Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo

Abstract

Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drug-resistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and urea-functionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102 nm with narrow size distribution, were stable in serum-containing solution over 48 h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96 h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. In vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis.

Introduction

The number of cancer-related deaths worldwide is in the increase in spite of the cutting-edge advances made in drug discovery and drug delivery [1], [2], [3]. Various treatment strategies like surgical resection, radiotherapy and systemic therapies are being used. However, the response to the treatments is low, and there is a high rate of recurrence and metastasis [4]. The capability of cancers to recur and metastasize is attributed to the presence of a side population of cancer cells (SP cells, or cancer stem cells) with the ability to self-renew, enhance drug resistance and initiate tumor [5], [6], [7]. Cancer stem cells (CSCs) have been well studied and documented in different types of cancers, and they express various unique surface markers [8], [9], [10]. A number of studies have shown that cancer stem cells were more resistant to chemotherapeutic drugs, and worsened the overall survival [11], [12], [13], [14], [15], [16]. Therefore, an effective treatment strategy should be able to target both cancer cells and CSCs, thereby preventing recurrence and metastasis, and enhancing the survival rate of patients.

Most conventional anti-cancer drugs have little effect on CSCs. This is potentially due to the inherent resistance of CSCs, their over-active efflux pumps that remove the drugs out of the cells and enhanced DNA repair capability [17]. Interestingly, other classes of drugs like anti-psychotics [18], anti-diabetics [19] and anti-helminthics [20] have been found to be effective in eradicating CSCs. Biguanides are a class of anti-diabetic drugs, of which metformin and phenformin have been frequently studied [21]. Both of these drugs have recently been shown to exhibit an antineoplastic effect against several types of cancers especially cancer stem cells [16], [22], [23]. Metformin is a highly prescribed drug for Type-2 diabetes mellitus (T2DM), and is preferred over phenformin because of lesser incidence of lactic acidosis in treated patients [24]. Compared to metformin, phenformin is 50 times more potent than metformin against different cancer cell lines [25]. Like many other drugs, phenformin is metabolized in the liver. The drug was associated with increased incidences of lactic acidosis [26], and was subsequently withdrawn by FDA in 1976 as an anti-diabetic drug [27]. Nanoparticles have been used to deliver drugs and reduce non-specific toxicity towards healthy tissues by exploiting the enhanced permeability and retention (EPR) effect of nanoparticles at leaky tumor tissues [28]. Among various types of nanoparticles, polymeric micelles are most commonly explored for drug delivery due to their unique core/shell structure having a hydrophobic core for encapsulating the drug and a hydrophilic corona that prolongs drug circulation in the blood, and ease of installing functionalities to the core for enhancing interactions with the drug and increasing drug loading level. We hypothesized that formulating phenformin into polymeric micelles would enhance its anti-cancer efficacy and reduce the CSC population without causing toxicity to the liver.

Recently, we reported functional micelles self-assembled from a mixture of poly(ethylene glycol) (PEG)/acid-functionalized polycarbonate diblock copolymer (PEG-b-PAC) and PEG/urea-functionalized polycarbonate diblock copolymer (PEG-b-PUC) via hydrogen-bonding [29]. These micelles had excellent stability due to the presence of hydrogen-bond (urea–urea and urea-acid), exhibiting preferable accumulation at tumor tissues based on the EPR effect [30]. In addition, they had high loading capacity for basic drugs such as doxorubicin [29], [30], [31]. Phenformin is a basic drug, and has two guanidine groups in the molecule, which can form hydrogen-bond with urea group and ionic interaction with acid group in the micellar core. In this study, PEG-b-PUC and PEG-b-PAC were synthesized by organocatalytic ring-opening polymerization (ROP) using methoxy-PEG (M_n 10 kDa) as the macroinitiator. Phenformin was loaded into micelles via self-assembly of PEG-b-PUC and PEG-b-PAC mixture. The phenformin-loaded micelles were characterized for drug loading level, particle size, size distribution, zeta potential, in vitro drug release, and stability in serum-containing solution. According to World Health Organization's 2014 statistics, lung cancer is the leading cause of cancer deaths worldwide in both men and women with 27% of all cancer deaths. Lung cancer also has the lowest 5-year survival rate (16.6%) compared to all other high-incidence cancers like colon (64.2%), breast (89.2%) and prostate (99.2%) (National Cancer Institute. SEER Cancer Statistics Review, 1975–2010). In addition, lung cancer is commonly metastatic, predominantly metastasizing to adrenal gland, bone, brain, liver and the other lung [32]. Therefore, in this study, lung cancer was chosen as a model cancer using H460 human lung adenocarcinoma cell line as an example. Cytotoxicity of phenformin and phenformin-loaded micelles against H460 cells was evaluated by MTS, and their non-specific cytotoxicity was also examined against human dermal fibroblasts (HDFs) and WI-38 human fibroblast like fetal lung cell line. SP (CSCs) and non-SP cells were sorted from H460 cells by flow cytometry, and identified by immuno-staining with anti-CD133-PE antibody. Inhibitory effect of free phenformin and phenformin-loaded micelles on SP and non-SP cells was investigated, and their anti-cancer efficacy was studied in an H460 xenograft mouse model. The population of CSCs in the tumor tissue after treatment with free phenformin and phenformin-loaded micelles was also analyzed.