Balancing the effect of corona on therapeutic efficacy and macrophage uptake of lipid nanocapsules
Introduction
The rapid advancement of nanomedicine has promoted the development of numerous nanosystems that can deliver drugs and other therapeutic agents to target tissues and that often possess complex structures and surface functionalizations [1], [2], [3].
Oil-in-water nanoemulsions, also known as lipid nanocapsules, have been studied in the pharmaceutical and medical fields because they show promise as drug carriers for their controlled and sustained release properties, subcellular size, inexpensive and easy-to-scale production, independence of dilution, and biocompatibility [4]. Generally, these lipid nanocapsules consist of a hydrophobic inner core which can incorporate lipophilic drugs, and a hydrophilic outer shell that provides stabilization and offers the possibility of chemical modifications, among other important properties [5]. Lipid nanocapsules have become increasingly important for drug delivery in the field of cancer treatment, showing greater advantages over other lipid-based nanosystems in terms of leakage and drug loading [6], and they have been shown to be effective carriers for delivering hydrophobic drugs such as docetaxel [7], [8], [9]. Due to their nanoscale size, they can accumulate in tumor tissues more than in surrounding healthy tissue through enhanced permeability and retention effect (EPR) [10], and they can also be engineered and actively targeted with different molecules against overexpressed receptors of cancer cells [11], [12], [13].
Following intravenous administration, a nanoparticle is exposed to an evolved combinatorial system containing thousands of different proteins alongside lipids and sugars which can reconfigure nanoparticle dispersion and surface characteristics, forming a “corona” [14], [15], [16], [17], [18], [19]. Recently, it has been shown that a biofunctional nanoparticle, quite basic in design, while showing specific recognition of biological receptors under model in vitro conditions, can lose uptake and receptor specificity as the complexity of the environment is increased by introducing human plasma [20]. The proteins adsorbed onto the original nanoparticle surface can mask targeting ligands and furthermore can interact with specific plasma membrane receptors on monocytes and various subsets of tissue macrophages, promoting rapid recognition and removal of the intravenously injected nanoparticles. Therefore, pharmacokinetics and biodistribution of nanoparticles are to a large extent governed by their surface properties, which in turn depend on the shell composition [21] and can be strongly altered upon interactions with complex biological environments [22].
To prolong their half life in the bloodstream by means of avoiding recognition by the mononuclear phagocyte system (MPS), different grafting materials have been tailored in drug delivery systems by using a variety of polymers such as poly (ethylene glycol) (PEG) and triblock copolymers (poloxamers and poloxamines). PEG is a flexible, electrically neutral, and hydrophilic polymer that has been commonly used to coat nanoparticles, to decrease the interaction of the surface with serum components, and to prolong particle circulation [23], [24], [25], [26]. Poloxamers (Pluronic®) and poloxamines (Tetronic®) have been used for the same purpose with different levels of success [27], [28], [29], [30]. They are amphiphilic nonionic block polymers composed by hydrophobic propylene oxide (PO) fragments and hydrophilic ethylene oxide (EO) branches. Poloxamers consist of a central poly-propylene oxide (PPO) backbone that is adsorbed onto hydrophobic interfaces, which is flanked on both sides by two hydrophilic chains of polyethylene oxide (PEO) that remain extended in the hydrophilic phase, yielding structures of the (PEO)a-(PPO)b-(POE)a type [31]. Another important property presented by poloxamer and poloxamines is the inhibition of multidrug resistance [32], [33]. Most applications of triblock copolymers are based on their spontaneous self-assembly, leading to structures with a hydrophobic core (PPO) and a hydrophilic shell (PEO) [34], [35], which however still present problems of low stability and degradation. Thus, an emerging application of poloxamers is their use as protective coating for nanocarriers (such as the oil-in-water nanoemulsions presented here), with the central POP block anchored onto the surface of the particles via hydrophobic interactions [36]. However further knowledge concerning the characteristics of pluronics as emulsifiers as well as their interactions with physiological media is needed to provide their rational use in the design of lipid nanocapsules.
Within this scenario the goal of the present study was to synthesize and physicochemically characterize lipid nanocapsule systems with different coatings, in order to study how the surface physicochemical properties of these colloidal particles influence protein adsorption, macrophage association, and uptake by cancer cells. Thus, four lipid core-shell nanosystems have been designed using a simple synthesis process. In all cases the hydrophobic core was constituted by olive oil, while the hydrophilic shell had a different composition. The commercially available and biocompatible surfactants composing the shell were Pluronic F127, also known as poloxamer 407, which has been chosen mainly because of its properties of long-term circulation [37], and Epikuron 145V (a mixture of phospholipid molecules), which provides a negative charge to the nanocapsule surface.
Isothermal titration calorimetry (ITC) is a known method to characterize the thermodynamic parameters of interactions between molecules in solution, but only more recently a few publications have described the use of ITC to study the binding thermodynamics of nanoparticles with proteins [38], [39], [40], [41]. In the present study, ITC has been used to assess the binding thermodynamics of proteins onto the shells of the different lipid nanosystems developed. For this purpose binding studies have been performed using both a simplified model solution of bovine serum albumin (BSA) and a more complex biological fluid containing fetal bovine serum (FBS), to mimic the biological environment to which these nanocapsules are exposed. This allows to study in detail how addition of poloxamer into the formulation controls corona formation, thus also affecting nanoparticle uptake and therapeutic efficacy.
An uptake study in the human macrophage-like U937 cell line was also performed to determine the resistance of these nanocapsules to clearance by these cells depending on their surface composition. For these studies, coumarin 6 was encapsulated in the hydrophobic core of the nanocapsules and experiments were performed both under serum-free (SF) conditions as well as in the presence of serum (complete medium) (cDMEM); also, docetaxel-loaded nanosystems were prepared to perform a cytotoxicity assay in the A549 human lung cancer cell line.
Overall, the approach presented here, where physico-chemical characterization of the different nanocapsules in biological fluids is combined with the assessment of uptake and efficacy in relevant cell systems, allows us to explore how different formulations are processed by cells and the role of the different components in these interactions.
Section snippets
Particle size
The synthesized nanoemulsions were stable under storage conditions –pure water and 4°C– for at least 3 months. The average diameter and PDI of the nanocapsules (see Fig. 1) were, respectively, 158 ± 5 nm and 0.108 for nanocapsules exclusively composed by lecithin (EP nanocapsules), 163 ± 3 nm and 0.110 for ME nanocapsules composed by a mixture of lecithin and poloxamer with a predominance of the first one, 215 ± 29 nm and 0.125 for nanocapsules with both surfactants with a predominance of
Materials
Poloxamer 407 (Pluronic F127), purchased from Sigma–Aldrich (Spain), is a triblock copolymer based on poly (ethylene oxide)-block - poly (propylene oxide)-block - poly (ethylene oxide) structure, expressed as PEOa–PPOb–PEOa being a = 100 and b = 65. The central hydrophobic block of PPO faces the oil phase while the two hydrophilic chains of PEO remain in the aqueous environment. Coumarin 6, sulforhodamine-B, phorbol myristate, acetate, and olive oil were also purchased from Sigma, and all were
Conclusions
In this study, different techniques were used to investigate how the surface composition of lipid nanocapsules influences the protein adsorption and, subsequently, the uptake by cancer cells, therapeutic efficacy, and uptake by macrophages. ITC and electrophoresis were used to characterize the interactions between proteins and the different nanoparticle surfaces, showing that the presence of poloxamer, a non-ionic surfactant, on the surface of lipid nanocapsules significantly reduced the
Acknowledgments
The authors gratefully acknowledge Dr. Mattia Bramini for insightful discussions and technical support with confocal microscopy and Dr. Gustavo Ortiz Ferrón for assistance with flow cytometer. This work was supported by the projects MAT2010-20370 and MAT2013-43922-R (European FEDER support included, MICINN, Spain), PI10/02295 (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, FEDER funds) and P07-FQM2496, P10-CTS-6270 and P07-FQM3099 (Junta de Andalucía, Spain).
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