Heterogeneous Fenton Reaction Enabled Selective Colon Cancerous Cell Treatment

A selective colon cancer cell therapy was effectively achieved with catalase-mediated intra-cellular heterogeneous Fenton reactions triggered by cellular uptake of SnFe2O4 nanocrystals. The treatment was proven effective for eradicating colon cancer cells, whereas was benign to normal colon cells, thus effectively realizing the selective colon cancer cell therapeutics. Cancer cells possess much higher innate hydrogen peroxide (H2O2) but much lower catalase levels than normal cells. Catalase, an effective H2O2 scavenger, prevented attacks on cells by reactive oxygen species induced from H2O2. The above intrinsic difference between cancer and normal cells was utilized to achieve selective colon cancer cell eradication through endocytosing efficient heterogeneous Fenton catalysts to trigger the formation of highly reactive oxygen species from H2O2. In this paper, SnFe2O4 nanocrystals, a newly noted outstanding paramagnetic heterogeneous Fenton catalyst, have been verified an effective selective colon cancerous cell treatment reagent of satisfactory blood compatibility.

SCIEnTIfIC REPORTS | (2018) 8:16580 | DOI: 10.1038/s41598-018-34499-0 efficacy toward colon cancer cells with deeper insights derived from relevant biomedical characterizations. For instance, this iron based paramagnetic nanomaterial may exhibit strong contrasts in MRI imaging, one of the most powerful diagnostic tools in medicine. In addition, the blood compatibility of the functional nanomaterial is a vital prerequisite for its usage in bio-imaging, drug delivery system, and gene treatment. In this study, these SnFe 2 O 4 NCs were used for the selective treatment of colon cancerous cells. The SnFe 2 O 4 NCs were farbricated through a single-step carrier solvent assisted interfacial chemical reaction procedure 11 . These SnFe 2 O 4 NCs went through a certain extent of aggregation when dispersed in saline for cell treatment applications, depending on whether or not sonication was applied and the concentration of the suspension 12 . First, the effect of the size of the SnFe 2 O 4 aggregates on the treatment efficacy was investigated. As expected, smaller-sized SnFe 2 O 4 aggregates, obtained from sonication treatment at an appropriate suspension concentration, were advantageous in cellular internalization of the SnFe 2 O 4 nano-agregates and following yielding of hydroxyl radicals via heterogeneous Fenton reactions. The successful cellular internalization of the SnFe 2 O 4 aggregates into cells, has been proven with confocal laser scanning microscopy (CLSM) previously, and the paramagnetic property of the SnFe 2 O 4 aggregates was elucidated with a superconducting quantum interference device (SQUID) and magnetic resonance imaging (MRI) technique 13,14 . The blood compatibility of the SnFe 2 O 4 aggregates was also studied. Furthermore, the concentrations of the hydroxyl free radical and catalase in both normal and colon cancer cells were quantified with an fluorescent staining method 15 , confirming the proposed characteristic differences between normal and cancer cells in terms of H 2 O 2 and catalase concentrations as described above. Finally, the efficacy of the SnFe 2 O 4 NC-triggered heterogeneous Fenton reaction cell treatment was confirmed with cell viability measurements. The treatment was proven to be effective at eradicating colon cancer cells, whereas was benign to normal colon cells, thereby extending this selective therapy to colon cancer cell eradication.

Results and Discussion
Low levels of catalase activity were characterized in most cancer cells including the colon cancer samples examined. These cancer cell samples were thus more vulnerable to oxidative stresses induced by ROS-generating reagents. Thus, elevating ROS levels provides a rational means to abolish cancer cells, without appreciably damaging normal cells because of the presence of high levels of endogenous catalase in normal cells. Much research effort has been focused on developing strategies aiming at creating cytotoxic oxidative stresses for cancer therapy [16][17][18] .
The heterogeneous Fenton reaction is a critical reaction in which the lattice ferric ions of the solid-state Fenton catalyst convert hydrogen peroxide into very toxic hydroxyl free radicals that raise ROS stresses for colon cancer cell eradication. The present study is to apply SnFe 2 O 4 NCs in the bio-pharmacological field and investigate, using a fluorescent imaging approach, their in vitro efficacy in producing ROS, paramagnetic property, blood compatibility, and subsequent cytotoxicity toward colon cancer cells.

Characteristics of SnFe 2 O 4 aggregates.
In the bio-pharmacological field, major studies have highlighted the importance of controlling the particle size, shape, and chemistry for drug delivery efficiency. In many cases, agglomeration/aggregation among solid particles is caused by prevailing attractive forces (van der Waals). Physical breakup, for example sonication, was identified as being a convenient way to achieve mechanical separation to lessen the extent of aggregation/agglomeration in the suspensions 19 . Besides, elevated particulate densities in solution tend to favor serious aggregation/agglomeration. Therefore, it is essential to adjust the concentration of the particulates and to apply sonication when preparing the SnFe 2 O 4 NC suspensions in physiological fluids.
Heterogeneous Fenton reactions make hydroxyl radicals via redox reactions on the surface lattice ferric ions of the solid-state catalys and absorption H 2 O 2 molecules 20 . It is therefore expected that SnFe 2 O 4 aggregates of large reactive surface areas will efficiently produce hydroxyl radicals in colon cancer cells. A convenient bio-probe as marker of intracellular reactive oxygen species is 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). It is a good indicator for hydroxyl radicals but is insensitive toward H 2 O 2 21-23 . Additionally, intracellularly endogenous catalase can efficiently scavenge hydrogen peroxide 24 , consequently suppressing the production of cytotoxic hydroxyl radicals.
As shown in Fig. 1, for the without catlase case, the amount of hydroxyl radicals created was positively correlated with the concentration of the SnFe 2 O 4 aggregate, at 0.05~1 mmol/L in the presence of H 2 O 2 (500 mM) and with application of sonication. This was expected since more SnFe 2 O 4 was available to generate ROS such as hydroxyl radicals with an increasing SnFe 2 O 4 agrregate concentration. Nevertheless, once the concentration of the SnFe 2 O 4 aggregates was further increased to reach 2 mmol/L, the catalytic efficiency of the hydroxyl radical generation decreased.
We speculated that in this case, the effective catalytic surface area of the SnFe 2 O 4 agrregates had in fact diminished because of the severe aggregation of the SnFe 2 O 4 NCs. It was also interesting to note the lack of hydroxyl radicals when there was present of catalase with SnFe 2 O 4 . This confirms that the source of the hydroxyl radicals was the catalytic conversion of H 2 O 2 by SnFe 2 O 4 . Figure 2a shows the TEM image of the SnFe 2 O 4 aggregates obtained under sonication for 1 h at 37 °C at two particulate concentrations of 1 and 2 mmol/L. It is evident that the SnFe 2 O 4 NCs went through aggregation process in physiological saline solution owing to the decrease of the electrostatically repulsive interactions caused by the presence of free counter-ions of Cl − and Na + offered by the solution of saline. Aggregate sizes around below 20 nm, however, were obtained at 1 mmol/L, much smaller than those obtained at 2 mmol/L, which were micron-sized. The data verify our conjecture for the decreased hydroxyl radical level at 2 mmol/L as compared to that at 1 mmol/L. Higher particle concentrations cause more severe aggregation, resulting in decreases in the effectively exposed catalytic surface areas for generating hydroxyl radicals from H 2 O 2 . According to a previously published article, smaller particulates enable greater intra-cellular internalization compared to larger ones, and thus smaller particulates can be more effectively uptaked by the cells 25  SnFe 2 O 4 aggregates can be readily internalized into colon cells, normal or cancerous, and produce large amounts of hydroxyl radicals in colon cancer cells to significantly raise the ROS stresses to kill the colon cells. Here, the crystalline structure of these SnFe 2 O 4 aggregates was studied with XRD. As shown in Fig. 2b, the diffraction pattern of the SnFe 2 O 4 aggregates is in good match with that of the SnFe 2 O 4 nanocrystals of ref. 9 , confirming the  composition of the catalyst to be SnFe 2 O 4 . Furthermore, SnFe 2 O 4 is a paramagnetic material, responsive to externally applied magnetic fields, and its paramagnetism was verified with the long moment vs. temperature curve presented in Fig. 2c, which was measured with a SQUID magnetometer. Bio-functional traits and cellular uptakes of particulates in active substance delivery are highly dependent on the geometrical and structural features, such as size and shape, of the particulates 26,27 . Typically, particles with spherical morphology can be more swiftly internalized by the cells than particles with irregular shape 28 . The particle size was also found to be associated with the cell internalization behavior and their endocytic pathway, crucially dictating the intracellular fate and consequent biologic effects of the particles. It has been shown that particles with a dimension of below 500 nm could accomplish significantly higher cellular uptake than could larger particles 29 . We have verified that the present SnFe 2 O 4 aggregates were successfully internalized by living cells 10 . As proposed in literature, cellular uptakes of nanoparticles of sizes below 200 nm would be observed to involve specific clathrin-coated pits 30 . In a physiological environment, the metal oxide materials taken in can be gradually degraded within the lysosomal space and are eventually converted into free metal ions that could be rapidly urinated via bladder. In practical circumstances, this designed formulation could be applied to carry out an in vivo study through an intravenously administrated route for colon cancer treatments, in which the SnFe 2 O 4 aggregates accumulate within the colon cancer cells through either the retention (EPR) effects and enhanced permeability 31 or magnetically guided drug targeting (MGDT)method 32 .
MRI evaluation. MRI has been considered a useful medical imaging technique in radiology and physiological processes for the anatomy of the human body 33 . MRI scanners operate radio waves, robust magnetic fields, and field gradients to generate living images of the body. Furthermore, magnetic particle imaging (MPI) has been considered a novel imaging modality using paramagnetic iron based particles as a substance of tracer. This newfangled tomography of radiation-free imaging technique offers quick, sensitive, background-free, straight quantifiable 4 dimensional (4D) reports concerning the spatial distribution of the magnetic substance at very high temporal resolutions, ultra-sensitivity, and excellent spatial resolutions.
MRI enables sensitive and specific detection of (para)magnetic nano-carriers in biological systems 34 . Here, it was applied to quantify the SnFe 2 O 4 aggregates. To evaluate their T2-enhancing capability, SnFe 2 O 4 aggregate suspensions of increasing concentrations were examined by T2-weighted MRI. The acquired outcomes suggested that the paramagnetism of the SnFe 2 O 4 aggregates was promptly detectable by MRI. Among increasing amounts of the SnFe 2 O 4 suspensions, the signal intensity of MRI decreased (Fig. 3a,b). As well known, MR imaging is a non-invasive approach that has become the most vital noninvasive diagnostic means in many medical applications. MRI not only provides excellent morphological information but also possesses the ability to provide the best soft tissue contrast compared to all techniques of clinical imaging.
Immunofluorescence of DCFH-DA. The majority of colon cancerous cells in general possess extraordinarily few anti-oxidative bio-enzymes. Interestingly, the levels of intracellularly endogenous catalase in healthy normal colon cells are meaningfully greater than those examined through confocal in cancer cells (Fig. 4). One possible explanation for the observed outcomes may be that catalase may affect at either the protein level or mRNA throughout the progressing period of the cancerous cells 35 (Fig. 4). The yielding of hydroxyl radicals by the SnFe 2 O 4 aggregates was suppressed in normal cells because of the presence of sufficient amounts of catalase, which was at considerably greater concentrations in normal colon cells than in colon cancerous cells. In addition, it has been recognized that a catalase protein is capable of decomposing millions of H 2 O 2 molecules into oxygen (O 2 ) and water (H 2 O) in short period (one second). Aiming at cancer cells, the SnFe 2 O 4 aggregates could convert excessive levels of intracellular hydrogen peroxide into a considerable level of ROS which possibly is mainly hydroxyl radicals. An important impact of this designed method is inhibiting heterogeneous Fenton reaction by using catalase via disintegration of hydrogen peroxide. In addition, it is acknowledged that the expression of catalase in normal cells has been considered as mediator at the protein, polypeptide, delivering message, and bio-actively molecular levels. The cancerous cells applied in this study have minimal catalase active levels 36 . Swiftly actively growing cells, for instance cancerous cells, make aberrantly large amounts of hydrogen peroxide (H 2 O 2 ). This would enhance the oxidative stresses experiencing transformation of neoplastic and consequently improve the therapeutically targeting of cancerous cells through differences in levels of catalase.
Hemolysis Study. The hemolysis (destructing red blood cells) in vivo would be associated with jaundice, anemia, or other undesired pathological circumstances, thus the hemolytic potential of all pharmaceuticals of intravenous administration should be estimated. Drug carrier system and nanomaterial-based devices are emerging as replacements to traditional therapeutic drugs, and in vitro test of their biocompatibility with blood substances is an essential part of the primary pre-clinical development. The unique physicochemical properties of nanomaterials may lead to bio-interactions with erythrocytes to differ from those detected for traditional pharmaceuticals, and may also lead to interfering with regulated in vitro tests. The results of the test samples with different amounts of SnFe 2 O 4 incubated with harvested red blood cells from rats suggested that no destructed red blood cells were observed (Fig. 5). However, the red blood cell is placed in pure distilled water (a hypotonic solution) in which the water molecules are in a high concentration external to the red blood cell and water can thus move into the red blood cell, causing rupture possibly due to the different osmotic pressure.

Cytotoxicity.
For producing an effect of cytotoxicity, hydroxyl radicals would destroy the DNA backbone of sugar phosphate by receiving hydrogen (H atoms) from deoxyribose and then damaging bases of DNA by the addition of generated OH onto the double bonds of the purine ring. Once DNA has noted to be disturbed by these harmful hydroxyl radicals, this reacting procedure has to be involved in the close DNA vicinity 37 . As is well recognized, hydroxyl radicals (OH) are expressively more energetic and therefore much more toxically offensive than H 2 O 2 38 .
As evident from Fig. 6, normal colon cells survived well in the treatment of the SnFe 2 O 4 aggregates, showing high cell viability. This was owing to the presence of high catalase levels, notably suppressing death of apoptotic cell induced by the SnFe 2 O 4 aggregates. On the contrary, the SnFe 2 O 4 aggregates imposed a pronounced cytotoxic bio-action in colon cancer cells (Fig. 6). These fluorescently imaged observations revealed that heterogeneous based Fenton reactions, bio-performing via the prepared SnFe 2 O 4 nano-aggregates, greatly intensify the amount of ROS for initiating damage of colon cancer cells. These SnFe 2 O 4 aggregates, however, has been considered as safe toward normal colon cells. The corresponding quantitative cellular viability outcomes were examined and The high concentration suspensions (group of caco-2 cell, 2 mmol/L) will experience severe particle aggregation as shown in the TEM graph, which limits efficacy of the cellular uptake due to the huge particle dimension. This observed phenomenon was consistent with the MTT cytotoxicity results. As well known, this metal based SnFe 2 O 4 aggregates are likely transported away through the compartment of vessel as dissolved metal oxide ionic species and then cleaned through the bladder and kidneys. Accordingly, this prepared SnFe 2 O 4 aggregates should very feasibly to be both drug delivery carrier system and a therapeutically active substance for H 2 O 2 -rich aims, for instance cancerous cell microenvironments.    Fig. 7. On the contrary, normal colon cells were secured by high levels of catalase which scavenges the hydrogen peroxide molecules before the heterogeneous Fenton reaction should proceed to generate hydroxyl radicals, as illustrated in Fig. 7. These consequences validate the efficacy of the developed SnFe 2 O 4 aggregates for anti-colon cancer treatment. This developed SnFe 2 O 4 NCs are paramagnetic, verified by MRI and SQUID experiments, and have blood compatibility. In the future, further studies are on-going to explore the physiological interactions between the SnFe 2 O 4 aggregates and real organs for acquiring bio-information on the in vivo anti-tumor efficacy, biodistribution, in vivo toxicology, and excretion rate form kidneys.

Materials.
All reagents and chemicals used were of analytical grade and were obtained from Sigma-Aldrich  The morphological change and dimension of the SnFe 2 O 4 aggregates were observed and imaged with transmission electron microscopy (TEM) (Hitachi H-600, Tokyo, Japan). The X-ray diffraction (XRD) and the superconducting quantum interference device (SQUID) study were also performed to characterize the crystalline structure and paramagnetism of the Hemolysis test. Briefly, after centrifugation (4 °C, 3500 rpm) to get precipitated intact red blood cells from rat blood, a 10-fold-volumal PBS was added to the red blood cells. Next, 0.3 mL of the above solution was mixed with 1.2 mL of different concentrations of the SnFe 2 O 4 aggregates dispersed in PBS (DI water as positive control). The mixture was centrifuged (3000 rpm) and then the amount of released hemoglobin to the supernatant PBS was spectrophotometrically recorded with a microplate reader (500-640 nm).
Cytotoxicity. Cells attached to a confocal dish were stained by with a Live/dead ® viability/cytotoxicity kits (Molecular Probes, Eugene, OR, USA). Ethidium homodimer (Eth-D) and acetoxymethylester of calcein, (calcein-AM) stock chemical compounds were adjusted to their final working concentrations in PBS as suggested by protocol. The samples were then incubated in these blends at room temperature for half hour. Fluorescent image were recorded by using a CLSM.
Cell viability assay. Cells (at a density of 2 × 10 4 cells/mL) were distributed into 96-well plates with cultured growth media in a moistened incubator at 37 °C with an atmosphere of 5% CO 2 overnight for allowing cell attachment. Cell growth medium solution was then substituted by 200 μL of a SnFe 2 O 4 aggregate suspension (1 mmol/L). After incubation for 12 h, the spent medium was withdrawn and newly cultured media (200 μL) including 20 μL of an MTT regaent (5 g/L in PBS) were added for further cultivation (within 4 h, 37 °C). Next removing the above cultured solution, the formazan reaction products were dissolved into a 150 μL of dimethylsulfoxide (DMSO) for reacting 20 min and then were extracted for microplate reader study. The absorbent optical density OD value at given 490 nm was detected by a microplate reader.
Statistical analysis. ROS production and MTT experimental results were expressed as average ± standard deviation (SD). For comparing the means of group pairs, Student's t-test was selected to analyse data. Differences result were considered as significant in case p < 0.05.