Dual-targeted casein micelles as green nanomedicine for synergistic phytotherapy of hepatocellular carcinoma
Graphical abstract
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third cause of cancer-induced mortality in the world [1]. Patients with early HCC should be considered for curative treatments including surgical resection, liver transplantation, and percutaneous ablation that may cause complete cure and increase the patient survival [2]. Unfortunately, the patients with advanced progressive disease accounts for >80%, where only palliative approaches are suitable. The most commonly used therapeutic approach is the systemic chemotherapy for improving the survival of HCC-patients. However, conventional chemotherapeutic agents have many challenges in treatment of HCC due to their severe side effects and multidrug resistance [3].
Recently, green chemotherapy in combination with tumor-targeted nanocarriers have attracted much interest to enhance the efficacy and overcome the challenges of conventional chemotherapy [4]. Accordingly, in our study, green phytomedicine was developed by co-encapsulating two phytochemicals with potential anti-cancer effects, diosmin (DSN) and berberine (BRB), in natural protein micelles for treatment of HCC [[5], [6], [7], [8]]. DSN (diosmetin 7-O-rutinoside), a natural flavone glycoside, is known as a potent chemopreventive agent against hepatocarcinogenesis. It is reported as a supportive treatment for many cancer types such as hepatocellular and bladder with advantages of less toxicity and lower cost in comparison with cytotoxic chemotherapy [9]. DSN reduces tumor promotion through inhibiting the proliferation markers and also suppresses the expression of inflammatory markers such as COX-2 and iNOS through knock-down of NF-κB expression in HCC-induced rats [10]. Another herbal drug, berberine chloride (BRB), is an isoquinoline alkaloid with the ability to selectively reduces the viability of HCC cells without affecting normal hepatocytes. BRB has been shown to effectively induce cell cycle arrest at the G1/S phase [11]. Moreover, it causes autophagic death of hepatoma cells through inhibiting the expression of the anti-apoptotic factor Bcl-2 and knock-down of mTOR-signaling [12].
Combination strategies play a crucial role in cancer therapy, wherein multi-target therapy combines a cocktail of drugs, or other novel agents having different mechanisms of action. Based on the reported anticancer effects of DSN and BRB and their pharmacological pathways for the inhibition of liver cancer cells [10,12], we aimed to combine both natural phyto-medicines in a dual-targeted nano-formulation for targeting HCC.
Hence, single-targeted drug carriers may have unsatisfactory therapeutic responses due to the inefficient specific ligand-receptor interaction with the target tumor cells. For instance, HCC-patients usually suffer reduced binding efficacy for ASGPR-based single targeted therapy mainly because of the down-regulation of asialoglycoprotein receptors (ASGP-Rs) by hepatoma cells [13]. Therefore, in our study, we successfully developed dual-targeted CAS micelles using LA and FA as ligands for targeting ASGP-R and FA-R, respectively.
However, DSN has poor solubility in water and most organic solvents resulting in its poor bioavailability [14]. On the other hand, BRB has aqueous solubility of 1 mg/ml, showing high initial burst release in water and aqueous buffers. To avoid premature berberine drug release in to circulation before reaching the tumor site it would be beneficial to develop an efficient controlled release nanoformulation. Therefore, novel tumor-targeted delivery systems are needed to enable i.v. administration of DSN and controlled release of BRB and enhance tumor-targeting efficiency resulting in decreased toxicity and improved clinical utility of both drugs. Among naturally occurring protein nanocarriers, casein (CAS), the major milk protein, has high self-assembling tendency into spherical nano-size micelles [15,16]. CAS micelles were successfully harnessed for delivering hydrophobic anticancer drugs including mitoxantrone, paclitaxel, and cisplatin [17]. In our laboratory, cross-linked CAS micelles have been successfully developed allowing controlled drug release via modulating the crosslinking density [18,19]. Recently, we have developed phospholipid bilayer-enveloped CAS micelles for combined delivery of the herbal drug resveratrol and the fungal-derived monascus yellow pigments as green therapy of breast cancer [20].
In this study, we propose LA/FA dual-targeted CAS micelles co-loaded with DSN and BRB as a completely green therapy of HCC. First, to overcome the high hydrophobicity and enable injection of the poorly soluble drug DSN, pH modulated loading approach was utilized to entrap the drug within micellar core. Second, to reduce the initial burst of BRB and prolong its release, both hydrophobic ion pairing with SDC and micellar crosslinking with genipin were exploited to increase the drug lipophilicity and hence facilitate its incorporation within the hydrophobic core of micelles. Third, both drugs were co-encapsulated within CAS micelles for improving their anti-tumor efficacy. Finally, for maximal tumor-targeting, the surface of CAS MCs was functionalized with LA and FA for respectively targeting asialoglycoprotein and folic acid receptors overexpressed HCC cells while reducing non-specific uptake by normal hepatocytes. The developed delivery system was thoroughly investigated in vitro and in vivo to prove the anti-tumor superiority of the combined drug nanocarriers compared with free drugs.
Section snippets
Materials
Casein (CAS), berberine hydrochloride (BRB), sodium deoxycholate (SDC), lactobionic acid (LA), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC), fetal bovine serum (FBS), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), bovine serum albumin, rhodamine B isothiocyanate (RBITC), dimethyl sulfoxide (DMSO), ethylene diamine tetraacetic acid (EDTA), Triton X100, haematoxylin solution, and eosin solution were from Sigma-Aldrich Co. Ltd.,
Fabrication & physicochemical characteristics of CAS MCs
By virtue of the hydrophobic and hydrophilic amino acids composing its structure, the amphiphilic nature of CAS induces its self-assembly into spherical micelles (CAS-MCs) [17]. Therefore, CAS MCs were successfully used to enable solubilization and i.v. injection of poorly soluble anti-cancer drugs by incorporation within their hydrophobic core. In our study, BRB and DSN were co-loaded into CAS-MCs via different mechanisms. First, for encapsulation of the poorly soluble DSN, the drug was
Conclusion
In this study, a combination of two phyto-medicines BRB and DSN was formulated into a GRAS (generally recognized as safe) protein (CAS) micelles as a completely green HCC therapy. pH-modulation strategy was utilized for efficient solubilization of the poorly soluble drug DSN within the hydrophobic core of CAS MCs whereas combined strategies of both hydrophobic ion pairing and genipin-mediated chemical crosslinking of CAS MCs have been exploited to prolong the release of BRB from the
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- 1
Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
- 2
Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA