الفهرس 
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Abstract
Doxorubicin (DOX) is a widely used, powerful chemotherapeutic agent for treatment of a wide variety of malignancies. Regrettably, it induces a dose-dependent cardiotoxicity. Over the last few decades, many remedies have been tried to protect from this life threating adverse effect. Resveratrol (RSV), a natural antioxidant, is one of the novel materials used to protect from DOX-induced cardiotoxicity. Despite the effectiveness of RSV, its low bioavailability represents an obstacle for its use. To overcome this difficulty, many nanocarriers have been studied to enhance its bioavailability. Liposomes, lipid-nanocarriers, are currently preferred in drug delivery due to their biocompatibility and biodegradability, and fewer side effects of the carried drugs.
The purpose of the present study was to validate the possible protective efficacy of liposomal-loaded RSV on DOX-induced cardiotoxicity in adult male albino rats. And to compare between the protective efficacies of RSV loaded on two different liposomal carriers; multilamellar and PEGylated unilamellar liposomes. The changes were documented histologically and by the biochemical analysis of serum cardiac enzymes.
The liposomes were prepared at Pharmaceutics department, Faculty of Pharmacy, Alexandria University. Their characterization was performed by transmission electron microscope (TEM), Nano Zeta particle analyzer, Entrapment efficiency and in vitro drug release studies.
The present study was conducted over 5 successive weeks on 50 adult male albino rats, 6-8 weeks old and weighing 150-200 gm which were assigned into 2 equal groups:
group I: twenty-five rats were randomly subdivided into five equal subgroups:
• Subgroup IA: received intraperitoneal (I.P) injection of physiological saline (1 ml/kg B.W.) twice per week.
• Subgroup IB: received daily oral suspension of unloaded multilamellar liposomes (0.01 μmol phospholipids/kg B.W) by oral gavage.
• Subgroup IC: received daily oral suspension of unloaded PEGylated unilamellar liposomes (0.01 μmol phospholipids/kg B.W).
• Subgroup ID: received daily oral suspension of RSV-loaded multilamellar liposomes (20 mg/kg B.W) by oral gavage.
• Subgroup IE: received daily oral suspension of RSV-loaded PEGylated unilamellar liposomes (20 mg/kg B.W) by oral gavage.
group II: 25 rats were randomly subdivided into five equal subgroups:
• Subgroup IIA: received I.P injection of DOX only at a dose of 2 mg/kg B.W. twice per week for 5 successive weeks to reach a total cumulative dose of 20 mg/kg B.W.
• Subgroup IIB: received I.P injection of DOX (2 mg/kg B.W) twice a week simultaneously with daily oral suspension of unloaded multilamellar liposomes (0.01 μmol phospholipids/kg B.W).
• Subgroup IIC: received I.P injection of DOX (2 mg/kg B.W) twice a week simultaneously with daily oral suspension of unloaded PEGylated unilamellar liposomes (0.01 μmol phospholipids/kg B.W).
• Subgroup IID: received I.P injection of DOX (2 mg/kg B.W) twice a week simultaneously with daily oral suspension of RSV-loaded multilamellar liposomes (20 mg/kg B.W).
• Subgroup IIE: received I.P injection of DOX (2 mg/kg B.W) twice a week simultaneously with daily oral suspension of RSV-loaded PEGylated unilamellar liposomes (20 mg/kg B.W).
The experiment continued for five successive weeks. By its end, blood samples were collected from all rats before sacrifice for estimation of serum biomarkers; creatine kinase (CK-MB) and cardiac troponin I (cTnI) levels. Then, rats of all subgroups were sacrificed by decapitation after ether anesthesia. Their chest walls were opened, and the hearts were rapidly excised. Heart apices were dissected and processed for the following studies:
I. Histological study:
A. Light microscopic examination:
• Hematoxylin and eosin (H&E) stain.
• Masson’s trichrome stain.
B. Electron microscopic examination: using TEM.
II. Histomorphometric study:
The area percentage of collagen in Masson’s trichrome-stained sections, and some mitochondrial parameters in TEM photomicrographs were measured. This was followed by statistical analysis of the biochemical and histomorphometric results.
Examination of subgroups IB, IC, ID, and IE revealed similar biochemical and histological results to the control subgroup IA; normal levels of the cardiac enzymes, classical myocardial structure, and similar morphometric measurements. This confirmed the safety of the multilamellar and PEGylated liposomes, both the empty and RSV-loaded types.
Regarding DOX-treated rats of subgroup IIA, the biochemical markers; CK-MB and cTnI were significantly increased as compared to the control subgroup IA. Moreover, H&E-stained sections showed prominent structural alterations of the cardiac muscle fibers. For instance, waviness and disorganization as well as interruption of the muscle fibers where encountered. Some muscle fibers showed foci of pale eosinophilic sarcoplasm, while others showed hypereosinophilic areas with deeply stained nuclei. Wide interstitial spaces and focal areas of cellular infiltration were noticed as well. This was supported by examination of trichrome-stained sections that revealed evident collagen fibrous deposits in the interstitial spaces between the muscle fibers and around the blood vessels. It was further confirmed by histomorphometric analysis where the area percentage of collagen exhibited a significant increase as compared to the control subgroup IA. On the ultrastructural level, myocardial injury was manifested as scalloped sarcolemma and interrupted myofibrils along with some sarcoplasmic vacuoles and areas of rarefied sarcoplasm of the cardiomyocytes. The mitochondria showed abnormalities in size, shape, and arrangement.Many nuclei exhibited increased chromatin condensation, while others were peripherally located. Notably, several autophagosomes were further encountered, some of which enclosed degenerated mitochondria. Other detectable lesions included dilated T-tubules, disrupted intercalated discs (ICDs), and noticeable foci of interstitial collagen deposition. Additionally, telocytes with dense nuclei were frequently seen between the cardiomyocytes. Histomorphometric analysis of mitochondria displayed significantly lowered mean aspect ratio (AR) and form factor, whereas significantly higher mean circularity value was depicted with respect to the control subgroup IA. Such values indicate that the mitochondria in this subgroup were globular and fragmented, while their counterparts in the central subgroup IA were tubular and elongated.
Administration of unloaded multilamellar and PEGylated liposomes in conjunction with DOX in subgroups IIB and IIC respectively displayed no differences with respect to subgroup IIA regarding all the studied biochemical, histological, and histomorphometric parameters. This denotes absence of any protective effect of the unloaded liposomes.
As for subgroup IID, the biochemical and histological findings revealed that RSV-loaded multilamellar liposomes had a satisfactory protective role against DOX-induced cardiotoxicity as judged by the evident decrease in the extent and severity of myocardial damage. The mean values of serum cardiac enzymes displayed significant decrease as compared to DOX-treated rats of subgroup IIA. H&E-stained sections generally showed intact cardiac muscle fibers. Nevertheless, some muscle fibers revealed small foci of hypereosinophilic sarcoplasm with deeply stained nuclei, while others showed scattered patches of pale eosinophilic sarcoplasm. Trichrome-stained sections revealed mild increase in interstitial collagen deposition at limited fields. The area percentage of collagen was significantly decreased compared to subgroup IIA, with no significant difference with the control subgroup IA. Ultrastructurally, considerable preservation of the cardiomyocytes was noticed. Most of the cardiomyocytes exhibited almost normal appearance. Yet, some myocytes showed noticeable structural alterations such as scalloped sarcolemma, myofibrillar loss, some sarcoplasmic vacuoles, and eccentric lobulated nuclei. Clusters of numerous bizarre-shaped mitochondria were encountered as well. Moreover, intact telocytes with no signs of apoptosis were seen. Histomorphometric analysis of mitochondria revealed significantly lowered AR with respect to the control subgroup IA, whereas no difference was depicted regarding the other two parameters: form factor and circularity. It reflected that some mitochondria in this subgroup maintained their elongated appearance, while others had a globular shape.
Upon administration of RSV-loaded PEGylated unilamellar liposomes with DOX in subgroup IIE, better amelioration of DOX-induced biochemical and histological alterations with respect to subgroup IID was depicted. The biochemical parameters revealed significant decrease in the serum cardiac enzymes; CK-MB and cTnI versus subgroup IIA with no significant difference compared to subgroup IA. H&E-stained sections showed that most of the muscle fibers retained almost normal histological picture except for limited foci of hypereosinophilic sarcoplasm and wavy muscle fibers. Trichrome-stained sections revealed minimal collagen fibrous deposits among the muscle fibers. The area percentage of collagen was significantly lowered with respect to subgroup IIA. On the ultrastructural level, most of the cardiomyocytes exhibited nearly normal appearance with well-organized myofibrils, straight sarcolemma, and intact ICDs. Although only few myocytes showed mild structural alterations in the form of irregular sarcolemma and interrupted myofibrils. Clusters of mitochondria were encountered at the sites of myofibrillar loss and subsarcolemmal. Additionally, telocytes of nearly normal appearance were seen in the interstitial spaces. Histomorphometrically, the studied parameters of mitochondria did not show any significant difference in comparison to subgroup IA. This indicated that they were tubular and elongated as their control counterparts