Elsevier

Journal of Controlled Release

Volume 287, 10 October 2018, Pages 78-93
Journal of Controlled Release

Dual-targeted casein micelles as green nanomedicine for synergistic phytotherapy of hepatocellular carcinoma

https://doi.org/10.1016/j.jconrel.2018.08.026Get rights and content

Abstract

In recent years, green nanomedicines have made transformative difference in cancer therapy researches. Herein, we propose dual-functionalized spray-dried casein micelles (CAS-MCs) for combined delivery of two phytochemicals; berberine (BRB) and diosmin (DSN) as targeted therapy of hepatocellular carcinoma (HCC). The nanomicelles enabled parenteral delivery of the poorly soluble DSN via its encapsulation within their hydrophobic core. Moreover, sustained release of the water soluble BRB was attained by hydrophobic ion pairing with sodium deoxycholate followed by genipin crosslinking of CAS-MCs. Dual-active targeting of MCs, via conjugating both lactobionic acid (LA) and folic acid (FA), resulted in superior cytotoxicity and higher cellular uptake against HepG2 cells compared to single-targeted and non-targeted CAS-MCs. The dual-targeted DSN/BRB-loaded CAS-MCs demonstrated superior in vivo anti-tumor efficacy in HCC bearing mice as revealed by down regulation of cell necrosis markers (NF-κB and TNF-α), inflammatory marker COX2, inhibition of angiogenesis and induction of apoptosis. Histopathological analysis and immunohistochemical Ki67 staining confirmed the superiority of the dual-targeted micelles. Ex-vivo imaging showed preferential liver-specific accumulation of dual-targeted CAS-MCs. Overall, this approach combined the benefits of traditional herbal medicine with nanotechnology via LA/FA-CAS-MCs loaded with BRB and DSN as a promising nanoplatform for targeted HCC therapy.

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

References (68)

  • B. Stella et al.

    Design of folic acid conjugated nanoparticles for drug targeting

    J. Pharm. Sci.

    (2000)
  • W. Lin et al.

    Preparation and characterisation of rose Bengal-loaded surface-modified albumin nanoparticles

    J. Control. Release

    (2001)
  • A.O. Elzoghby et al.

    Swellable floating tablet based on spray-dried casein nanoparticles: near-infrared spectral characterization and floating matrix evaluation

    Int. J. Pharm.

    (2015)
  • A.O. Elzoghby et al.

    Ionically-crosslinked milk protein nanoparticles as flutamide carriers for effective anticancer activity in prostate cancer-bearing rats

    Eur. J. Pharm. Biopharm.

    (2013)
  • A.O. Elzoghby et al.

    Superiority of aromatase inhibitor and cyclooxygenase-2 inhibitor combined delivery: hyaluronate-targeted versus PEGylated protamine nanocapsules for breast cancer therapy

    Int. J. Pharm.

    (2017)
  • F. Ungaro et al.

    Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers

    J. Control. Release

    (2012)
  • X. Zhen et al.

    Cellular uptake, antitumor response and tumor penetration of cisplatin-loaded milk protein nanoparticles

    Biomaterials

    (2013)
  • M. Nasr et al.

    Improved antitumor activity and reduced cardiotoxicity of epirubicin using hepatocyte-targeted nanoparticles combined with tocotrienols against hepatocellular carcinoma in mice

    Eur. J. Pharm. Biopharm.

    (2014)
  • X. Zhao et al.

    Doxorubicin and curcumin co-delivery by lipid nanoparticles for enhanced treatment of diethylnitrosamine-induced hepatocellular carcinoma in mice

    Eur. J. Pharm. Biopharm.

    (2015)
  • L. Zhang et al.

    Glycyrrhetinic acid-graft-hyaluronic acid conjugate as a carrier for synergistic targeted delivery of antitumor drugs

    Int. J. Pharm.

    (2013)
  • S.H. Choi et al.

    Hydrophobic ion pair formation between leuprolide and sodium oleate for sustained release from biodegradable polymeric microspheres

    Int. J. Pharm.

    (2000)
  • T. Imsombut et al.

    Genipin-cross-linked silk fibroin microspheres prepared by the simple water-in-oil emulsion solvent diffusion method

    Powder Technol.

    (2010)
  • Y.-J. Li et al.

    Folate-decorated anticancer drug and magnetic nanoparticles encapsulated polymeric carrier for liver cancer therapeutics

    Int. J. Pharm.

    (2015)
  • S. Wang et al.

    The effect of casein as a spray-drying additive on the sorption and crystallization behavior of lactose

    Dry. Technol.

    (2010)
  • P. Shi et al.

    Release and cellular acceptance of multiple drugs loaded silk fibroin particles

    Int. J. Pharm.

    (2011)
  • H.-z. Zhang et al.

    Pullulan acetate nanoparticles prepared by solvent diffusion method for epirubicin chemotherapy

    Colloids Surf. B: Biointerfaces

    (2009)
  • N. Elgindy et al.

    Biopolymeric microparticles combined with lyophilized monophase dispersions for controlled flutamide release

    Int. J. Pharm.

    (2011)
  • C. De Kruif et al.

    κ-Casein as a polyelectrolyte brush on the surface of casein micelles

    Colloids Surf. A Physicochem. Eng. Asp.

    (1996)
  • S. Narayanan et al.

    Sequentially releasing dual-drug-loaded PLGA–casein core/shell nanomedicine: Design, synthesis, biocompatibility and pharmacokinetics

    Acta Biomater.

    (2014)
  • T.-C. Chou

    Drug combinations: from laboratory to practice

    J. Lab. Clin. Med.

    (1998)
  • T.-C. Chou et al.

    A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems

    J. Biol. Chem.

    (1977)
  • C.S. Boosani et al.

    Regulation of COX-2–mediated signaling by α3 type IV noncollagenous domain in tumor angiogenesis

    Blood

    (2007)
  • T.D. Dung et al.

    Diosmin induces cell apoptosis through protein phosphatase 2A activation in HA22T human hepatocellular carcinoma cells and blocks tumour growth in xenografted nude mice

    Food Chem.

    (2012)
  • M. Wei et al.

    Hepatocellular carcinoma targeting effect of PEGylated liposomes modified with lactoferrin

    Eur. J. Pharm. Sci.

    (2012)
  • Cited by (76)

    View all citing articles on Scopus
    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

    View full text