cancer cells. The antibody can be efficient in cell uptake of the carrier system and nanoparticles thorough the internalization ability of Anti-HER-2 (Moore and Cobleigh 2007; Wuang et al. 2008). First, dextran-spermine was synthesized and the amine content was evaluated as described in the literature (Azzam et al. 2002). The dextran-spermine magnetic nanoparticles (DMNPs) were prepared using ionic gelation method. The size, zeta potential and morphology of the nanoparticles were analyzed. Finally, the antibody was conjugated to the nanoparticles. The anti-HER2 conjugated dextran-spermine magnetic nanoparticles (ADMNPs) were characterized and compared to DMNPs and iron oxide nanoparticles (MNPs) in terms of biocompatibility, cell uptake and in vitro hyperthermia for cancerous SKBR3 and normal fibroblast cells.
Materials and methods
Characterization of the synthesized polymer
H-NMR analysis and TNBS method were used to determine the formation of the primary amine groups. For TNBS method, dextran-spermine was dissolved in 0.1 M sodium bicarbonate solution (pH 8.5) to obtain solutions ranging 20–200 µg/mL. A standard calibration curve for l-lysine was plotted. 1% TNBS was added to 0.1 M sodium bicarbonate solution and 500 µg of the resulting solutions was added to 1 mL of each sample. The samples were incubated for 2 h at 37 °C. Finally, 500 µg sodium dodecyl sulfate (10% w/v) and 250 µL HCl (1 N) were added to each sample to stop the reaction. The absorbance of the samples was recorded at 345 nm using a spectrophotometer (UV–Vis-CARY50). The primary amine content was calculated according to the calibration curve. H-NMR spectra were recorded on a BRUKER DRX500 AVANCE (500 MHz) instrument using DDW as solvent. Values were recorded as ppm relative to internal standard (TMS).
Preparation of SPION-loaded dextran-spermine nanoparticles
Characterization of the MNP-loaded nanoparticles
DLS analysis was carried out to determine the size distribution and zeta potential of the DMNP using a particle size analyzer (PSA) (Malvern, 3000 HAS, England). The DMNPs were suspended in purified water and sonicated to produce a homogenous suspension for measurement. The zeta potential of the nanoparticles was also measured to confirm their surface charge. TEM imaging (Zeis-EM10C-80 kV) was also used to evaluate the size, morphology and encapsulation of the nanoparticles. A drop of well-dispersed nanoparticle suspension was placed on a copper grid and then dried at ambient condition before its attachment to the sample holder of the microscope.
Conjugation of the antibody to the MNP-loaded nanoparticles
Characterization of antibody-conjugated nanoparticles
Size distribution and zeta potential of antibody-conjugated MNP-loaded dextran-spermine nanoparticles (ADMNP) were also measured using a particle size analyzer. Comparing these results to those of the DMNP can confirm the antibody conjugation. Antibody conjugation to the nanoparticles was also evaluated using Bradford assay. The Bradford solution was prepared by adding 100 mg of Coomassie brilliant blue G250 to 50 mL ethanol, then mixing by 100 mL phosphoric acid (85% w/w) and finally adding them to DDW to reach the volume of 1 L. Then 5 mL of the solution was added to different concentrations of the antibody ranging from 0 to 1 mg/mL and their absorbances at 600 nm were measured. These values were plotted to form a standard curve. The amount of the antibody in the supernatant of the centrifuged antibody-conjugated nanoparticles was determined by comparing to the standard curve.
Immortalized primary human fibroblast and breast cancer SKBr3 cells obtained from cell bank (Stem cell technology research center, Tehran, Iran) were grown in T75 cell culture flasks in 3 mL of complete medium. The medium consisted of Dulbecco’s Modified Eagles Medium (DMEM), with 10% of fetal bovine serum (FBS) and Penicillin/Streptomycin. The cells were incubated in an incubator (RS Biotech Galaxy) at 37 °C with a 5% CO2 atmosphere until they reach the suitable confluence.
In vitro cytotoxicities of MNPs, DMNPs and ADMNPs against breast cancer SKBR3 and normal fibroblast cells were evaluated based on the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays. SKBR3 and human fibroblast cells with cell density of 5000 per well were dispensed in 96-well plates in triplicate, and 200 µL of fresh medium was added to each well and incubated for 48 h. Then, the cells were treated with different concentrations of the nanoparticles (50, 80 and 150 µg/mL medium). A control group containing no particle was also considered for each cell type. The samples were incubated for 24 and 48 h. After the incubation, the wells were washed with 200 µL PBS and overlaid with 50 µL fresh media containing 0.5 mg/mL of MTT reagent and incubated for 4 h. Subsequently, the media were aspirated and formazan crystals were solubilized in 200 µL dimethyl sulfoxide (DMSO). The averaged absorbance at 570 nm for each treatment group was normalized to the zero-time viability values.
Cellular uptake was evaluated using iron staining method. For this purpose, the cultured SKBR3 and fibroblast cells were seeded in 96-well plates with cell density of 5000 per well and 200 µL of fresh media was added. The cells were left to incubate for 24 h, prior to addition of nanoparticles. Three sample groups (MNP, DMNP and ADMNP) and a control were considered for each cell type. The samples were overlaid by 200 µL of fresh media containing 80 µg/mL nanoparticles and after 6 h of incubation, the culture medium was removed and the cells were fixed upon addition of 500 µL 10% neutral buffered formalin. After 20 min, the formalin was replaced by 500 µL Prussian blue solution, containing 5% w/v potassium ferrocyanide and 5% v/v HCl and left till blue stains appeared. Prussian blue is a prototype of mixed-valence transition-metal hexacyanoferrates with the general formula of Fe4[Fe(CN)6]3 (Cheng et al. 2014). The cells then were washed twice by PBS and examined by microscope.
For in vitro hyperthermia study, SKBR3 and fibroblast cells were seeded in the 96-well plates with the concentration of 5000 cells per well and 200 µL of fresh media was added. After 48 h of incubation, the media was replaced by 200 µL of fresh media containing optimum concentration of 80 µg/mL (Attar et al. 2016) of three magnetic nanoparticle groups (MNP, DMNP and ADMNP). A control group with no magnetic substance was also considered for each cell type. After 8 h of incubation, the wells were washed with 200 µL PBS and overlaid with 200 µL fresh media. The cells were left to incubate overnight prior to initiating the test. The samples were placed in the copper coil of a radio frequency generator at 80 kHz frequency with 150 kA/m field. The temperature was monitored over the test.
All experiments were performed at least in triplicate. Microsoft office excel was used for t tests (paired t test with unequal variances) to determine any significance in the observed data. The P value <0.05 was considered statistically significant.
Results and discussion
Characterization of the synthesized polymer
1.4405 (m, 4H, dextran-CH2 NH (CH2)3 NH CH2CH2 CH2CH2 NH (CH2)3 NH2), 1.6106 (m, 4H, dextran-CH2 NH CH2CH2CH2 NH (CH2)4NH CH2CH2CH2 NH2), 2.6303 (m, 14H, dextran-CH2 NH CH2CH2CH2 NH CH2CH2CH2CH2 NH CH2CH2CH2 NH2), 3.4150–3.5860 (m, glycoside hydrogens) and 4.6822 (m, 1H, anomeric hydrogen).
Characterization of the MNP-loaded nanoparticles
Comparison of average size and zeta potential of the DMNPs, the antibody and the ADMNPs
Average size (nm)
Zeta potential (mV)
Dextran-spermine encapsulated Iron oxide nanoparticles (DMNPs)
Anti-HER2 conjugated dextran-spermine encapsulated iron oxide nanoparticles (ADMNPs)
Characterization of the antibody-conjugated nanoparticles
DLS analysis was carried out to confirm the conjugation of the antibody to DMNPs. As reported in Table 1, the mean size of ADMNPs and the antibody was 85.6 and 7.5 nm, respectively, with corresponding zeta potentials of +3.47 and −12.5 mV. These particles are still within optimal size range and with a cationic surface charge, can be easily uptaken by cancer cells. These results indicate that the average size of DMNPs increased upon antibody conjugation; but the extent of their positive charge decreased due to the negative charge of the antibody. These observations confirm the successful conjugation of the antibody to the DMNPs.
Bradford assay was also used to quantitatively evaluate the antibody conjugation. A standard absorbance curve was plotted for the antibody concentration. The supernatant of the centrifuged antibody-conjugated nanoparticles suspension was analyzed using Elisa reader and conjugation of 24.1 µg antibody to 1 mg of the nanoparticles was obtained. Higher antibody conjugation that resulted in negatively charged nanoparticles have been reported; but these negatively charged nanoparticles did not show good cell uptake characteristics (Nahta and Esteva 2006; Purushotham and Ramanujan 2010; Rao et al. 2013).
As shown in Fig. 8a, the control groups for SKBR3 and fibroblast cells, having no magnetic content, did not show any change in temperature. The Fibroblast samples did not experience any significant raise of temperature except for the DMNP sample, which came close to the hyperthermia region. The reason is that these cationic nanoparticles with size <100 nm are capable of entering the cells and as they do not have any specific targeting ability (antibody), they enter any cell type indiscriminately (Cole et al. 2011).The MNPs and DMNPs, due to significant uptake into the SKBR3 cells, were able to heat them to 40 and 41 °C, respectively. The most temperature elevation was observed in SKBR3 cells containing ADMNPs. This group is the only one that reached the hyperthermia temperatures, so the most toxicity was expected for this group.
Figure 8b shows that the cytotoxicity in all fibroblast groups was negligible, so none of the groups were able to generate sufficient amount of heat to damage the cells. However, the ADMNPs, having targeting ability, did not enter the fibroblast cells and the cellular uptake of the MNPs and DMNPs was not enough to induce hyperthermia effect. Figure 8a shows that fibroblast groups did not reach the hyperthermia temperatures and no significant cytotoxicity was expected. The only group which was able to kill some of the fibroblast cells was the DMNPs group. Although the uptakes of MNP and DMNP groups were not significantly different for SKBR3 and fibroblast cells, cytotoxicity was higher for cancer cells due to more sensitivity of the cancer cells to high temperatures (Javidi et al. 2015). Finally, the ADMNPs group with excellent targeting ability and good temperature elevation under AFM was able to kill over 65 percent of cancerous cells without affecting normal fibroblast cells.
Dextran-spermine polymer with 1.43 mM amine groups was successfully synthesized. Magnetic nanoparticles of dextran-spermine (DMNPs) with a size of 67.3 nm and zeta potential of and +30 mV were prepared. These characteristics make the particles ideal for biomedical applications. EDC-NHS method was used to conjugate anti-HER2 antibody to DMNPs that resulted in nanoparticles with a size and surface charge of 85.6 nm and +3.47 mV, respectively. These antibody-conjugated magnetic nanoparticles (ADMNPs) were compared to DMNPs and MNPs in terms of cytotoxicity, targeting ability and hyperthermia efficiency. The results showed that ADMNPs were not toxic to normal cells, while they showed a slight toxicity to SKBR3 cells. However, they can target HER2-expressing cancer cells and enter them effectively. In vitro hyperthermia confirmed the ability of ADMNPs in targeting cancer cells and heating them up to hyperthermia range while more than 63% of cancer cells were destroyed over a 20-min treatment course. Based on the in vitro results, ADMNPs have a great potential for breast cancer treatment by hyperthermia.
Compliance with ethical standards
Conflict of interest
Authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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