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Micro and Nano Systems Letters
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Effects of MRI magnetic iron oxide nanoparticles on the structural and enzymatic properties of liver-related enzymes

Micro and Nano Systems Letters volume 12, Article number: 13 (2024) Cite this article

Abstract

Cancer survivors undergo meticulous examinations, including regular magnetic resonance imaging (MRI) scans, to monitor the risk of disease recurrence. The use of magnetic iron nanoparticles (MNPs) enhances MRI accuracy. However, post-injection, MNPs exhibit a notable affinity for binding with proteins and biomolecules, forming a dynamic protein coating called a protein corona (CORONA). While there are reports of its elimination in the liver and kidney metabolism system, patients undergoing this method have shown symptoms of liver problems and related enzyme alterations. This study aims to discern whether the impact of MNPs on liver enzymes significantly contributes to liver damage. The investigation focuses on the effects of magnetic nanoparticles (MNPs) on selected enzymes, including alanine aminotransferase (ALT), aspartate transaminase (AST), α-amylase, and lipase. Employing 104 experiments over a central composite design (CCD), the study evaluates the effects of agents on MNP and enzyme structure, stability, and properties: enzyme assay, electron microscopy, and circular dichroism of secondary structure after interaction with MNPs. The study’s findings unveil the intricate relationship between MNPs and liver enzymes, providing valuable insights for clinical practices and refining the safety profile of MRI. This comprehensive exploration contributes to our understanding of potential implications and aids in optimizing the use of MNPs in medical imaging for cancer survivors.

Introduction

Cancer survivors are at risk of cancer recurrence. They often undergo regular Magnetic Resonance Imaging (MRI) scans every six months to monitor disease progression, early detection of recurrence, assessment of treatment efficacy, identifying new lesions, patient reassurance, guiding post-treatment care, assessing treatment side effects and clinical trials [1,2,3]. This monitoring is particularly vital for cancers with a potential for recurrence or metastasis. A high-resolution MRI every six months allows intervals for cancer patients to serve as a proactive and comprehensive approach to their ongoing care [4, 5]. These scans are instrumental in monitoring disease status, detecting early signs of recurrence, assessing treatment efficacy, guiding post-treatment care, and contributing to both patient reassurance and advancements in cancer research [6,7,8].

In recent years, the integration of nanotechnology and high-resolution MRI has revolutionized medical diagnostics, offering unprecedented insights into cellular and molecular structures [9, 10]. At the core of this imaging innovation lies Magnetic Nanoparticles (MNPs), nanoscale wonders endowed with superparamagnetic properties [11]. MNPs, serving as contrast agents in MRI, play a pivotal role in enhancing image contrast and sensitivity, reshaping the landscape of diagnostic medicine [12]. While MNPs hold immense promise, concerns have surfaced regarding their potential toxic effects on the human body [13]. Studies have illuminated adverse outcomes associated with nanoparticle exposure, spanning from mitochondrial and chromosomal damage to protein denaturation [14]. Despite these findings, the intricate mechanisms governing these toxic functions remain elusive [15]. As MNPs are injected as contrast agents, their strong affinity for proteins and biomolecules, driven by their small size and high surface energy, sets the stage for intricate interactions within the body [16].

Upon injection into the bloodstream, MNPs exhibit a remarkable affinity for binding to proteins and other biomolecules [18], forming a dynamic protein coating known as the protein corona (CORONA) [17, 19, 20]. This protein-NP interaction extends to the formation of a protein coating (CORONA), with varying compositions based on nanoparticle characteristics. Previous research has explored the toxic effects of various metal and metal oxide nanoparticles on biomolecules, cells, and tissues [21,22,23].

Extending beyond theoretical studies, considerable research has delved into the toxic effects of exposure to diverse metal nanoparticles, including gold, silver, and metal oxide nanoparticles like ZnO and TiO2. Reports of elevated amylase, lipase, AST, and ALT levels in patients undergoing frequent high-resolution MRIs due to controlled cancers prompted our interest [24,25,26]. Our goal is to address whether the effects of MNPs on AST and ALT enzymes contribute to liver damage and symptoms of liver disease in individuals undergoing frequent high-resolution MRIs. Additionally, we aim to unravel the patterns of enzyme variations and their changes upon direct exposure to nanoparticles. As we embark on this investigative journey, it is crucial to contextualize the broader landscape of nanoparticle research. Previous studies have meticulously explored the toxic effects of various metal and metal oxide nanoparticles, shedding light on their impact on biomolecules, cells, and tissues. The intricate dance between nanoparticles and proteins has been the subject of intense scrutiny, revealing a spectrum of potential consequences.

Our ultimate goal is twofold: firstly, to study whether the MNPs has direct influence on liver enzymes, and secondly, comparing the selected enzyme direct reacting to these magnetic nanoparticles. These questions underscore the critical need to understand the nuanced interplay between MNPs and liver enzymes, potentially unlocking insights that could reshape our approach to high-resolution MRI protocols. Our findings aim not only to contribute to the growing body of knowledge in nanomedicine but also to inform clinical practices and enhance the safety profile of a widely employed diagnostic tool–MRI. Through this study, we aspire to bridge the realms of cutting-edge nanotechnology and clinical implications, offering a comprehensive perspective on the multifaceted relationship between MNPs, enzymes, and liver health.

Experimental

Materials

The enzymes including alanine aminotransferase (ALT- EC 2.6.1.2), aspartate transaminase (AST- EC 2.6.1.1), α-amylase (EC 2.6.1.2) and Lipase (EC 2.6.1.3) were purchased from Sigma-Aldrich Co. (Germany). HPLC purity water was used in all stages. The chemicals FeCl3·6H2O, FeSO4.7H2O, NaOH, ammonia, and citric acid were purchased from Merck Co. (USA). Enzyme measuring kits were prepared by Pars Azmoon Co. (Iran). Starch, phosphate buffer, 3,5-Dinitrosalicylic acid, and DMSO were purchased from Sigma-Aldrich Co., (Germany).

Nanoparticle synthesis and characterization methods

The synthesis reactions were performed in two-neck flasks with a capacity of 50 ml and a final reaction volume of 30 ml. The required solutions for the synthesis reaction include ferrous iron salt solution (245 μl of 36% hydrochloric acid and 1.65 g of FeSO4.7H2O added to 1.5 ml of distilled water, with a final volume of 3 ml), ferric iron salt solution (2.43 g of FeCl3.6H2O dissolved in water with a final volume of 2 ml), and 1 M NaOH (1 g of NaOH salt dissolved in 25 ml of distilled water) were freshly prepared just before the synthesis reaction. The reverse co-precipitation was conducted in all the treatments by suddenly adding the separately prepared iron salt solutions to the stirring alkaline solution. To this end, 25 ml of alkaline solution (ammonia 25% or 1 M NaOH) was charged in the reactor flask. The desired reaction temperature was adjusted to the required temperature (80 °C) while the solution was stirred under a steady flow of N2 atmospheric. The ferrous and ferric iron salt solutions were simultaneously added to the alkaline solution in the reactor, and the solution was then mixed by a magnetic mixer at a speed of 900 rpm. After adding the iron solutions to the alkaline medium, the reaction proceeded for 30 min under 900 rpm magnetic stirring. Thereafter, iron oxide particles were separated with a permanent magnet and the resulting supernatant was removed. The remaining precipitate was washed 3 times with 25 ml of ethanol (96% aqueous solution). For each washing step, the container was placed on a magnetic mixer at 900 rpm and 80 °C for 10 min. The magnetic particles were then separated by placing the reaction container on a permanent magnet and the resulting supernatant was removed and discarded using a pipette. The washing process for non-magnetic products was accomplished by centrifugation. After washing, 65 ml of 0.02 g ml−1 citric acid solution was added to the precipitate and the reaction pH was set to 5.4 by adding 25% ammonia. The temperature was raised to 80 °C under continuous stirring at 900 rpm for 90 min. At the end of the 90 min, a small amount of precipitate was collected and discarded. The pH of the obtained suspensions was adjusted to 7 by adding 25% ammonia, and the resulting suspensions were stored at 4 °C.

Statistical design of experiments

To study the influence of magnetic nanoparticles (MNPs) on selected enzymes, 104 experiments were to evaluate the two numerical factors of enzyme concentration (Unit/mL); A and Time (min); B and two categorical factors of MNP size distribution (nm); C and Enzyme type; D were designed using the central composite design (CCD) algorithm in 5 levels (Table 1). As can be seen in this table, five study levels have been considered for two quantitative factors a and b. The selection of these levels is to be able to examine their quantitative effect more precisely. This value is considered in the case of the concentration of the wires used to be 2–60 units/ml compared to the enzyme. In general, an MRI is performed 10 to 30 min after the injection of nanomaterials, and these materials interact with the body’s environment for a long time. The high intensity of this interaction is during the first two hours. The interaction time (factor B) is considered from 10 to 100 min. Injectable nanoparticles are generally smaller or larger than 100 nm. Since some reports of liver and enzyme-related problems have been reported in 6 month-evaluated patients, enzymes related to the liver system have been considered as a qualitative factor (D). For interaction experiments (Conditions that can be checked in Table-1-supplementary), a certain amount of enzyme and MNP were mixed in phosphate buffer (0.4% v/v) and incubated at 37 ℃ for a specified time (the times detailed mention in Table-1-supplementary). The sample was then used to study the detection of enzyme or nanoparticle changes. To optimize the factors, a rotatable and orthogonal CCD was applied using Design Expert® version 7.0.0. This design permitted the response surface to be modeled by fitting a quadratic polynomial equation. Forty-center point and sixty-four non-center point experiments were designed for a total of 104 experiments (Table-1-supplementary) performed to determine the effects of the factors on the MNP and the enzymes’ structure, stability, and features. To minimize the effects of uncontrolled variables, the experiments were performed based on the randomization law. In each designed experiment, the enzyme assay was used as a response.

Table 1 Factors together with their notations and levels for the central composite design. (α: 1.41)
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Enzyme analysis methods

Enzyme assay

Four types of enzymes and diabetes-related enzymes (mentioned in Sect. “Materials”) were examined in this study. One of the studies performed on enzymes after interaction with MNP is to investigate the percentage change in their activity. The measurement method is based on the consumption of NADH and its conversion to NAD+ (corresponding to the activity of the AST or ALT enzyme). For AST and ALT enzymes assay IFCC kit (Man Co., Iran) was used. For this measurement, in summary, 30 μL of oxoglutarate substrate (12.5% w/v) and 30 μL of alanine (or aspartate) substrate (8% w/v) were added to 30 μL of the mixture from the designed experiments (Table-1-supplementary). The basis of this reaction without the use of pyridoxal phosphate is as follows [27].

$$2 - {\text{Oxoglutarate }}\xrightarrow{{\begin{array}{*{20}c} {L - Aspartate + AST} \\ {or} \\ {L - Alanine + ALT} \\ \end{array} }}{\text{Oxaloacetate}}$$
$${\text{Oxaloacetate}}{\mkern 1mu} {\text{ + }}{\mkern 1mu} {\text{NADH}}{\mkern 1mu} {\text{ + }}{\mkern 1mu} {\text{H}}^{ + } \xrightarrow{{{\text{MDH}}}}{\text{L - Malate }} + {\text{NAD}}^{ + }$$

The NADH coenzyme concentration decrease is directly related to enzyme activity. The control medium in this experiment contains all substances except the enzyme and the standard sample used enzyme activity without treatment. The sample was kept at 37 °C for 5 min and then the absorbance value was checked at 340 nm [27].

For the α-amylase inhibition assay, 100 µl of each sample was mixed with 200 µl of the starch substrate solution (10% w/v starch and 17 mmol/L NaCl in phosphate buffer 20 mmol/L at 20 °C and pH 6.9. Then, 200 µl of α-amylase solution (2 mg/ml in phosphate buffer 20 mmol/L) was added. The mixture was incubated for 3 min at 20 °C. After adding 200 µl of DNS solution, the vials were incubated at 100 °C for 15 min, and finally, 1100 µl of distilled water was added to all vials. The absorbance of 250 µl of each sample (in 3 repetitions for each sample) was studied at 540 nm. The DMSO was used as a negative control. The amount of α-amylase inhibition was measured by Eq. 1 where Abln and Asmp represented the absorption of negative control and sample average, respectively [28, 29].

$$\alpha -\text{amylase inhibition \% }=\frac{{A}_{bln}- {A}_{smp}}{{A}_{bln}} \times 100$$
(1)

The Lipase activity was studied over the colorful reagent produced by the release of para-nitrophenol in the hydrolysis of 12 μM pNPP at pH 7.5 and 37 °C. For that, 20 μl of the lipase-nanoparticle was added to 460 μl phosphate buffer, containing 20 μl substrate. One unit was defined as the amount of enzyme necessary to hydrolyze 1 μmol of pNPP per minute per ml. The final mixture absorbance was analyzed at 410 nm as a lipase assay [30].

Enzyme second structure analysis

In addition to studying the enzyme activity, their secondary structure and CD spectrum variations of enzymes after interaction with MNP were evaluated. For this 215Model-Circular Dichroism Spectrometer (AVIV Biomedical, Inc., USA) containing a sample container with a length of 1 cm in the range of 4 nm to study the changes in the polarized light pattern was used.

Nanoparticle analysis methods

The nanoparticles before and after interaction with enzymes were characterized through the investigation of suspension appearance, ultraviolet–visible (UV–Vis) spectrum (Rayleigh instrument, Model UV2601, China, in the range of 190 nm to 1100 nm with a resolution of 0.3 mm), dynamic light scattering (DLS) measurements were performed at 25 °C using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., UK, equipped with a helium–neon laser and a scattering angle of 173°), field emission surface electron microscopy (FESEM) study (SU3500, Hitachi Co., Japan), vibrating sample magnetization (VSM) analysis (Ametek Inc., Model No. 155, USA), and 2-theta X-Ray powder diffraction (XRD) analysis ( RINT/DMAX 2200 H/Pc, Japan).

Three-D structure and electrostatic potential analyses

The 3D structure of the alanine aminotransferase (ALT EC 2.6.1.2), aspartate transaminase (AST EC 2.6.1.1), α-amylase (EC 2.6.1.2) and Lipase (EC 2.6.1.3) were analyzed by using the SWISS-MODEL program [https://swissmodel.expasy.org/ (accessed on 10 April 2023)]. Positive and negative potentials are drawn in blue and red, respectively.

Results

In this comprehensive study, magnetic nanoparticles (MNPs) were synthesized and their intricate interactions with body enzymes were meticulously investigated. Utilizing advanced statistical experimental software, we systematically examined the multifaceted changes and forecasted variations in design conditions. The analytical results, elucidating the complex interplay, can be found in Table 2 and Fig. 1.

Table 2 Analysis of variances (ANOVA) of Enzyme-MNP interaction
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Fig. 1
figure 1

Response Surface diagram of enzyme assay due to the simultaneous effect of interaction time and the ratio of MNPs to enzyme (A, B) in the case of MNPs larger (A–D) and smaller (E–H) than 100 nm in case of and ALT (A , E), amylase (B, E ), AST (C, G) and lipase (D and H). The activity percentages of ALT (I), AST (J), amylase (K), and lipase (L) in the period of 10–100 min in the conditions without interaction (●), interaction with MNPs larger (■) and smaller ( ×) than 100 nm

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To imprisonment these variations, characteristics of MNPs such as size, size distribution, crystallinity, and magnetism were scrutinized. Concurrently, enzymes underwent thorough analysis, assessing alterations in secondary structure and enzymatic activity. Subsequently, molecular enhancers were explored to deepen our understanding of the intricate enzyme-nanoparticle interaction. Before delving into the reciprocal effects of nanomaterials and enzymes, the synthesized nanoparticles underwent meticulous characterization. Based on the results, two batches of MNPs were successfully synthesized: one with a size smaller than 100 nm (Fig. 2A) and another with a larger size (Fig. 2F). Based on the DLS results, the statistical values of the obtained peaks are 110 nm (98.1%, ± 0.1 nm, PDI: 0.01) and 61.98 nm (96.2.1%, ± 0.1 nm, PDI: 0.02). These particles exhibited uniform distribution and distinct crystal lattice, aligning with prior crystallographic findings (Fig. 2K, L). Notably, both types of synthetic particles displayed a spherical shape and smooth surface morphology without pores, as depicted in Fig. 3A, F. Additionally, both particle types exhibited permanent and reversible magnetization, with the magnetization range for smaller nanoparticles (100 nm) being 0.2 emu wider than their larger counterparts, as illustrated in Fig. 3K, L. This nuanced exploration sets the stage for a comprehensive understanding of the dynamic interplay between MNPs and enzymes, offering insights into potential applications in various fields.

Fig. 2
figure 2

DLS results of synthesized MNPs smaller (A) and larger (F) than 100 nm before interaction with enzyme and after interaction over AST (B , G), ALT (C, H), amylase (D, I) and lipase (E, J). X-ray crystallographic results of MNPs smaller (K) and larger (L) than 100 nm

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Fig. 3
figure 3

SEM results of synthesized MNPs larger (A) and smaller (F) than 100 nm before interaction with enzyme and after interaction over AST (B, G), ALT (C, H), amylase (D, I) and lipase (E, J). The MNPs magnetization (VSM) features and variations of smaller (K) and larger (L) than 100 nm over enzyme interaction

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To explore the intricate changes occurring in nanoparticles and enzymes upon direct interaction, an extensive experimental design using Design Expert 0.0.7 was employed. A total of 104 experiments (Table-1-supplementary) were meticulously crafted based on the factors outlined in Sect. "Statistical design of experiments". The resulting analysis of variance (ANOVA) data, as presented in Table 2, establishes the model’s reliability and significance at a confidence level of 99.5%, with a remarkably low P-value of 0.0001. Significant single-factor parameters include the interaction time of nanoparticles with enzymes (A), the ratio of nanoparticles to enzymes (B), the size of nanoparticles (C), and the type of enzyme (D). Among the six two-factor parameters, the most crucial factors influencing the nanoparticle-enzyme interaction are the interaction of nanoparticles with enzyme and the type of enzyme (AD) and the time interaction of nanoparticles with enzyme and the type of enzyme (BD). The central composite design method’s advantage is evident in these two-factor parameters resulting from the interaction of single-factor parameters. Table 2 delves into the model’s proportionality and accuracy. The lack of significant lack of fit indicates the absence of one-sided errors. Additionally, the close alignment between the degree of conformity and data convergence (R-Squared: 92.05%) and the conformity of the data with the designed and predicted theoretical model (Adj R-Squared: 91.62%) underscores the model’s robustness and reliability. The presented equation in this table serves as a mathematical model for predicting laboratory results. Based on this model, the effective laboratory conditions influencing the interaction of MNPs and enzymes were predicted and thoroughly investigated, providing valuable insights for future experimental endeavors.

The primary and initial impact of MNPs on enzymes is reflected in their activity (Fig. 1A, H). Results indicate that, within the first 30 min of interaction, lipase activity increases, while other enzyme activities decrease (Fig. 1L). Notably, amylase shows a slight increase during short-term interactions with MNPs smaller than 100 nm, which may be considered negligible (Fig. 1K). Upon extending the interaction time to approximately 1 h, a more detailed analysis reveals a significant decrease in the activity of both aspartate amino transferase (AST) and alanine amino transferase (ALT) (Fig. 1I, J). Contrarily, amylase activity increases over the prolonged interaction period. Lipase exhibits diverse behavior, with its activity showing an increase for MNPs smaller than 100 nm and a decrease for those larger than 100 nm over an extended period (Fig. 1D, H).

The activity of enzymes, except for lipase, decreases with prolonged interaction. Lipase, however, initially experiences a more severe decline in activity, which is later compensated over time (Fig. 1D, H). Increasing the nanoparticle-to-enzyme ratio intensifies the decrease in AST and ALT enzyme activities, with these trends persisting regardless of particle size. The activity of all four enzymes decreases post-interaction with nanoparticles, and over time, enzyme activity further diminishes. The most substantial decrease in enzyme activity after nanoparticle interaction is observed with the AST (Fig. 1C, G), while the lowest decrease is associated with the amylase (Fig. 1B, F). This study enhances our understanding of the interaction interplay between nanoparticles and enzymes and offering valuable insights related to this interaction.

Following the interaction of MNPs with enzymes, the overall distribution range of particles increases (Fig. 2A–J). This is much more the case for MNPs smaller than 100 nm (Fig. 2G–J). This change and dispersion is more in interaction with lipase and then AST (Fig. 2G, J). Nanoparticles smaller than 100 nm show less distribution change in interaction with lipase and amylase (Fig. 2D, E). More changes occur in nanoparticles smaller than 100 nm in interaction with ALT (Fig. 2C). Both nanoparticle sizes experience the most significant decrease in magnetism after interacting with the AST enzyme, with ALT and lipase being the most effective enzymes for smaller and larger nanoparticles, respectively (Fig. 2A–J). Vitiations on the crystal structure (Fig. 2K, L) and magnetization of MNPs (Fig. 3K, L) following interaction with enzymes, respectively. Both nanoparticle types are influenced after interaction with AST and ALT enzymes. Nanoparticles smaller than 100 nm retain some crystalline structure after interacting with AST and ALT, while they become entirely amorphous after interaction with lipase and amylase enzymes (Fig. 2L). Larger nanoparticles (> 100 nm) exhibit a slightly crystalline structure after lipase interaction but generally become amorphous and irregular when interacting with other enzymes (Fig. 2K).

In the examination of particle size changes, scanning electron microscopy (SEM) was employed to analyze alterations in the nanoparticle surface structure (Fig. 3A–J). Notably, the nanoparticles exhibit adherence to AST and ALT enzymes post-interaction. Larger nanoparticles (> 100 nm) undergo significant morphological changes and adhesion when interacting with amylase and lipase enzymes (Fig. 3A–E). In contrast, nanoparticles smaller than 100 nm show less severe changes in interaction with these enzymes (Fig. 3F–J). The last change in magnetism occurs after interaction with the amylase (Fig. 3K, L). In general, MNPs larger than 100 nm undergo more aggregation and accumulation after interaction than the larger types (Fig. 3A–E). Among the types of enzymes, amylase and lipase (Fig. 3D, E) have the most interaction, influence, and effectiveness related to amylase and lipase. The AST enzyme has the least change in their activity after this interaction on MNPs (Fig. 3B).

Circular dichroism (CD) spectroscopy was employed to monitor secondary structural variations in enzymes following optimal interaction conditions (Fig. 4A–D). The results illustrate the structural alterations in the enzyme secondary structure post-interaction with nanoparticles. Lipase undergoes displacement in the α-helix structure, accompanied by drastic variations in β-sheets when interacting with MNPs smaller than 100 nm (Fig. 4A). For amylase, there is a slight reduction in β-sheets intensity and displacement under treatment with MNPs smaller than 100 nm, with this displacement being more intense compared to larger nanoparticles (Fig. 4B). In the case of AST, interaction with MNPs larger than 100 nm results in intense β-sheets variations (Fig. 4C). Moreover, MNPs smaller than 100 nm lead to decrements in α-helix and β-sheets intensity in ALT (Fig. 4D). Overall, the secondary structural variations across all four enzymes reveal that MNPs smaller than 100 nm exert a more intense influence on enzymes compared to their larger counterparts. This nuanced exploration enhances our understanding of the intricate interplay between MNPs and enzymes, shedding light on potential applications and avenues for further research.

Fig. 4
figure 4

Circular dichroism (CD) of lipase (A), amylase (B), AST (C) and ALT (D) before and after interaction with MNPs larger (dashed dot) and smaller (dashed line) than 100 nm and the in-silico analysis of polarization and surface charge of lipase (E), amylase (F), AST (G) and ALT (H) using SWISS-MODEL

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Discussions

To investigate the effect of the interaction of nanoparticles with enzymes in their activity, we can pay attention to how the active centers of these enzymes are located in the second structure. Studies have shown that the active site of the lipase enzyme is located between the beta plates and is protected in the outermost layer by alpha helices. In the structure of this enzyme, the lip domain is located near the alpha helices. In amylase, the active part is the endogenous enzyme and is not readily available by nanoparticles. In AST and ALT enzymes, the active part is in the alpha helix. As reported in the results of Fig. 1, most of the lipase enzyme changes are in the range of beta plates and complete alpha-helix displacement. Thus, it is not far from the mind that the activity of this enzyme changes drastically due to its interaction with nanoparticles. However, the enzyme amylase has an active part that is out of reach, and therefore, in general, its effectiveness will be less than nanoparticles. Both AST and ALT enzymes are subject to alpha-helix and beta-sheet shifts. AST enzyme interacts with larger nanoparticles and ALT enzyme interacts with smaller nanoparticles. In general, among these four enzymes studied, lipase showed the most severe structural changes after interaction with nanoparticles. The general results of the study of enzyme activity in 30 min after interaction with nanoparticles showed that in a short time, the activity of AST and ALT amylase decreased and lipase activity increased. However, increasing the reaction time to about one hour does not affect the pattern of changes in the activity of AST and ALT enzymes and we are still facing a decreasing trend in the activity of these enzymes. Also, with increasing the duration of interaction, the activity of the amylase enzyme increases, and the activity of the lipase enzyme decreases. This is also consistent with the findings of crystallography and magnetization of nanoparticles. These results showed that the amylase enzyme had the least effect on the magnetization of nanoparticles smaller than 100 nm. While the two enzymes AST and ALT have the greatest effect on the magnetization changes of nanoparticles. This was also confirmed in Fig. 1 and the study of the activity of enzymes. The amylase enzyme showed the lowest activity of nanoparticles. The results of the study of nanoparticle size in interaction with enzymes were performed by DLS and SEM methods. Figures 3 show that nanoparticles undergo strong interaction with the ALT and AST enzymes and accumulate and change their morphological structure. However, nanoparticles are less susceptible to lipase and amylase, but their adhesion and morphological change cannot be ignored.

The MNPs have a positive electric charge. One of the hypotheses in the interaction of these nanoparticles with enzymes is their interaction with the positively or negatively charged part of the enzymes. It can be assumed that if the active part of the enzyme is in the negatively charged part, the probability of being affected by the positively charged magnetic nanoparticles and the decrease in enzyme activity will be higher. In Fig. 4E–H, the positively charged and negatively charged parts of the studied enzymes are marked bioinformatically in blue and red, respectively. As can be seen in the amylase enzyme, the polarized positively and negatively charged parts are dispersed proportionally throughout the molecule, making serious interaction of the nanoparticles with this enzyme less likely than the negatively charged part. The lipase enzyme has two polarizations and the negative part is completely separated from the positive part. Therefore, there is a high probability of a positively charged nanoparticle reacting with a negatively charged portion of this enzyme. Other results showed that lipase and nanoparticles had the greatest effect during interaction with each other. AST enzyme has more negative charge centers than ALT enzyme. While ALT enzyme has separate parts of positive and negative charge and the active part of this molecule is inside the positive part. Thus, the effect of the AST enzyme and magnetic nanoparticles on each other is more possible and more intense than the ALT enzyme.

Conclusions

The journey into the realm of nanoparticle-biomolecule interactions has unveiled a wealth of information, yet significant knowledge gaps persist. Future research endeavors should focus on elucidating the specific mechanisms of enzyme damage, refining our understanding of the protein corona’s dynamic nature, and exploring strategies to modulate these interactions for enhanced safety and efficacy. In conclusion, the integration of Magnetic Iron Oxide Nanoparticles in medical imaging not only transforms diagnostic capabilities but also unravels a complex nexus of interactions within the human body. The formation of protein corona and potential enzyme damage underscore the need for a nuanced understanding of these processes. ALT and AST, as sentinel enzymes for liver health, provide a clinical lens through which to view the intricate interplay of MIONs in cancer survivors.

As we embark on further exploration, it is essential to bridge the knowledge gap, refining our understanding of the molecular consequences of nanoparticle-biomolecule interactions. The future holds the promise of safer and more effective medical imaging practices, where the transformative potential of MIONs can be harnessed while ensuring the utmost care for patient well-being. Based on the results, it could be concluded that:

  1. 1.

    Magnetic nanoparticles in interaction with AST, ALT, lipase, and amylase enzymes cause changes in the secondary structure and activity of these enzymes.

  2. 2.

    Enzymes change the crystal structure, size, morphology, and magnetization of these nanoparticles during their interaction with magnetic nanoparticles.

  3. 3.

    It can be said that the degree of effect after the interaction of magnetic nanoparticles and enzymes on each other is related to the charged and available levels of enzymes and their type of charge.

Also, considering the effectiveness of enzymes studied in this study with magnetic nanoparticles, it can be assumed that these nanoparticles after injection increase the resolution of MRI images, affect the profile of blood enzymes, and cause structural and functional damage.

Data availability

The data that support the findings of this study are available on request from the corresponding author (fateme.mirzajani@gmail.com). Also “Table 1-supplementary” can be used as an online validated statistical support.

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Acknowledgements

The results presented in this article relate to the research project at Shahid Beheshti University with the number of Sad/600/1449. The authors have no conflict of interest.

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Authors and Affiliations

  1. Protein Research Center, Shahid Beheshti University, Po Box 1983969411, Tehran, Iran

    Fateme Mirzajani, Azam Rostamzadeh & Zepiur Tahmasian

  2. Department of Medical, Hammurabi College of Medicine, University of Babylon, Hillah, Iraq

    Heydar Sudan Abbas Obaid

  3. School of Chemistry, University of New South Wales, Sydney, NSW, 2052, Australia

    Seyed Mohammad Motevalli

  4. Knowledge University, College of Science, Erbil, 44001, Iraq

    Fateme Mirzajani

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  1. Fateme Mirzajani
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Correspondence to Fateme Mirzajani.

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Mirzajani, F., Rostamzadeh, A., Tahmasian, Z. et al. Effects of MRI magnetic iron oxide nanoparticles on the structural and enzymatic properties of liver-related enzymes. Micro and Nano Syst Lett 12, 13 (2024). https://doi.org/10.1186/s40486-024-00200-6

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