Elsevier

Journal of Controlled Release

Volume 196, 28 December 2014, Pages 87-95
Journal of Controlled Release

Cytotoxicity and internalization of Pluronic micelles stabilized by core cross-linking

https://doi.org/10.1016/j.jconrel.2014.10.001Get rights and content

Abstract

A UV-cross-linkable agent was incorporated and polymerized in Pluronic micelle core to create an interpenetrating polymer network (IPN) of poly(pentaerythritol tetraacrylate). This stabilization prevented micelle disruption below the critical micelle temperature (CMT) and concentration (CMC), while maintaining the integrity of the PEO corona and the hydrophobic properties of the PPO core. The prepared stabilized spherical micelles of Pluronic P94 and F127 presented hydrodynamic diameters ranging from 40 to 50 nm.

The stability of cross-linked Pluronic micelles at 37 °C in the presence of serum proteins was studied and no aggregation of the micelles was observed, revealing the colloidal stability of the system. Cytotoxicity experiments in NIH/3T3 mouse fibroblasts revealed that the presence of the cross-linking agent did not induce any further toxicity in comparison to the respective pure polymer solutions. Furthermore, stabilized micelles of Pluronic P94 were shown to be less toxic than the polymer itself. A hydrophobic fluorescent probe (Nile red) was absorbed in the cross-linked core of pre-stabilized micelles to mimic the incorporation of a poorly water-soluble drug, and the internalization and intracellular localization of Nile red was studied by confocal microscopy at different incubation times. Overall, the results indicate that Pluronic micelles stabilized by core cross-linking are capable of delivering hydrophobic components physically entrapped in the micelles, thus making them a potential candidate as a delivery platform for imaging or therapy of cancer.

Introduction

Polymeric micelles composed of amphiphilic block copolymers have been shown to be promising nanocarriers for tumor imaging and drug delivery applications. Above their critical micelle temperature (CMT) or concentration (CMC), the block copolymers self-assemble into supramolecular structures composed of an inner hydrophobic core and an outer hydrophilic shell. Therefore, the CMT or CMC are defined as the minimum temperature or concentration, respectively, at which single polymer chains (unimers) self-assemble to form micelles.

The inner hydrophobic domain formed in the micelles has been used to incorporate hydrophobic anticancer drugs in order to increase their solubility, and improve the in vivo drug bioavailabiliy [1], [2], [3], [4], [5], [6], [7]. Several nanocarriers have been engineered to meet these goals, but only a few have received regulatory approval [7], [8]. Among those, the Pluronic-based delivery system has been shown to be very promising due to its nonionic and biocompatible properties, as well as its ability to incorporate hydrophobic drugs in the core [3].

Pluronics are triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO–PPO–PEO) that can self-assemble into micelles in aqueous medium. However, unlike some kinetically stable polymeric micelles with ‘glassy’ cores (e.g. poly(styrene)-poly(ethylene oxide)), micelles composed of Pluronic block copolymers are thermodynamically stable (‘soft’ cores) which leads to micelle aggregation or dissociation upon variations in concentration and/or temperature.

Therefore, the main concern regarding the clinical use of these polymeric micelles is the extreme dilution they undergo once they are in the bloodstream. This dilution can easily reach a total concentration below the CMC, which will result in micelle dissociation into unimers in vivo [1], [2], [3], [4], [5], [9], [10], [11], [12]. Due to this fact, the in vitro and in vivo studies of Pluronic micelles as supramolecular carriers have been limited. Through the use of different concentrations of polymer solution (in order to be below or above the CMC), researchers have shown that the association state, unimers versus micelles, has a very important effect on the circulation half-time life, biodistribution and intracellular fate of the carriers [10], [11], [12], [13], [14]. Thus, Pluronic micelles require further stabilization to prevent dissociation caused by severe dilution in the bloodstream after intravenous injection.

To control this effect, the stabilization of Pluronic micelles has been addressed by different groups in recent years. To our knowledge, Rapoport and co-workers were the first to explore three different mechanisms to stabilize Pluronic micelles: direct radical crosslinking of the micelle core with a hydrophobic radical initiator (benzoyl peroxide), introduction of low concentrations of a vegetable oil, and formation of an interpenetrating polymer network (IPN) [9]. The third route was the most successful and was based on the polymerization of a temperature-responsive low critical solution temperature (LCST) hydrogel (N-isopropylacrylamide, NiPAAm) inside the micelle core. The diameters of the micelles ranged from 30 to 400 nm depending on Pluronic P105 concentration. This formulation was named Plurogel® [9].

Pruitt et al. in collaboration with Rapoport used the same principle to polymerize N,N-diethylacrylamide (NNDEA) in the Pluronic P105 micelle core. The size of the stabilized micelles depended on the Pluronic concentration and temperature. The diameters ranged from 120 to 490 nm at 25 °C, and from 50 to 390 nm at 37 °C [15].

The major problems of this cross-linking method are related to the toxicity of the acrylamide derivatives, the toxic potential of the other cross-linking agents and radical initiators used, the large increase in the micellar size compared to the pure Pluronic micelles, and the limited stability of the obtained micelles (days to weeks) [5], [9], [16], [17].

More recently, Petrov et al. used the same approach to create an IPN of poly(pentaerythritol tetraacrylate) (PETA) in the micelle core [18]. Stabilized micelles of Pluronic F68 had average diameters ranging from 32 to 50 nm depending on the concentration of Pluronic and cross-linking temperature. Yoncheva et al. in collaboration with Petrov also stabilized Pluronic F38 micelles using the same approach to deliver the anticancer hydrophobic drug Paclitaxel. In this study, the micelle diameters obtained were considerably larger (180 nm) [19].

Other groups have focused on the chemical modification of the PEO [20], [21], [22], [23], [24], [25] or PPO [26], [27] blocks in order to covalently cross-link the micelle structure. However, in some of these studies, the introduction of chemical modifications appears to influence the micellization properties of Pluronics, since the reported sizes are much larger than the pure micelles, which are not consistent with the structure of a core/shell Pluronic micelle.

A key point in choosing a strategy to stabilize the micelle structure is to assure the integrity of the PEO corona. In the first stage, this will provide colloidal stability to the system. In a later stage, the shielding properties of the PEO corona will be preserved and will prevent unwanted in vivo interactions such as opsonization and uptake by the reticuloendothelial system (RES) [1], [2], [9]. Therefore, the ideal stabilization strategy should be confined to the micelle core without compromising the structure and flexibility of the PEO corona.

In this work, we have used a non-covalent approach and adapted the protocol developed by Petrov et al. [18]. Two polymers with different molecular weight and different PEO:PPO composition were chosen (Pluronics F127 and P94). Pluronic F127 was selected due to its known biocompatibility, wide use in pharmaceutical formulations and FDA approval for intravenous use in humans. Pluronic P94 has a lower molecular weight and a much higher PPO content than Pluronic F127. In this way, the applicability of the cross-linking process on stabilizing Pluronic micelles with different molecular weight and PPO:PEO composition was assessed. The efficiency of the cross-linking technique was evaluated by studying the physical stability of the pure and cross-linked micelles below the CMT and CMC, and in the presence of serum proteins by light scattering. The cytotoxicity of the cross-linked micelles on mouse NIH/3T3 fibroblasts was evaluated by the MTT assay and compared with the raw polymers. To evaluate the stabilized micelle potential to load and deliver a hydrophobic molecule, pre-stabilized micelles were loaded with a highly hydrophobic fluorescent dye (Nile red) to mimic a hydrophobic anticancer drug, and the intracellular localization of Nile red was evaluated by confocal microscopy at different incubation times.

Section snippets

Materials

Pluronic P94 (Mn = 4600 gmol 1, 60% PPO) was kindly supplied by BASF Ltd. Pluronic F127 (Mn = 12600 gmol 1, 30% PPO) was obtained from K. Morawska (Ghent University, Belgium). Pentaerythrol tetraacrylate (PETA), Dulbecco's Modified Eagle's Medium (DMEM) with and without phenol red, Newborn Calf Serum (NCS), penicillin–streptomycin (10,000 units penicillin and 10 mg streptomycin/mL), 0.25% (w/v) Trypsin-EDTA solution, Dulbecco's Phosphate Buffered Saline (PBS), Thiazolyl Blue Tetrazolium Bromide (MTT),

Stabilized Pluronic micelles by core cross-linking

The stabilization of Pluronic micelles was obtained through incorporation of a hydrophobic cross-linking agent and subsequent photo-polymerization. A scheme of the procedure used is depicted in Fig. 1. A more detailed description of the photo-polymerization set-up can be consulted in the Supplementary information.

Firstly, a mixture between the Pluronic aqueous solution and the hydrophobic cross-linking agent (pentaerytrol tetraacrylate, PETA) was prepared below the CMT. Secondly, the

Discussion

In this study, Pluronic stabilized micelles were prepared by photo-cross-linking of a hydrophobic molecule. This compound was solubilized in the micelle core and polymerized to create an interpenetrating polymer network of poly(pentaerythritol tetraacrylate). The obtained stabilized micelles present hydrodynamic diameters ranging from 40 to 50 nm for both SPM-P94 and SPM-F127. The cross-linked micelles were stable upon temperature decrease below the CMT, upon dilution below the CMC, and in the

Conclusions

In this work, the stabilization of spherical Pluronic micelles has been achieved through the use of a hydrophobic photo-cross-linkable agent. Results reveal a monodisperse population of spherical micelles, with an optimal size range to avoid both rapid clearance of the nanocarriers by the kidneys and retention in the liver [35], [50]. Cytotoxicity and internalization studies demonstrated the non-toxicity of the PETA network and the intracellular localization of Nile red.

Especially for cancer

Acknowledgments

We would like to thank Philippe Mesini for clarifying discussions about photochemical reactions, and Jean-Philippe Lamps for his kind help in the chemistry lab.

The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. PITN-GA-2012-317019 ‘TRACE 'n TREAT’.

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