New functional degradable and bio-compatible nanoparticles based on poly(malic acid) derivatives for site-specific anti-cancer drug delivery

Abstract

Design of an efficient site-specific drug delivery system based on degradable functional polymers still remains challenging. In this work, we synthesized and characterized three degradable functional polyesters belonging to the poly(malic acid) family: the poly(benzyl malate) (PMLABe), the poly(ethylene glycol)-b-poly(benzyl malate) (PEG₄₂-b-PMLABe), the biotin-poly(ethylene glycol)-b-poly(benzyl malate) (Biot-PEG₆₂-PMLABe). Starting from these building blocks, we were able to prepare the corresponding well-defined degradable functional nanoparticles whose toxicity was evaluated in vitro on normal and cancer cell lines. Results have evidenced that the prepared nanoparticles did not show any significant cytotoxicity even at high concentrations. A model anti-cancer drug (doxorubicin, Dox) or a fluorescent probe (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, DiD oil) has been encapsulated into PMLABe, PEG₄₂-PMLABe or Biot-PEG₆₂-PMLABe based nanoparticles in order to evaluate, respectively, the in vitro cytotoxicity of Dox-loaded nanoparticles on normal and cancer cell lines and the ligand (biotin) effect on cellular uptake in vitro using mmt 060562 cell line. Dox-loaded PMLABe, PEG₄₂-PMLABe or Biot-PEG₆₂-PMLABe nanoparticles showed an in vitro cytotoxicity similar to that of free Dox. Moreover, the DiD oil loaded Biot-PEG₆₂-PMLABe based nanoparticles showed a better in vitro cellular uptake than ligand-free DiD oil loaded nanoparticles. Both results evidence the great potential of such degradable functional poly(malic acid) derivatives for the design of highly efficient site-specific anti-cancer nanovectors.

Introduction

Cancer, characterized by an abnormal and anarchical cell proliferation within a normal tissue of the body, is a very complex disease and a major cause of mortality (Misra et al., 2010). Over the past decade, the better understanding of the molecular basis of cancer unveiled new therapeutic targets especially proteins involved in cell signalling pathways regulating cell division and/or programmed cell death. In the meantime, considerable efforts to prevent and treat this disease have lead to the identification of new drugs with protective effects or anti-cancer properties through the inhibition of cell division and/or induction of apoptosis. Administration of these new potent anti-cancer therapies is however often associated with severe side effects such as hair loss, damages to liver, kidney, bone narrow and heart (Misra et al., 2010, Jaracz et al., 2005, Torchilin, 2006), which limit their use for clinical applications. In this context, numerous research works are focused on the search of optimal cancer therapeutic strategies decreasing significantly the dramatic side effects of the actual chemotherapies (Misra et al., 2010, Jaracz et al., 2005, Torchilin, 2006, Hoffman, 2008, Malam et al., 2009). Therefore, an objective of research in the pharmaceutical and biomedical fields is concerned with the development of drug delivery systems able to protect effectively the drug from non-specific degradation and/or biodistribution, and to target the drug to its site of action (cells, tissues, organs) (Hoffman, 2008, Malam et al., 2009). This concept should allow decreasing not only the administrated doses of drugs but also the non-specific drug distribution. Several kinds of drug delivery systems have been reported, among which the most frequent ones are liposomes formed by the association of lipids (Misra et al., 2010, Malam et al., 2009) and nanoparticles consisting of polymers which may be (bio)degradable or not (Misra et al., 2010, Torchilin, 2006, Hoffman, 2008, Malam et al., 2009, Liu et al., 2009).

While certain PEGylated liposomes containing anti-cancer drugs have reached the market, most of the polymeric nanoparticles are still in the preclinical phase of development (Misra et al., 2010, Torchilin, 2006, Hoffman, 2008, Malam et al., 2009, Liu et al., 2009). However, it is noteworthy that two polymeric nanoparticles formulations are in phase II of clinical trials: the Doxorubicin Trandrug^® produced by the society BioAlliance Pharma (http://www.bioalliancepharma.com) and the Mitoxantrone-poly(alkylcyanocrylate) nanoparticles (Zhou et al., 2009). Improvement of several properties of these polymeric nanoparticles needs to be reconsidered, such as (i) the drug loading efficiency, (ii) the targeting of the site of action, (iii) the particle in vivo stability and the control of drug release, (iv) the improvement of nanovector biocompatibility and its elimination from the organism after tumor treatment.

Within this context, we have focused our attention on the following objectives: (i) synthesis of polymers combining biologically active molecules to a biocompatible and degradable macromolecular backbone, (ii) formulation and characterization of the corresponding biodegradable nanoparticles, (iii) in vitro and in vivo toxicity and a favourable biodistribution, (iv) anti-cancer drug encapsulation for site-specific delivery to liver.

Towards this goal, we have first synthesized a degradable and biocompatible nanoparticle that is derived from poly(malic acid), PMLA shown in Fig. 1.

This polymer is synthesized by certain fungi and slime molds (Rathberger et al., 1999) and has been shown to be non-toxic and biodegradable into malic acid under physiological conditions (Vert and Lenz, 1979). PMLA was originally synthesized for applications in the biomedical field. Moreover, PMLA has been successfully used as platform in the synthesis of nanovectors (Abdellaoui et al., 1998, Osanai and Nakamura, 2000, Cammas et al., 2000, Cammas-Marion and Guérin, 2000, Martinez Barbosa et al., 2004) or as a constituent in macromolecular conjugates bearing several functionalities to treat human brain and breast tumors in mouse models (Fujita et al., 2006, Fujita et al., 2007, Ljubimova et al., 2008, Ding et al., 2010). In all of these investigations it has been concluded that PMLA was a promising building block for the design of efficient drug delivery systems.

PMLA and its derivatives having well-defined structures are accessible either from natural PMLA extracted from fungi or myxomycetes, for example, from the slime mold Physarum polycephalum (Ljubimova et al., 2008) or by anionic ring opening polymerization (ROP) of β-substituted β-lactones (MLAR) synthesized from aspartic acid (Cammas et al., 1996) or malic acid (Cammas et al., 1993). In the frame of the present work, we have chosen to prepare the selected poly(malic acid) derivatives by anionic ROP of MLAR synthesized from aspartic acid. Besides the bio-compatibility, current research in drug delivery aims at designing new nanoparticles that exhibit stealth properties, i.e. a prolonged circulation time in the bloodstream without being recognized by the reticulo-endothelial system, and that preferentially target a specific cell type. For stealth properties, we selected the poly(ethylene glycol), PEG, which is the most widely used material for achieving such steric stabilization (Romberg et al., 2008). We have also selected the biotin (Biot) as a targeting moiety because this vitamin (vitamin B₇ or H) is a promoter of cell growth in many cell types and expression of its receptors is increased in several tumoral cells (Yang et al., 2009).

In this paper, we report the synthesis and characterization of three copolymers of PMLA: (i) a lipophilic polymer, poly(benzyl malate) (PMLABe), (ii) a amphiphilic block copolymer, poly(ethylene glycol)-b-poly(benzyl malate) (PEG₄₂-b-PMLABe), and (iii) a amphiphilic block copolymer possessing a targeting moiety (biotin) at the hydroxyl terminal end of PEG (the hydrophilic block) (Biot-PEG₆₂-b-PMLABe). All these copolymers self-assembled into nanoparticles. These have been characterized and their in vitro cytotoxicity has been evaluated on healthy and cancer cell lines. Moreover, an anti-cancer model drug, doxorubicin (Dox) or a fluorescent probe, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD oil), have been encapsulated into PMLABe, PEG₄₂-b-PMLABe and Biot-PEG₆₂-b-PMLABe nanoparticles. Results obtained for their in vitro cytotoxicity on healthy and cancer cells as well as their in vitro cellular uptake using the mouse breast cancer mmt 060562 cells overexpressing biotin receptor are reported.