Cardioprotective role of biosynthesized gold nanoparticles compared to an iodinated-based x-ray contrast agent in Male Wistar Rats

INTRODUCTION

As clinical experiences broaden human knowledge, changes in diagnosis and treatment are required, thus imaging is employed to accurately diagnose health problems [1, 2]. However, the similarity in the physical characteristics of some anatomical features may make those features not to be distinguished radiographically from their surrounding tissues. Hence, to assist medical practitioners in efficiently visualizing abnormality and to achieve high-resolute images, contrast agents (CAs) are used in different x-ray imaging modalities, with the existing CA (ECA) based on iodine and barium molecules [1 - 4].

Upon administration, the distribution of CA is regulated by the cardiovascular system [5], however, its circulation may affect and perturb the hemodynamic of the cardiovascular system [6] and other associated organs through which the substance traverses. An action that may be followed by destruction in free radicals, upsurge in the activity of cardiac indicators, and pro-inflammatory cytokines [7], as well as oxidative stress, calcium overload, injury to the myocardial and endothelial cells, contractile dysfunction, necrosis, and/or apoptosis-induced cell death [8]. Hence, cardiovascular disease (CVD) is the leading cause of death worldwide, claiming about 17.9 million lives a year [9] and this is a major cause of concern regarding the use of CA. More so, reports from previous studies have it that, over the last three decades, there has been no fundamental improvement in the currently used CAs [10], despite their limitations in medical imaging [11].

With the incorporation of nanomaterials into medicine, several progresses have been made in the development of materials and devices suitable for medical applications. Of utmost interest in this study is the call for the use of nanoparticles (NPs) as CAs in x-ray imaging to overcome the limitations of the ECAs [11, 12]. One of the NPs that has gained significant attention as a promising CA is gold nanoparticles (AuNPs). This is because AuNPs is very easy to synthesize, its properties can be controlled, high attenuation coefficient at the diagnostic energy level, and biocompatibility. Gold (Au) has a high atomic number (Z = 79) compared to iodine (Z = 53) and barium (Z = 56) and thus absorbs more x-rays at specific energy levels [13]. Various methods of synthesizing AuNPs are documented in the literature in terms of control over size, morphology, and surface chemistry, most of which involve the use of physical or chemical methods. However, the use of expensive technologies and non-biocompatibility challenges associated with the physical and chemical methods have resulted in studies suggesting the use of biological methods [14].

The biological method involves the use of microorganisms and plants, with plants and plant materials having a superior advantage over other biological processes. This is because synthesizing NPs from plants eliminates the process of maintaining microorganisms, the process do not need multi-step procedures [15, 16], and can be easily scaled up for large-scale production [17]. In addition, the abundance of several phytochemicals and antioxidants present in Psidium guajava (guava)) and Corchorus Olitorius (Jute mallow) have been found to be beneficial medically [18, 19]. Other studies have also demonstrated that these plants exhibit anti-cancer, anti-inflammatory, and anti-proliferative activities in various in-vitro and in-vivo evaluations [18 - 20].

This study, therefore, synthesize AuNPs using the extract of P. guajava and C. olitorius and their properties were determined using different spectroscopy and microscopy techniques. The performance of an existing iodine-based CA and the synthesized AuNPs were thereafter evaluated on cardiac biomarkers of rats exposed to different tube voltage of x-rays. The novelty of the study lies in the use of orthodox plants (P. guajava and C. olitorius) that are readily available, cheap, and of high medicinal value in synthesizing AuNPs for use as a contrast agent, in order to disperse the fear arising from the cellular toxicity, mutagenicity, or genotoxic effects of chemically prepared AuNPs in medical imaging applications. The synthesis of AuNPs using plant extract has been reported for different biological applications such as cardioprotective [21], antiparasitic [22, 23], antioxidant [24, 25], anticancer activities [26 - 29], antibacterial [25, 30] and many more. However, to the best of our findings, this is the first study comparing the cardioprotective role of biosynthesized AuNPs and an existing iodinated CA.

MATERIALS AND METHODS

The procedure followed in conducting the experiment are as shown in Fig. 1.

Preparation of extracts

Leaves of P. guajava and C. Olitorius were obtained from the Teaching and Research Farm of Ladoke Akintola University of Technology, Ogbomoso. The collected plant samples were rinsed with distilled water, air dry, pulverized, and passed through a 2 mm mesh. 10 g of each powdery sample was dissolved in 200 ml of 70% ethanol solution and stirred for 5 mins, the mixture was thereafter sealed with aluminium foil and heated for 5 min at 50 ͦC to aid the formation of aqueous extract. The resulting solution was allowed to cool for 24 h and then filtered through Whatman No. 1 filter paper, and stored in a cleaned bottle pending further usage.

Synthesis

1 mL of each prepared extract was added to 20 mL 1 mM HAuCl₄ (prepared from the stock salt of tetrachloroauric (III) acid trihydrate (HAuCl₄.3H₂O), Sigma-Aldrich and used as received), and stirred using a magnetic stirrer. The color change was monitored before the reaction was further subjected to UV-Vis spectrophotometry analysis to investigate its absorbance. The reaction was modulated by further increasing the amount of the extract stepwise from 2 – 5 mL against that of the precursor (20 mL). The most optimized of each synthesized solution was thereafter subjected to other characterization.

Characterization

The prepared solutions were subjected to absorption spectroscopy using BIOBASE^® UV-Vis spectrophotometer in the scanning range of 200 – 800 nm. The functional biomolecules was identified using Nicolet iS10 FTIR spectrometer at the range of 400 – 4000 cm⁻¹. The crystalline structure was determined using Rigaku MiniFlex Benchtop X-ray diffractometer operated at a voltage of 40 kV and current of 20 mA with a CuK∝ (λ = 1.54056 Ǻ) radiation source. The elemental composition of the samples was undertaken under a scanning electron microscope JOEL JSM-7600F equipped with an energy-dispersive x-ray EDAX spectrometer operated at 15 kV. The morphology of the synthesized AuNPs was identified under JEOL JEM-2100 transmission electron microscopy (TEM), operated at 300 kV.

Animals

One hundred and fifty-six (156) male Wistar rats (270 ± 25 g) were used in this study. The animals were kept at room temperature of twelve hours (12 h) natural light-dark cycle and fed daily with animal pellet feed and water ad libitum. The rats were allowed to acclimatize. The Research Ethics Committee, Ministry of Health, Oyo State, Nigeria approved the experimental procedures and the animals received humane care in compliance with laid down guidelines as outlined in the guide for the care and use of laboratory animals [31].

Experimental Design

The rats were categorized into two: the first category is the unexposed and tagged group A. The rats in the second category were exposed and it consists of four (4) groups tagged: B, C, D and E. Group A served as the baseline control (BC) and consist of twelve (12) rats; the rats in this group were not administered any of the substances nor exposed to x-ray. Groups B, C, D, and E consist of twelve (12) rats in triplicate. The rats in group B serve as the experimental control (EC), as no substances were administered to the rats. The rats in group C were injected with the existing iodinated contrast agent (Urografin 76%, Berlimed S. A.) while those in groups D and E were administered the synthesized plant-based AuNPs from the extract of Psidium guajava and Corchorus Olitorius respectively. The substances were administered intraperitoneally at 6 mL/kg body weight of the rat [32 – 35].

X-ray irradiation

The rats in groups B, C, D and E were restrained inside an acrylic rectangular box and irradiated at the tube voltage of 40, 80 and 120 kV of x-ray respectively, at an exposure current 8 mAs with the distance from source to the subject (rats) of 1 m using MARS Fixed X-ray, Allengers Medical Systems Ltd.

Collection of Samples

Six rats from each group/replicate were sacrificed on day 1 of the treatment and four weeks post-treatment (day 30) through cervical dislocation and samples of blood were collected via cardiac puncture. 3 mL of the blood was dispersed into lithium heparin tubes, allowed to clot for 45 min at room temperature, retracted and centrifuged at 3000 rpm for 15 minutes using a centrifuge to obtain serum. The heart was excised and divided into two equal halves. One portion of the tissue was immediately fixed in 10% formal saline to preserve the organ pending histopathology examination. The remaining half was washed in normal saline, homogenized on ice with 10 mM phosphate buffer (pH 7.4) at 5 ml of tissue weight, and thereafter centrifuged at 10,000 rpm to obtain supernatant. The serum and tissue supernatant was stored at -80°C pending analysis for the level of biomarkers activity.

Evaluation of Biomarkers activity

Samples of serum and tissue homogenate were retracted, and an aliquot of each sample was investigated for the markers of oxidative stress (superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA)); cardiac (creatine kinase (CK), troponin T (TnT), lactase dehydrogenase (LDH)) and inflammatory (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF- α) and caspase-3 (CAP3)). Each parameter was analyzed with appropriate commercially available kits following the protocols provided by the manufacturer.

Histopathology evaluation

Histopathological examination of the fixed heart tissue was undertaken to determine any abnormalities in the morphology of the heart and this was carried out following standard histopathology procedure.

Data Analysis

GraphPad Prism software (version 9.1.0 (221)) was used for the analysis of the data obtained. The results were presented as mean ± standard error of the mean (SEM). The data were also subjected to analysis of variance (ANOVA) with Dunnett’s multiple comparisons post-hoc test at 0.001, 0.01, and 0.5 levels of significance.

RESULTS AND DISCUSSION

Absorbance spectra

The absorbance spectra revealed that there is a rapid decrease in the intensity of the AuNPs capped with P. guajava extract as the amount of the extract increases from 1 – 5 mL (Fig. 2a), while a gradual increase in the intensity of the C. Olitorius capped AuNPs (Fig. 2b) was obtained until a stable peak was reached at 3 mL before the intensity began to fall. 1 mL of P. guajava and 3 mL of C. Olitorius extract were found to be sufficient for the completion of bioreduction and therefore found to be the optimum for the formation of AuNPs. The surface plasmon resonance (SPR) peak of 534 nm and 538 nm was obtained for the AuNPs capped with the extract of P. guajava and C. Olitorius respectively, both of which fall within the characteristic range 510 – 560 nm of SPR band for AuNPs [36, 37].

Functional biomolecules

The spectra obtained for the biomolecules responsible for capping the AuNPs revealed the presence of different functional groups and their involvement in the formation of nanomaterials (Fig. 3). For the AuNPs capped with P. guajava (Fig. 3a), the peak observed at 3482 cm⁻¹ could be assigned to stretching vibrations of –OH groups signifying the presence of alcohols, phenols, and flavonoids.  The The peak is not in the spectrum; the peak at 1648 cm⁻¹ is due to the C=O stretching of the carbonyl and carboxylic groups present in the amide linkage of the proteins, the peaks at 1508 could be assigned to the symmetrical stretch of carboxylate group; and the peaks at 1154 cm⁻¹ is attributed to the C–O stretching of polyols, ether and alcoholic groups, and bending vibration of C–N. For the AuNPs capped with C. Olitorius (Fig. 3b), the major stretching appeared at 3000 – 3500 cm⁻¹ and indicate the presence of -OH groups. The peaks at 2422 cm^-1 indicate the presence of the amine linkage of protein and the peak at 1646 cm^-1 was assigned to the C=O stretch. The C-C aromatics stretch was observed for both spectra at the region of 1416 cm⁻¹. Finally, C-O-C stretch can be found in the region of 1302 cm⁻¹. The peak at 1070 cm^-1 could be assigned to the -OH vibrations of the proteins in the plants.

Crystalline structure

The diffractogram revealed a diffraction pattern of three (3) peaks at 2θ = 38.45˚ (111), 44.46˚ (200), and 64.75˚ for the AuNPs synthesized from P. guajava (Fig. 4a), while four (4) peaks at 2θ = 38.23˚ (111), 44.42˚ (200), 64.44˚ (220), and 77.39˚ (311) was obtained for the AuNPs synthesized from C. Olitorius (Fig. 4b). All the peaks revealed the structure of synthesized AuNPs to be face-centered cubic (FCC) structure of elemental gold as conformed with the standard database file of the Joint Committee on Powder Diffraction Standards (JCPDS, file number 00-004-0784). These peaks also conformed with the FCC structures of other green synthesized AuNPs reported in different studies [19, 38 – 42].

Elemental composition

Fig. 5 presents the EDX spectra obtained for the two different AuNPs. The spectra revealed the elemental composition of the synthesized AuNPs consisting of elemental gold (Au) which accounted for over 70% of the weight in both the analyzed samples. The spectra also revealed the presence of other elements such as C, O, Si, K, and Na, all of which may be associated with the elemental contents of the P. guajava (Fig. 5a) and C. Olitorius (Fig. 5b). Thus, the elemental composition obtained reflected the composition of the AuNPs. This elemental composition also aligned with the crystal structure obtained for the nanoparticle, both of which confirmed that AuNPs have been truly synthesized.

Morphological feature

Fig. 6 revealed the micrograph for the shape and the histogram distribution of the particle size for the synthesized AuNPs capped with the extract of P. guajava and C. Olitorius respectively. The micrographs revealed the synthesized AuNPs are spherical in shape. The image analysis of the micrograph revealed the particle size to be in the range 5 – 11 nm with an average diameter of 8.23 ± 1.03 nm for the AuNPs capped P. guajava (Fig. 6a) and 3 – 10 nm with an average size of 6.57 ± 1.62 nm for the AuNPs capped C. Olitorius (Fig. 6b), both of which showed highly distributed nanoparticles.

Effect on biomarkers of heart

The results obtained for the biomarkers of the rat’s heart on day 1 and 30 are presented in Tables 1 to 3 respectively. It was observed that at each of the x-ray tube voltage (40, 80 and 120 kV), there was a significant difference in the level of oxidative stress markers (Superoxide Dismutase (SOD), Catalase (CAT), Glutathione (GSH), Malonaldehyde (MDA)); cardiac markers (Creatine Kinase (CK), Lactate Dehydrogenase (LDH) and Troponin T (TnT)) as well as the inflammatory markers (Interleukin-6 (IL-6), Tumour Necrosis Factor-alpha (TNF-α); Caspase-3 (CAP3)) compared to those of the normal control (group A), experimental control (group B, x-ray only) and group C (Urografin) respectively.

Exposure of the heart to a foreign substance, whether toxic or not may produce oxygen-derived free radicals termed reactive oxygen species (ROS). These species namely hydroxyl radical (OH⁻), superoxide radical anion (O²⁻), and hydrogen peroxide (H₂O₂) cause severe damage to macromolecules, tissues, and organs through the process of lipid peroxidation, protein modification, and deoxyribonucleic acid (DNA) strand breaks [43, 44]. An imbalance between the production of ROS and the inability of the antioxidant systems to readily detoxify these reactive intermediates results in oxidative stress.

The importance of oxidative stress is commonly emphasized in the pathogenesis of various degenerative diseases, such as cardiovascular disorders or neurodegenerative diseases, diabetes, cancer [45 – 47]. Protective actions against ROS are performed by enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). Thus, whenever, the level of these antioxidants system decreases, the level of inactivated ROS rises, resulting in the ROS impairing the function of cardiac muscle cells, as it induced an energy deficit by affecting the function of proteins involved in energy metabolism and ultimately result in cardiac dysfunction and potential heart failure [48].

Lipid peroxidation is also a significant determinant of the degree of free radical generation, with MDA being one of the products and an important marker of the process of oxidative stress [49, 50]. Lipid peroxidation leads to the generation of free radicals which cause cell damage and lead to the release of marker enzymes. When the liver and kidney cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released into the bloodstream. Thus, forcing the cardiac muscle to be significantly stressed.

Cardiac markers are endogenous substances released during circulation whenever the heart is damaged or stressed. The CK is mainly found in all muscle (cardiac and skeletal) and brain tissues. It plays an important role in energy storing mechanism of the tissues. Increased levels are found in myocardial infarction (MI), muscular dystrophy, cerebrovascular-disease, pulmonary infarction, electrical shocks and hypothyrodisim. The LDH enzyme is found in all organ cells, but especially in cardiac and skeletal muscle, liver, kidney and red blood cells (RBCs). Elevated levels are also found in MI, liver diseases, hemolytic anaemias, pernicious anaemia, leukemia and pulmonary diseases.

Troponin is the most sensitive and specific test for myocardial damage because it has increased specificity compared with CK and LDH. Troponin is released during MI from the cytosolic pool of the myocytes and its release is prolonged with the degradation of actin and myosin filaments. The differential diagnosis of TnT elevation includes acute infarction, severe pulmonary embolism causing acute right heart overload, heart failure, and myocarditis [51]. Inflammation is a normal biological response of the body to physical or chemical agents, tissue damage, and infections in which the production of inflammatory mediators such as cytokines, and ROS is triggered. If not controlled, these inflammatory mediators are over-produced which can induce pathological processes linked to several chronic conditions [52].

Photomicrograph of the organs

The photomicrograph of the rat’s heart tissue sections stained by hematoxylin and eosin (H & E) are shown in Fig. 7. The cellular structures of the heart showed a normal epicardial layer, with the myocardial layer showing a focal area of mild degeneration and inflammatory cells in Group A and moderate congestion in Group B compared to those of groups C, D and E. All the groups had normal myocytes. The photomicrograph of group C revealed moderately infarcted myocytes in the myocardial layer while those of Groups D and E revealed normal epicardial layer, myocardial layer and myocytes.

Thus, it was observed that the antioxidant defense mechanisms of the rats in group C is low while those of groups D and E is high compared to those of group A (baseline control). Likewise, it was also observed that the SOD and MDA level of the rats in group B (experimental control) has the highest value. Similar pattern was observed in the cardiac and inflammatory markers of the rats injected with the synthesized AuNPs compared to those of groups B and C. This is an indication that x-ray induces stress on the myocardium by producing more ROS an action that may lead to myocardial ischemia or infarction. However, the administered substances reduce the stress by producing antioxidants to detoxify the free radicals with the rats administered with the plant-based AuNPs producing more antioxidants compared to those administered with the iodinated CA (Urografin). 

Thus, with the higher atomic number and absorption coefficient of gold (Au) compared to those of iodine (I), Au provides about 2.7 times greater contrast per unit weight than I, and to which there was no evidence of toxicity at 6 mL/kg body weight of the rat on the cardiac cells. The rats injected with the plant-based AuNPs showed positive significant antioxidant, cardiac and inflammatory effects than those injected with the iodinated CA (Urografin) by protecting the heart. Hence, it can be inferred that the cardioprotective role of biosynthesized AuNPs may be attributed to the abundance of antioxidants present in the leaves of P. guajava and C. Olitorius [53]. The findings in this study conformed with the report of Vinodhini et al reporting the cardioprotective potential of biobased AuNPs from proanthocyanidin [21] and Mehanna et al reporting a significant decrease in antioxidant and inflammatory indicators when investigating the effects of AuNPs shape and dose on immunological, hematological, inflammatory, and antioxidants parameters in male rabbits [54].  The present study also confirms the hypothesis that the biomedical applications of AuNPs vary according to the stabilizing molecule.

CONCLUSION

Thus, while several studies are proposing the utilization of AuNPs as a novel CA for x-ray imaging, the findings in this study demonstrated that biosynthesized AuNPs will be useful as CAs and offer significant advantages over the existing iodinated CA without affecting the hemodynamics of the cardiovascular system. The single-step green process has proven to be effective for the synthesis and stabilization of biocompatible AuNPs as the phytochemicals and antioxidants present in the assayed plants protect the cardiac cells from all forms of cardiovascular injury. Hence, the incorporation of nanotechnology into medicine will be helpful in the development of new drugs for various biomedical applications. The findings in this study serve as a yardstick for further studies and will also aid in the use of biosynthesized AuNPs for several biomedical applications.

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

Conceptualization and study design: APS, IGA, AMK.

Methodology: APS, IGA, SWA, AMK, SYK

Data analysis: APS, IGA, SWA, AO, AAG.

Visualization: APS, IGA, SWA, LMK and AAG.

Supervision: IGA, AMK, and SWA.

Funding acquisition: APS, IGA.

Intellectual input: APS, IGA, SWA, SYK, AO, AAG.

Project administration:  IGA, SWA, AMK, AO

Writing (original draft): APS, IGA, SWA

Writing (review and editing): All authors.

ACKNOWLEDGMENTS

          The authors appreciate the Radiographer and Technical Staff at the Radiography Unit of Ladoke Akintola University of Technology Health Centre for their vital contributions towards the actualization of the research.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

FUNDING

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.