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Nano-pharmaceutical Formulations for Targeted Drug Delivery against HER2 in Breast Cancer Sams M.A. Sadat, Soodabeh Saeidnia, Adil J. Nazarali and Azita Haddadi* Division of Pharmacy, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada Abstract: Nanotechnology has revolutionized fundamental opportunities for higher specific drug delivery with minimum side effects. Since its inception, the goal of nanotechnology has been to advance effective and reliable systems for precise anti-cancer therapy and diagnosis. To accomplish this goal, bio-conjugation strategies of therapeutic agents loaded nanoparticles with monoclonal antibodies or their analogues have demonstrated a targeted approach both in vitro and in vivo. In this review, we primarily focus on the specific recognition of HER2 receptors of HER2 overexpressed tumor cells, and evaluate anti-HER2 monoclonal antibody as an effective tool for active targeting. Currently, a variety of nanoparticle systems are under both preclinical and clinical trials for targeting to HER2 positive breast cancer. Different nanotechnology scaffolds including liposomes, dendrimers, micelles, polymeric and inorganic nanoparticles that have higher flexibility for macromolecular synthesis and versatile functionalizing properties have been reviewed in this study. Continuing advances in anti-HER2 functionalized nanoparticles have good potential to lead to the development of nano-therapy against HER2 positive breast cancer.
Keywords: Breast cancer, dendrimer, HER2, liposome, micelles, nanoparticle, PLGA INTRODUCTION Cancer occurs when genes are damaged or altered. This genetic alteration is usually associated with an unregulated proliferation of cells in the body. As a result of a higher proliferative rate of cancerous cells compared to normal healthy cells, cancerous cells require more nutrients and exhibit a higher metabolic elimination rate or biotransformation of toxicants. Hence normal healthy cells surrounding the cancerous cells are not capable of competing for nutrients and are displaced by tumor cells, which continue to have uncontrolled cell division [1]. Cancer continues to remain one of the most prevalent and predominant diseases causing death. Worldwide more than 10 million people each year are diagnosed with cancer [2]. Breast cancer is rated as the second leading cause of cancer deaths among all cancerous diseases in women [3]. Approximately 25% of all types of breast cancers are found to be associated with human epidermal growth factor receptor (HER2) gene amplification. HER2 is a key biomarker for pathogenesis of breast cancer and has a good potential for application in the design of a targeted anticancer drug delivery system [6] HER2 positive breast cancer is found in one out of every four women after primary diagnosis [7]. However, specific symptoms are not notable in the initial stages of breast cancer in women and only HER2 screening test is able to identify the disease [8].
very common effective treatment options to tackle breast cancer. However, these traditional cancer therapies have a number of drawbacks and limitations [9]. Research has been successful in developing treatment options with antibodies that impede breast cancer cell growth [10, 11]. Chemotherapy is the most common breast cancer therapy where cytotoxic drugs interfere with the cellular processes in a nonspecific manner [12]. However, normal healthy tissues are inevitably exposed to these chemotherapeutic agents leading to adverse drug effects [13]. Poor bioavailability of high molecular weight chemotherapeutic agents and drug resistance are two of the most important drawbacks of chemotherapy [14]. Inefficient and non-selective drug delivery system can lead to the development of drug resistance. Additionally drug entry (influx and efflux), metabolic alteration, drug sequestration or repossession, disruption of apoptotic pathways, alteration of signal transduction pathways, microenvironment and activation of DNA repair mechanisms are also responsible for the manifestation and development of drug resistance [15]. To overcome these limitations, more effective sitespecific drug delivery systems need to be developed, which can significantly improve the efficacy of the therapeutic agent with minimal toxicity. To advance research towards this goal, nanotechnology has attracted a considerable attention from researchers to establish the syntheses of nano scale biodegradable drug carriers that can specifically target tumor sites [16].
Currently surgery, radiation therapy, hormone therapy, immunotherapy and chemotherapy are well established as
ROLE OF HER2 IN TARGETING
*Address correspondence to this author at the 3D01.01, D Wing Health Sciences, 107 Wiggins Road, Saskatoon SK, S7N 5E5, Canada; Tel: (306) 9666495; Fax: (306) 966-6377; E-mail:
[email protected]
The transmembrane human epidermal growth factor receptors (HERs) play a key role in transmission of cell signals regulating cell growth and proliferation. This receptor family is comprised of four homologous members
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including HER1, HER2, HER3 and HER4. Among these, only HER2 (Fig. 1) does not have an identifiable ligand on its extracellular domain [17]. The HER2 receptor (also known as Neu, ErbB2, CD340 or cluster of differentiation 340, p185 or 185-kDa plasma membrane phosphoprotein) is a transmembrane glycoprotein encoded by ErbB2 gene on the long arm of chromosome-17 in humans [18]. Structurally, HER2 has three parts including the N-terminal cysteine rich extracellular domain (ECD), a single α-helix transmembrane lipophilic domain and an intracellular tyrosine kinase domain in cytoplasmic sites [19]. HER2 receptor’s dimerization depends on the binding of selective ligands with its extracellular domain while a conformational change occurs. This conformational change as a result of dimerization activates the cytoplasmic catalytic function, which in turn autophosphorylates the tyrosine residues within the cytoplasmic domain of the receptor. The induced autophosphorylation initiates a complex variety of signal transduction pathways (Fig. 2) [20-22]. Although HER2 regulates cell development stages in normal cells, signaling of HER2 through various pathways stimulates cell proliferation and blood vessel growth to nourish tumor angiogenesis as well as to induce metastasis. The common mechanism of HER2 protein overexpression due to increased HER2 protein synthesis is associated with HER2 gene amplification and transcriptional dysregulation. This overexpressed HER2 protein concentration results in the activation of HER2 dimerization in the absence of ligand binding leading to uncontrolled cell proliferation and tumorigenic transformation [23].
preferential heterodimers for other HER receptors. These ‘preferential’ heterodimers have long survival on the cell surface, which induces enhanced transduction signaling and high malignant survival growth [24]. Monoclonal antibodies have been employed as an effective active targeted therapy since these antibodies can specifically recognize the overexpressed HER2 positive tumor cells and internalize through receptor mediated endocytosis [6, 25].
During normal cell growth, HER2 receptor is expressed at normal levels; but in cancerous cells, overexpression of HER2 receptors occurs. This overexpression facilitates free ligands to bind with excessive HER2 receptors to become
Liposomes
NANO-FORMULATIONS THAT TARGET HER2 Ligand free nano-formulations generally possess passive targeting property with little tissue specificity. In order to address these limitations, research has been continued to advance active or specific targeting to enhance the efficacy of anticancer therapeutic agents as well as to reduce the toxicity to non-targeted healthy tissues. This review will describe some of the key experimental strategies adopted by the researchers to design pharmaceutical formulations employed for targeted drug delivery against HER2 overexpressed or positive breast cancer. More precisely, it will focus on the steps to functionalize the nanoparticles prepared using lipid based or amphiphilic materials (e.g. liposomes, polymersomes, dendrimers, micelles) and non-amphiphilic or polymeric materials (e.g. poly-lactic-co-glycolic-acid or PLGA). Several other types of nano-formulations (such as solid lipid nanoparticles, inorganic nanoparticles) conjugated with antiHER2 ligand and targeting HER2 receptors with the aim of treating HER2 positive breast cancer are summarized in the Table 2.
To deliver therapeutic drugs or pharmaceuticals, liposomes have been validated as an important biocompatible
Fig. (1). N-terminal cysteine rich extra cellular domain (ECD) structural configuration of HER2 (wikimedia.org).
Nano-pharmaceutical Formulations for Targeted Drug Delivery
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Cell Survival PI3K-mTOR Dependent Pathways Cell-Cycle Progression Normal HER2 Signaling Pathway
Dimerization/ Activation of HER2 Cell Proliferation/ Gene Transcription STAT-MAPK Dependent Pathways Cell Motility/ Cytoskeletal Organization
Fig. (2). HER2 signaling pathways.
carrier in medicine and biological science since their inception by A.D. Bangham in 1961 [26]. The key components of liposomes are amphiphilic unilamellar or multilamellar membranes of nontoxic phospholipids (Fig. 3). Liposomes can be produced by different methods such as heating, sonication, extrusion, solvent injection, and reversephase evaporation in the presence of surfactants [27, 28]. Liposomes form small nanoparticles that are generally found in a range from 80 to 100 nm in average size, and their amphipathic nature enables them to incorporate hydrophilic agents into their hydrophilic aqueous core and hydrophobic agents into the lipid bilayer region [29]. This unique property of liposomes brought the attention of researchers to design targeted drug delivery system to treat cancer. Generally, chemotherapeutic agents have a narrow therapeutic index, and are poor metabolizers with poor solubility profiles and exhibit higher side effects because of higher required treatment doses. Liposomes are small spherical vesicles that can improve the pharmacokinetic properties and the solubility profiles of chemotherapeutic drugs. In addition, the surface environment, composition, and pH of liposomes are the most important variable factors for the release kinetics of drugs from liposomes [30]. However, one significant limitation of liposomal formulations is associated with their stability, which can influence the release kinetics of drugs from the nano-system particularly after intravenous (IV) administration. In addition, optimal drug release from liposomes is an important consideration for liposomal formulation that is still under investigation. Moreover, due to enhanced permeability and retention (EPR) effect; liposomes can passively accumulate into the tumor cells through leaky vasculature of cancer tissue endothelium at higher concentrations in comparison to the normal cells, which are naturally protected by healthy tissue endothelium [31, 32]. Antibodies or ligands can be attached to the surfaces of the liposomes that recognize the receptor or tumor epitopes to block receptor mediated signaling pathways or bind with the tumor cell for subsequent receptor mediated
transport or endocytosis. In addition, proteins, peptides or drugs can be incorporated into the liposomes to regulate immune function [33]. However, the transportation of therapeutic agents using liposomes has been shown to be impacted due to the lack of receptor or vector mediation [34]. Although many studies show liposomes as suitable targeted vehicles, they still exhibit instability [35]. Quick elimination from blood is a rate limiting factor for liposomal formulations, however surface modification properties enable liposomes to reside in the circulatory system for a longer period of time to enhance controlled efficacy of released drugs [36]. Nobs et al. (2004) reported that thiol functionalized ligands (anti-HER2 monoclonal antibody) can be conjugated with the maleimide containing liposomes following simple adsorption technique to form immunoliposomes. Internalization of these antibody coupled immunoliposomes by HER2 positive breast cancer cell line (SK-BR-3) was observed with enhanced effects compared to the control liposomes [37]. Puri et al. (2008) demonstrated that thermo-sensitive liposomes could be coupled with HER2 specific stable biomolecule like affibody (ZHER2.342Cys) followed by covalent coupling-interaction with maleimide group of pegylated (PEG: poly ethylene glycol) lipids to prepare HER2 specific affisomes [38] to improve targeting ability for the treatment of breast cancer. In studies by Park et al. (2002) and Kirpotin et al. (1997) site-specific anti-HER2 immunoliposomes developed with a specific conjugation method can be successfully used as vehicles to carry cytotoxic drug (doxorubicin) to the HER2 overexpressing cells [39, 40]. The conjugation process to prepare the drug loaded anti-HER2 immunoliposomes is followed by the thio-ether linkages between maleimide group on surface of liposome and the free thiol adjacent to COOH ended fragment of the monoclonal antibody (MAb). In these studies, researchers demonstrated that the drug loaded immunoliposomes are long circulating liposomes that are stable due to polymeric coating of poly ethylene glycol (PEG) such that free thiol of monoclonal antibody (mAb) is
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Targeting Agent
Protected Layer Hydrophilic Core Hydrophilic Therapeutic Agent
Bilayer Phospholipid Hydrophobic Therapeutic Agent Fig. (3). The schematic structure of liposomes exhibiting their function as nanoparticles.
conjugated with maleimide group of PEG through PEG-mAb linkage [39, 40]. Researchers have also reported that in-vivo application of these drug-loaded immunoliposomes in animal models did not result in high localization to tumor cells. However, HER2 overexpressed breast cancer cells were able to internalize or uptake the anti-HER2 immunoliposomes at a significantly higher rate than the non-targeted sterically stabilized liposomes [41]. The reason for lower localization to tumor cells in animal models could be attributed to the nature of the tumor, which may not exhibit similar EPR effects. In addition, the penetrability of the circulatory vessels will differ between tumors [42]. Nielsen et al. (2002) revealed that doxorubicin containing F5-scFV antibody targeted liposomes actively attenuated the size and proliferative activities of ErbB2 (HER2/neu) overexpressed tumor cell lines compared to the non-targeted drug loaded liposomes [43]. In another animal model (female NCr nu/nu mice), Laginha et al. (2008) reported maximum cytotoxicity effects for tumor regression by doxorubicin loaded targeted liposomes compared to non-targeted liposomes. These researchers also reported the highest intracellular localization of the drug concentration through the receptor mediated endocytosis of targeted liposomes against HER2 overexpressed tumorigenic cell line BT-474 M3C5, a subculture of BT-474 cell line from tumors of nude mice [44]. Anti-HER2 affibody ligands are small sized strong mimics of specific monoclonal antibodies, which recognize protein sequences of the target HER2 receptor. Alexis et al. (2008) assayed paclitaxel loaded HER2 specific affibody fabricated nanoparticles (affisomes) to quantify toxicities against HER2 expressing cancer cell lines SKBR-3 (cell viability: 70±5% and p
