Qualitative analysis of XRD
The XRD spectra for QU nano, CHS, CHSNPs, and QU- CHSNPs are shown in Figure 1. For CHS, the XRD spectrum had a low diffraction signal intensity, which reflected its low level of crystallinity. With a d-spacing of 4.43, the highest peak at 19.9° represents a relative intensity of 100%. According to Eq 1, the calculated crystallite size of the pure CHS was 128.878 Å with a micro-strain of 1.72%. It is important to note that as the width of the peak increases, the crystallite size decreases.
The XRD spectrum of CHSNPs had a disordered arrangement of CHS chains, showing a wide diffuse hump peak at around 30o, which is a typical fingerprint of semi-crystalline chitosan [35]. Our findings were proportionate with those of a former study [36], which showed that TPP counter-ions induce cross-linking among CHS chains, and thus the construction of an opaque network that vanishes in the diffraction peaks of CHS, which form a single floppy peak. The broad peak indicated a decrease in crystallite size, reflecting a reduction in periodicity, i.e., the long-range order of atoms, ions, or molecules in the particles, and consequently, there was low ordering of the hkl planes, which decreased crystallinity. The low degree of crystalline perfection may also be attributed to the high number of defects in the nucleation and the growth rate of the crystals as a result of using polymer like chitosan [37].
According to the chemical structure of QU, scheme 1, the previously published XRD spectrum for QU confirmed its crystallinity state [38]. In our study, the nanoscale crystallinity of QU was determined by high-resolution XRD to enhance our understanding of its chemical action. The obtained XRD spectrum of QU nano had similar peaks to [38]; they were in slightly different positions. The XRD spectrum of QU nano had peaks at 8.9o, 9.7o, 10.7o, 12.4o, 13.1o, 13.6o, 16.6o, 23.1o, 24.8o, 26.1o, 27.4o, 28.1o, 28.5o, 38.6o, and 41.9o, respectively. There was a high basal diffraction peak at the diffraction angle of 13.6o with 100% relative intensity, d-spacing of 6.77 Å, a crystallite size of 485.6 Å, and a micro-strain of 0.69%. This peak was coordinated with prior research [39]. These distinct diffraction peaks indicate that the crystallinity of QU nano did not change, in agreement with previous research [40].
The same Fig 1 paraded the crystal form of QU-CHSNPs with a trick peak shifting to the left. The position of the diffracted peak can shift in response to several different factors, including substitution doping, temperature, and stress. Here, the shifting could be because of differences in interaction angles caused by changes in the QU's arrangement structure plans during the loading process. Another explanation for peak shifting was the changes in the interatomic distances of the QU-CHSNPs, which resulted from the entrapped QU within the CHSNPs. According to the chemical structure of CHS and QU (Scheme 1), we suggest that a chemical bonding occurred between the CHS’s amino group NH2 and the CO carbonyl and OH hydroxyl functional groups of the QU. The diffracted peak of QU disappeared, enforcing this suggestion. The shifting may also be because of the adsorption of QU on the surface of CHSNPs. Overall, the QU-CHSNPs spectrum showed languid diffracted signal changes in shape, position, and the relative integrated intensity between 2 theta angles, which is agree with Zhang et al. [41]. These alterations show that there was QU loading on the CHSNPs.
FTIR spectra analysis
In figure 2, comparing to the standard QU [38], the FTIR spectrum of QU nano presented a variation of sharp, intense, and faint peaks as well as essential practical groups that corresponded to the stretching vibrations of O-H, =C-H, -CH2, C=O, C-O, and C=C. While the spectrum exhibited peaks similar to those previously reported, there were shifts in position and intensity. The spectrum revealed a specific strong stretching peak of the carbonyl group C=O at 1660 cm−1, a weak peak referred to as the =C-H stretching vibration at 2937 cm-1, and a peak that indicates C=C aromatic stretching at 1511 cm−1. There was a distinct peak at 3283 cm-1 that coincides with the stretching vibration of hydroxyl group OH, and another at 880 cm−1 that corresponds to a benzene ring. The obtained results reflect the molecular structure of QU.
The FTIR spectra for CHS and CHSNPs are also presented in Fig. 2. Considering the chemical structure of CHS, the presented broad peak at 3437 cm−1 is attributed to the stretching vibration of the OH group. A polysaccharide peak of CHS, which is in-plane N-H bending vibration, appeared at 1637 cm-1. The C-O stretching vibration of the primary alcoholic group’s evidence is at 1385.34 cm-1, while a bending vibration peak of C-N is presented at1076 cm−1. Regarding the CHSNPs spectrum, the two spectra appeared to be similar and showed various characteristic peaks with a faint diversity in the wideness and non-considerable shifting of the peak position. The broad peak ranged from 3353.09–3172.46 cm−1, which corresponded to overlapping among the stretching vibrations of the O–H and N–H groups. The peaks at 2919.18 cm−1 were associated with aliphatic sp3 C-H stretching, those at 1636.91 cm−1 with in-plane N-H bending vibrations, and those at 1405.34 cm−1 with C-O stretching vibrations. The peak at 1020 cm−1, which appears in the FTIR spectrum for CHSNPs, shows a characteristic P=O stretching vibration from the phosphate groups of the TPP. A previous study [42] reported similar results for the formation of CHSNPs treated with TPP.
The pattern of intramolecular hydrogen bonds may explain the broadness divergence between CHS and CHSNPs. Furthermore, the observed hypochromic shifting in peak positions was due to the interactions between the NH3+ groups of the CHS and phosphate groups of the TPP. For this reason, the FTIR spectra were employed to verify TPP and CHSNPs. The decline in amide I band intensity for CHSNPs (1636 cm−1) when compared to CHS (1637 cm−1) enforced this interaction. The –CH2 wagging peak at1405 cm−1 was another substantial for CHSNPs.
The spectrum of QU-CHSNPs revealed significant variations in the peak patterns, intensities, and positions. For example, the broad band extended from 3500–3000 cm−1 due to the intermolecular hydrogen bonding, as well as the disappearance of some peaks. These variations reflected a type of interaction between CHSNPs and QU, confirming the loading process. These results were in accordance with those of previous research [43].
Surface morphology TEM and SEM study
Employing high-resolution transmission electron microscopy (HRTEM) to study the shape and particle size, the HRTEM images of CHSNPs and QU-CHSNPs are shown in Fig 3. An optimized spherical shape of the prepared CHSNPs with an average particle size of approximately 50 nm is shown in Fig 3 (A), while an HRTEM with a low field of view revealing stack clusters of the prepared QU-CHSNPs is shown in Fig 3 (B). The images display two different signal intensities, dark and gray, which might be due to a variation in the attenuation of the incident electron beam on the QU-CHSNPs. This attenuation is based on the variations in the electron densities of the QU and CHSNPs, affirming the loading process. In the HRTEM images, the appearance of darker regions inside the particles indicates the lipophilic QU in the matrix of the CHSNPs, and gray regions may indicate soluble CHS. The surface microstructures show that the sample was coated with gold to avoid charges and promote the signals needed for surface analysis with the SEM, Fig 3. The CHSNPs had a non-homogeneous, rough, and irregular surface texture, Fig 3 (C); this irregularity could be due to the complex's formation scheme, which includes incorporating two aqueous phases, one containing polymer CHS and the other containing poly-anion TPP. In contrast, the SEM image of QU-CHSNPs had a smooth surface pattern with morphology like a pineapple, Fig 3 (D).
Zeta sizer and potential measurements
The hydrodynamic size distribution of the QU nano and QU-CHSNPs was determined by the intensity distribution method via dynamic light scattering (DLS) technique, as shown in Fig 4 (A). The intensity-distribution and cumulant fit sizes were similar, and The Z average, or called cumulant mean, or log mean of the hydrodynamic diameter size distribution of QU nano was 150 nm. The polydispersity index PDI value (.992) predicted a wide width of the distribution peak, indicating a heterogeneous size distribution. The intercept value (1.09) indicated an excellent signal-to-noise ratio. For the QU-CHSNPs the reported hydrodynamic size, PDI, and the intercept were 329.4 nm, 0.541, and 0.983, respectively (Figure 4). The hydrodynamic diameter size distribution of QU-CHSNPs was slightly bigger than that identified using the HRTEM.
However, this discrepancy is reasonable as the distribution number (from electron microscopy) is expected by smaller than the distribution intensity (from DLS). In other words, the particle size determined using the DLS represents its hydrodynamic diameter, whereas that obtained using HRTEM represents its real diameter. The surface charge of the QU-CHSNPs formula was estimated by measuring the zeta potential. In total, a zeta potential value of greater than 30 mV is thought to be a standard value that will give ample repulsion force to limit particle aggregation. The repulsion among particles indicated that the low zeta potential value was -27.9 ± 6.8 mV, which indicates low physical stability. QU is a hydrophobic polyphenol with a pentahydroxy flavone and five hydroxy groups at the 3-, 3'-, 4'-, 5-, and 7-positions, (Scheme 1). The negative charge of the formula was due to the hydroxy group, indicating the successful entrapment of QU by cross-linked CHSNPs.
Entrapment efficiency and release profiles
The results from Eq. 3 showed that the average entrapment efficiency for the QU in CHSNPs was 92.56% ±7.72%. The release profiles for the free QU and the QU from the CHSNPs in the PBS solution at a constant temperature (37°C) and pH 6.8 for 48 h are shown in Fig 4 (B). A surge release of 47.2% was observed from free QU within the first 4 hours, whereas QU-CHSNPs showed a slaw burst release of 29.21%. Free QU was released faster than QU-CHSNPs, which could be attributed to the existence of QU in the CHS’s network. The initial rapid release of the loaded QU may be due to the rapid dispersion of the QU present on the surface of the CHSNPs, while at a later stage, QU may also be constantly released from the core of the CHS matrix because of CHS hydration and swelling. The drug was therefore released more gradually, and the rate of release was influenced not by polymer erosion but by drug diffusion through the amorphous territory of the polymer matrix. The free QU cumulative release in a saturation state after 12 h was approximately 70%, while that of QU-CHSNPs was 49% and it showed a pattern of sustained release, reaching 98% after 48 h. This was explained previously [44], as CHS was found to be a biodegradable polymer. However, its biodegradation is substantially slower than that of other degradable polymers. In addition, the deterioration of CHS was particularly limited in an aqueous medium because of its poor crystallinity and hydrophobicity.
Therefore, the only possible mechanism of QU release was diffusion, and not the degradation of the CHS polymer. Furthermore, it is worth noting that the diffraction pattern of the QU-CHSNPs showed that some of the QU crystal peaks had disappeared, suggesting the amorphous pattern of QU in the matrix of CHSNPs polymer or that it was dispersed in the amorphous region of the CHSNPs. Consequently, the cumulative drug percentages of the free QU were lower than those of QU-CHSNPs, attributing to the slow diffusion of QU from the CHSNPs matrix.
Biological study
Doxorubicin (brand name: Adriamycin) is a chemotherapy drug made from Streptomyces peucetiu. It is used together with other chemotherapy agents against malignant neoplasms, including breast cancer, lung cancer, leukemia, Hodgkin’s disease, Kaposi’s sarcoma, acute lymphoblastic, leukemia, lymphomas, and several metastatic tumors. Through the intercalation, DOX merges with DNA and hinders the macromolecular biosynthesis, restricting the DNA double helix formation and blocking the DNA replication process [45]. However, the long-term use of DOX produce severe effects, which restricts its clinical applications [46]. Cardiotoxicity is one of the extremely serious effects of DOX as it can lead to dilated cardiomyopathy, which results in congestive heart failure. The DOX aggregation dose controls the cardiomyopathy rate. Oxidative stress, downregulation of genes for contractile proteins, and p53-mediated apoptosis are the avenues of cardiomyopathy promoted by DOX [47].
Oxidative stress
There is abundant evidence that oxidative stress plays an essential role in the pathophysiology of DOX-induced cardiotoxicity. In cardiomyocytes, DOX can lose one electron through the metabolic efficiency of Nicotinamide Adenine Dinucleotide/Phosphate-cytochrome P 450 reductase. The DOX reduction forms semiquinone free radicals, which produce radicals of hydroxyl (•OH), hydrogen peroxide (H2O2), and proxynitrite (ONOO) [48]. These species trigger proteins and LPO via damaging the macromolecular cellular components of the cell membrane, inducing oxidative stress and starting cell apoptosis [49]. Antioxidants regulate the cellular oxidative stress that is induced via an inequality in the cellular production of ROS and reactive nitrogen species (RNS) [50]. Since the heart lacks antioxidant enzymes, this results in extensive destruction of the cardiac cellular mitochondrial membranes, nucleic acids, and endoplasmic reticulum [51].
Our investigation showed a marked elevation in cardiac nitric oxide NO levels in DOX-rats when compared with control rats (Fig 5), in agreement with previous research [52]. The capability of DOX to intercede the induction of NOS expression and NO release in the heart is the reason for the ascent in NO levels. Further, DOX causes a surge in e-NOS transcription and protein activity in cardiac endothelial cells by H2O2 and calcium influx triggering, leading to the synthesis of NO [53]. A recent study gives evidence of the upregulation of iNOS genes and protein expression in DOX-induced cardiomyopathy. The concomitant overabundance of NO and ROS yields excessive levels of peroxinitrite and RNS, which may invade and damage vital cellular biomolecules [54].
In our study, we investigated the protective effects of QU and QU-CHSNPs on DOX-induced cardiotoxicity and the underlying mechanisms of this protection [55]. As displayed in Fig 5, QU decreased NO levels with an even greater decrease in QU-CHSNPs. This may be because QU leads to the scavenging of free radicals, which decreases their interactions with nitric oxide and thus reduces the amount of damage [56]. The results suggest that QU provides cardiac protection against DOX by decreasing oxidative stress and damage. It is worth mentioning that the administration of QU-CHSNPs in normal rats showed a non-significant change when compared to the control, indicating safety.
Oxidative stress provides deleterious effects either by triggering LPO or by operating as a second messenger for primary free radicals that start LPO [57-58]. MDA levels were measured in the present study as an indicator for LPO [59]. In Fig 5, DOX administration caused a significant increase in MDA levels compared to the control, consistent with preceding research that employed an analogous drug regimen [60, 61, 62]. The initial targets of DOX-mediated free radical damage are cellular membranes that are rich in lipids susceptible to peroxidation. This radical damage produces stable and toxic aldehydes, such as MDA. These aldehydes can diffuse within the cell, or even cross the plasma membrane, and attack macromolecular targets far from where they were generated, thus acting as "second cytotoxic messengers." The treatment of DOX-injected rats with QU showed a decrease in MDA levels with QU-CHSNPs high pattern, Fig 5. The existing research supports the potency antioxidant activity of QU versus DOX-induced oxidative stress in vivo, as evidenced by the inhibition of ROS generation and the reduction in MDA LPO activity, which corresponds with the findings [63].
Furthermore, our results confirmed that DOX decreased the antioxidant content and performance of non-enzymatic (GSH), enzymatic GPx, SOD, and glutathione-S-transferase (GST), significantly, as shown in Fig 5. These results are in accord with preceding reviews [64, 65, 66], which supported the competence of DOX to provide ROS and overthrow the antioxidant defence policy. Our results connected the devaluation in cardiac GSH content to the enhanced responses of the GSH metabolizing enzymes. The first enzyme is GPx, which catalyzes the disintegration of H2O2 and organic peroxides through its four selenium co-factors, using GSH as a reducing agent. The Fig 5 revealed a decline in GPx in the DOX-rats, and this was because of the mopping up of GPx by the free radicals generated by DOX [67]. The second enzyme is GST, which employs GSH in the conjugation of DOX toxic metabolites [68]. GST detoxifies xenobiotics, drugs, and carcinogens and supports cells against redox cycling and oxidative stress. The heart possesses low levels of GST and an overwhelming generation of free radicals by DOX may result in low GST levels higher than natural levels.
The administration of QU and QU-CHS enhanced the activities of the antioxidants GSH, GPx, SOD, and GST against DOX, in agreement with previous research [69]. Thus, QU scavenges for superoxide radicals and reduces myocardial damage. Indeed, evidence indicated that QU has cardioprotective properties because of its antioxidant activity. QU is an excellent metal chelator that chelates transition metals such as iron, which can initiate the formation of ROS. The scavenging of free radicals and chelating effects are both involved in its cardio-treatment and protective effects [21].
Lipids
Lipids play a role in cardiovascular disease complications. DOX interferes with the lipid metabolism, changing the lipid profile. In Fig 6, the levels of T. cholesterol, TG, LDL-c, and VLDL-c were found to increase in the DOX-rats, consistent with previous research [70, 71]. The increase in the cholesterol concentration could be because of a reduction in HDL levels, as HDLs transport cholesterol from tissues to the liver for catabolism. In this context, we observed decreased levels of HDLs in the DOX-rats. We attribute the elevation in TGs to the low activity of lipoprotein lipase. These changes in lipid levels may be due to enhanced lipid biosynthesis by the cardiac cyclic adenosine monophosphate [72]. Lipid-lowering drugs preserve the myocardium from DOX-induced toxicity. [73].
The administration of QU and QU-CHSNPs decreases the concentration of total cholesterol, TGs, VLDLs, and LDLs and increases the concentration of HDLs in the DOX-induced rats. QU and QU-CHSNPs were both effective at ameliorating cholesterol levels. Other studies have shown the effectiveness of QU in minimizing dyslipidemia [74]. Furthermore, QU has anti-hypercholesterolemia, antihypertensive, vasodilator, and anti-atherosclerotic properties [75]. The presented results suggest that QU has a talent for acting as a cardioprotective agent against DOX-induced cardiotoxicity.
CK-MB, AST, and LDH activities
This investigation showed that DOX-induced cardiotoxicity was manifested by a significant elevation in serum CK-MB, AST, and LDH activities (Fig 7), in agreement with previous research [76-77]. A possible explanation is the DOX induced damage of the cardiomyocyte membranes, releasing these entities from the cytoplasm into the plasma. Further, elevation of the cardiac enzyme function was found to be associated with histological degradation such as distinct necrosis of cardiomyocytes and inflammatory leukocytic infiltration into cardiac tissues [78-79]. In this study, the QU and QU-CHSNPs treatments lessened the CK-MB, AST, and LDH in the DOX-injected rats. Our results significantly reinforce the conclusion of preceding research [80], which showed that the QU pretreatments induced a substantial decline in the CK-MB, AST, and LDH activities when compared with the toxic control rats. This may be because of the protective effects of the QU in regulating the leakage of CK-MB, AST, and LDH. QU possesses free radical scavenging and antioxidant activities, which could justify its competence to defend the myocardium from DOX-induced damage by blocking the leakage of cardiac LDH, AST, and CK-MB isoenzymes [21]. Based on these results, QU and QU-CHSNPs retain potent cardioprotective effects against DOX-induced heart injury.
Cardiac troponin (c Tn-I)
c Tn-I level function is a sensitive and specific marker for myocardial cell injury. This contractile protein is not normally present in the serum and is released only after myocardial necrosis. Elevated troponin I levels predict the risks of both cardiac cell death and infarction following infarction [81]. In this study, the development of cardiotoxicity in the DOX group was also evidenced by elevated c Tn‑I levels in the serum (Fig 7), which was in accordance with previous research [82]. However, the QU treatment reduced the c Tn‑I levels in the serum, which was also in agreement with previous research [83]. These decreases of the c Tn‑I level in the serum suggest that QU and QU-CHSNPs protects against DOX‑induced cardiotoxicity in rats.
Cytokines (IL-1B, TNF-α and IL-10)
Inflammation participates in the pathogenesis of DOX-induced cardiotoxicity. It is fully cited that the overabundance of free radicals enhances the output of inflammatory mediators and triggers cardiac inflammatory processes [84]. The progressive increase of pro-inflammatory and a cytokine level within heart tissues was identified as a potential pathological indication for DOX-induced cardiomyopathy. The results, in line with previous investigations, support the idea that inflammation plays a fundamental part in the pathogenesis of DOX-induced cardiotoxicity. The current study found a significant increase in serum TNF- and IL-1B in the DOX group, which is consistent with previous findings [85, 86] (Fig 8). The principal underlying mechanism promoting this elevation in inflammatory markers is not yet understood. However, it is possible that heightened levels of ROS, impaired tissue antioxidant capacity, and subsequent LPO are provoking factors for these variations.
Indeed, it was declared that rises in inflammatory mediators interacted with heightened oxidative stress, which is considered to provoke inflammatory reactions through the activation of the NF-κB pathway, resulting in the transactivation of cytokines [85]. QU and QU-CHSNPs administration inhibited the elevated TNF- in DOX-treated rats. In harmony with this finding, it has been shown that QU can inhibit NF-KB via its free radical scavenging and antioxidant activities [21]. According to this result, QU and QU-CHSNPs inhibit the elevated IL-1β in DOX-treated rats.
On the other hand, the results of this study have shown that DOX causes a significant decrease in serum anti-inflammatory cytokine IL-10 in DOX- rats (Fig 8), in agreement with previous research [87], while QU and QU-CHSNPs increased the IL-10 levels. The standing research asserted that QU has anti-inflammatory properties and acts by suppressing pro-inflammatory cytokines. While the mechanisms by which QU increases IL-10 levels are not clear [88].
Apoptotic markers
The oxidative stress generated by the DOX triggers many signalling pathways of cardiomyocyte apoptosis, including the activation of caspase-3 [85]. The caspase-3 protein is a cysteine-aspartic protease that is activated in apoptotic cells by the death ligand. The caspase-3 protein is thought to be a biomarker of cardiac tissue toxicity [89]. The present study showed that DOX causes a significant elevation of caspase-3 levels (Fig 8), in agreement with previous research [90]. Also, the results of this investigation agree with previous findings, which showed that DOX increases the levels of caspase-3, activates apoptosis by decreasing the expression of antiapoptotic proteins, and induces oxidative stress by increasing H2O2 production [91]. QU and QU-CHSNPs administration reduced the elevated caspase-3. These results are in accordance with previous research, which supports the hypothesis that the protection presented against DOX-induced cardiotoxicity by treatment with QU may involve the suppression of cardiac apoptosis [ 92].
Cardiac mRNA expression of annexin-V, NrF2 and PPAR-γ
The Annexin isoforms II, V, and VI are a particular group of membrane-associated, Ca2+-binding proteins that exist in heart tissue. Annexin V is present in both cardiomyocytes and non-myocytes and may play a part in regulating cellular ion fluxes, organization, and secretion. To determine alterations in the annexin V isoform that might arise in heart failure, we measured mRNA expression of this annexin. DOX administration caused a significant increase in cardiac annexin-V (Fig 9) compared with control, indicating heart failure. A possible explanation for this might be that DOX can disturb the homeostasis of intracellular calcium cycling in the heart, in agreement with previous research [93]. The DOX-rats treated with QU and QU-CHSNPs revealed a considerable decline in their cardiac annexin-V ascent levels, consistent with the preceding study [92]. It is essential to notice that QU-CHSNPs decreased the annexin-V elevation more than QU alone.
Nuclear factor erythroid 2 related factor 2 (Nrf2), a transcriptional factor, retains crucial protective effects against oxidative stress. In the oxidative stress stage, Nrf2 is triggered, enters the cell nucleus, stimulates antioxidant gene expression, and reduces oxidative damage. Preceding research showed that DOX-induced oxidative stress is correlated with cardiac damage [86]. In our study, DOX administration decreased active nuclear Nrf2 in the cardiac tissues (Fig 9), consistent with previous research [94]. The upregulation of Nrf2 is a unique way to limit DOX-induced cardiac injury [86]. The current study proved that QU and QU-CHSNPS protect against DOX-induced cardiomyopathy by increasing NrF2 expression, which stimulates the synthesis of antioxidant defenses. Furthermore, QU has free radical scavenging and antioxidant properties [75].
Peroxisome proliferation-activated receptor gamma (PPAR-γ) is a nuclear receptor that regulates the transcription of several genes that are primarily involved in fatty acid and energy metabolism [95]. PPAR-γ is also present in a variety of cell types, including cardiomyocytes and vascular cells. PPAR-γ activation reduces the production of inflammatory cytokines, and there is growing evidence that PPAR-γ ligands have anti-inflammatory, antioxidative, and antiproliferative effects on cardiomyocytes [96]. DOX treatment significantly decreases PPAR-γ levels. These results suggest that PPAR-γ may help to protect rats from DOX-related heart injuries [97]. Moreover, our results have indicated that PPAR-γ levels were increased by the treatment with QU and QU-CHS (Fig. 9). We have concluded that quercetin increased PPAR-γ and reduced inflammation due to its anti-inflammatory properties [88].
Cardiac DNA fragmentation
In this investigation, the DNA fragmentation of cardiomyocytes was significantly high after the DOX administration (Fig 10), which is a sign of oxidative stress. The reason is that DOX promotes mitochondrial ROS production, which in turn damages cardiomyocyte DNA and triggers apoptosis [98-99]. QU and QU-CHSNPs showed a reduction in DNA fragmentation. This agrees with other research [67], which reported QU attenuates DOX-cardiotoxicity by reducing oxidative stress, ROS levels, and DNA damage to maintain heart cell viability. Our results were harmonious with another investigation, which confirmed that QU limited the pernicious effects of DOX and cyclophosphamide on the kidney and liver, obstructing peroxidative destruction [100].
Electron microscope and histopathology
Mitochondria are membrane-bound cells, their potential saved in adenosine triphosphate (ATP). One of the key managers in DOX-induced cardiotoxicity is mitochondria. The reason is that DOX suppresses mitochondrial synthesis, stimulates fission, and impairs mitochondrial function, ending with heart collapse. In this investigation, the mitochondrial determinants of DOX cardiotoxicity were assessed, and the results showed that the size, shape, and integrity of the mitochondria were lost (Fig 11 B), while the QU-CHS preserved their membrane integrity but seemed to be compact (Fig 11 C) not elongated like the control (Fig 11 A). Understanding the critical role of mitochondria in DOX-induced cardiomyopathy is critical to reduce the barriers that severely limit the clinical success of this life-saving anticancer therapy.
Considering the histological changes of the heart in normal rats, no histopathological alterations were observed, and a normal histological structure of the cardiac muscles could be found (Fig. 12A). Severe degenerative changes and necrosis of the cardiac muscles were accompanied by focal lymphocytic infiltration in the DOX-treated group (Fig. 12B). Treatment of DOX-administered rats with QU showed moderate hyalinosis of the cardiac muscles and focal lymphocytic infiltration (Fig 12C). Treatment of the DOX-administered rats with QU-CHS was associated with mild to moderate degenerative changes of the cardiac muscles and minimal lymphocytic myocarditis (Fig 12D). Additionally, the normal group treated with QU-CHS showed minimal changes to the cardiac muscles and lymphocytic myocarditis (Fig 12E). In general, DOX administration caused severe histopathological lesions, which were characterized by severe inflammatory cell infiltration, hemorrhage, degeneration, and necrosis, and this was in agreement with previous research [ 101]. In this investigation, QU was found to have an anti-inflammatory effect as it improved the histopathological features of DOX-induced cardiotoxicity and decreased inflammation, degeneration, and necrosis of the myocardium, in agreement with others [ 102].
Overall, through the current study, one can see the protective effect of QU-CHSNPs was better than that of QU-free. An explanation for this may be that the QU encapsulation with CHSNPs could avoid enzymatic degradation in the gastrointestinal tract, limit the burst release, and sustain an adequate QU release rate pattern. Chitosan is a suitable absorption enhancer for poorly absorbable drugs because of its mucoadhesive properties, and it could boost the QU absorption across the intestinal mucosa, increasing its bioavailability. As was noted, the QU-CHSNPs has a positive surface charge, so it is possible to enter cardiac cells more rapidly through the endocytosis process when compared to QU-free. Another explanation is that the CHSNPs with high surface area could increase the loaded QU dose and enhance its therapeutic action.