The effective conjugation of chemotherapeutic drugs, imaging agents, and tumor-targeting ligands into nanoparticles has been extensively applied to provide diagnostic agents for selective cancer chemotherapy (Morales-Cruz et al., 2019, Navya et al., 2019). Generally, in DDSs, if the size of nanocarriers is less than 100 nm, it will be appropriate for the transit of pores on the cell surface formed by the regulation of osmolarity at a size of 10–500 nm (Gupta et al., 2014). Thermo and pH dual-responsive polymer nanoparticles have demonstrated considerable anticancer properties in the controlled release of drugs due to lower pH and higher tumor temperatures than normal tissues (Lanzalaco and Armelin, 2017, Oroojalian et al., 2018).
Likewise, the folate-targeted nanoparticles have been extensively used in active targeting for effective cancer treatment due to the folate-dependent cellular internalization mechanism by FOLR1 binding (Li et al., 2011, Li et al., 2015, Ndong et al., 2015, Ak et al., 2018). Recent studies have shown that if a folic acid is conjugated with a pH-sensitive nanocarrier, it could act as a targeting moiety to facilitate receptor-mediated endocytosis to cancer cells, improve targeted drug delivery, increase DOX accumulation and release in the tumor tissue, and ultimately reduce tumor growth and adverse effects following DOX chemotherapy (Banu et al., 2015, Ak et al., 2018, Kanwal et al., 2018).
DIC is characterized by a number of abnormalities, including myopericarditis, dysrhythmia, electrocardiographic repolarization changes, troponin elevation, and systolic or diastolic ventricular dysfunction, which may be predisposed to dilated cardiomyopathy (DCM) or congestive heart failure (CHF) several days to one year after chemotherapy (Jain et al., 2017). A previous study demonstrated that DOX-folate-conjugated poly (NIPAAm-co-IA) @Fe3O4 NPs and DOXpoly (NIPAAm-co-IA) @Fe3O4 NPs could significantly improve the antitumor effects in HeLa cell (FR-positive) and A549 cell (FR-negative) (Ghorbani et al., 2016). In this study, further in vivo studies were conducted on the breast cancer model to reduce the adverse effects of DOX chemotherapy by increasing selectivity and controlling release at lower pH and higher tumor tissue temperatures. Therefore, poly-MNPs and folate-poly-MNPs were used to evaluate whether passive and active tumor-targeting of DOX might protect cardiotoxicity while not affecting the antitumor activity of DOX in the breast cancer model.
The ECG is a simple, non-invasive, and accessible technique used for tracing cardiotoxicity in clinical practice (Drew et al., 2004). Previous studies have shown that the most common ECG changes in the DOX cardiotoxicity, including QRS complex, QT, and QTc interval prolongation, could predict a high risk of developing malignant ventricular arrhythmias (Cai et al., 2019). However, the findings of this study demonstrated that loading DOX into tumor-targeted DDSs showed a considerable improvement in ECG parameters, including heart rate, RR interval, PR interval, QRS complex, QTc interval, and QT interval compared to the DOX group.
DOX chemotherapy significantly attenuates cardiac function by inducing ROS generation, disruption of calcium homeostasis, and decreases in SA node excitability in the cardiac conducting system, leading to decreased cardiac function (Kim et al., 2006, Shi et al., 2018). To assess hemodynamic function, arterial blood pressure and left ventricular functions were analyzed in the study groups. However, DOX treatment with novel targeted nanocarriers improved SAP, DAP, MAP, min pressure, max pressure, mean pressure, the EDP index, contractility index, min dP/dt, and max dP/dt compared to the DOX group. The results of the present study were consistent with previous studies that examined DIC, ECG pattern abnormalities, left ventricular dysfunction, and decreased blood pressure parameters (Octavia et al., 2012, Razmaraii et al., 2016c). Moreover, it is generally accepted that DIC is associated with a reduction in the heart to body weight ratio (Ghibu et al., 2012, Swamy et al., 2012, Arafa et al., 2014, Razmaraii et al., 2016b). In contrast, the rats treated with DOX loaded into tumor-targeted DDSs experienced a significant increase in HWI and final body weight compared to the DOX group, but there was no statistically significant difference in the heart weight as compared to the control group (p > 0. 05).
Likewise, histopathological results of the left ventricular myocardium were consistent with prior studies that described DOX-induced cardiac abnormalities such as cytoplasmic vacuolization, hyaline degeneration, interstitial edema, inflammatory cell infiltration, mild cardiac fibrosis, focal to extensive hemorrhage, and cardiomyocyte necrosis following DOX administration (Razmaraii et al., 2016a). Notably, the histopathological findings and the cardiomyopathy severity scores confirmed that the tumor-targeting DDSs significantly reduced cardiac structural abnormalities, including interstitial edema, hyaline degeneration, and inflammatory cell infiltration compared to the DOX group. The results of this study revealed that hemodynamic function, HWI, and cardiomyopathy severity scores in the novel targeted DDSs were effective in protecting against DIC in the experimental groups.
Irreversible LV dysfunction and myocardial cell death were associated with DOXinduced cardiotoxicity following breast cancer therapy (Zhang et al., 2009, Kumar et al., 2012). A similar study suggested that cardiac health monitoring would be determined by a significant decrease in heart weight and increased apoptosis in the TUNEL assay (Adão et al., 2013). In this study, TUNEL staining confirmed that DNA fragmentation and apoptotic cells in the tumor-targeting DDSs decreased compared to the DOX group.
Previous studies have demonstrated that mitochondrial damage plays a critical role in the initiation of apoptosis in cardiomyocytes due to the release of cytochrome c, cleaved-caspase-3, cleaved-PARP1, and AMP-activated protein kinase. The cleavage of PARP (C-PARP1) is a prominent feature of cell death, occurring in two different pathways; a lysosomal protease cleaves PARP to a 55-kDa C-PARP1 protein in the necrotic pathway, and caspase-3 cleaves PARP to an 89-kDa C-PARP1 protein in the apoptosis pathway (Sosna et al., 2014, Shin et al., 2015, Morris et al., 2018). Several in vivo studies indicated that the accumulation of DOX with folate nanocarriers decreased in cardiac tissue due to the EPR effect and folate-receptor-mediated endocytosis while reducing the concentration of DOX in the bloodstream and maintaining a higher concentration of DOX in the tumor tissue (Yoo and Park, 2004, Li et al., 2011, Li et al., 2015, Ndong et al., 2015, Ak et al., 2018). In addition, the results of DIC studies confirmed that DOX treatment led to increased free radicals, decreased gene expression of Bcl-2, and increased gene expression of BAX, PARP1, caspase-3, and the BAX/Bcl-2 ratio (Yoo and Park, 2004, Sun et al., 2016).
As shown in Figures 4-6, the results of this study indicated that DOX-folate-poly-MNPs administration could inhibit apoptosis by reducing the protein expression levels of BAX, cPARP1, ccaspase-3, and the BAX/Bcl-2 ratio compared to the DOX group. Moreover, these novel tumor-targeting DDSs could prevent apoptosis in cardiac tissue by decreasing the ccaspase-3/caspase-3 and c-PARP1/PARP1 protein expression levels compared to the DOX group.
A previous study demonstrated that the inactivated PARP1 protected the heart against ROS generated, myocardial apoptosis, and cardiac dysfunction (Bartha et al., 2011). However, in the DOX-folate-poly-MNPs group, the protein expression of c-PARP1 (89kDa) was reduced compared to the DOX group. Besides, in a similar study, the biodistribution profiles of DOXFA-PEG-P (HB-HO) NPs, DOXP (HB-HO) NPs, and free DOX in cardiac tissue were measured for up to 48 hours. The cardiac accumulation of DOX was significantly reduced in DOX-FA-PEG-P (HBHO) NPs treated mice, whereas the DOX content in tumors and the halflife of DOX-P (HB-HO) NPs increased compared to free DOX (Zhang et al., 2016).
Recent research has shown that DOX treatment causes pyroptosis in cardiomyocytes, an inflammatory caspases-dependent programmed cell death via the NLRP3-caspase-1 pathway (Meng et al., 2019). The NLRP3 inflammasome is composed of a large protein complex of NLRP3, ASC, and pro-caspase-1. The adaptor ASC and pro-caspase-1 are recruited when the NLRP3 inflammasome is activated. Then, activated caspase-1 processes the maturation and release of IL-1β, IL-18, and gasdermin D. The N-terminal cleavage fragment of gasdermin D induces pyroptosis by forming plasma membrane pores, causing cell swelling, plasma membrane rupture, and the secretion of proinflammatory cytoplasmic contents, inflammatory responses, cardiomyocyte death, and cardiac damage (Meng et al., 2019, Zeng et al., 2020, Liu et al., 2021). As DOX induces cardiomyocyte by activating the NLRP3-caspase-1 pathway, our findings suggest that the novel targeting DDS causes less cardiotoxicity than the free DOX and inhibits NLRP3-mediated pyroptosis in vivo by downregulating ASC, c-caspase-1, c-IL-1β, and c-IL-18 protein expression levels compared to the DOX group. In addition, the c-caspase-1/pro-caspase-1 ratio in the DOX-folate-poly-MNPs group was reduced in comparison with the DOX group. Similarly, the findings of NLRP3 inflammasome-mediated inflammation following tumortargeting DDSs confirmed the histopathological results of the left ventricular myocardium, which showed that the novel targeted DDSs reduced inflammatory cell infiltration and cardiomyopathy severity scores as compared to the DOX group.
The results of this study are in agreement with previous in vivo studies, which suggested that the synergistic antitumor effects of passive and active targeting DDSs in the tumor tissues might be attributed to decreased DOX exposure in the cardiac tissues (Huang et al., 2016, Ma et al., 2017, Hyun et al., 2019). Therefore, the cardiac health monitoring following breast cancer therapy confirmed that administration of DOX-folate-poly-MNPs, a tumor-targeting DDSs, could decrease irreversible LV dysfunction, myocardial cell death, and DNA fragmentation in the cardiac tissue by decreasing the protein expressions of BAX, c-PARP1, c-caspase-3, and the BAX/Bcl-2 ratio compared to the DOX group. Finally, these facts suggest that the folatepolyMNPs could be a desirable nanocarrier for the tumor-targeting drug delivery of DOX, capable of reducing DIC and improving the efficacy of chemotherapy in a breast cancer model rather than commercial DOX therapy.