Pharmaceutical Nanotechnology
Purified and surfactant-free coenzyme Q10-loaded biodegradable nanoparticles

https://doi.org/10.1016/j.ijpharm.2007.07.001Get rights and content

Abstract

The intent of this work was to synthesize and comprehensively characterize ubiquinone-loaded, surfactant-free biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles in vitro. Surfactant-free, empty and ubiquinone (CoQ10)-loaded biodegradable nanoparticles were synthesized by nanoprecipitation, and the physicochemical properties of these nanoparticles were analyzed with a variety of techniques. Nanoprecipitation consistently yielded individual, sub-200 nm, surfactant-free empty and CoQ10-loaded nanoparticles, where the physical and drug encapsulation characteristics were controlled by varying the formulation parameters. CoQ10 release was sustained for 2 weeks but then plateaued before 100% CoQ10 release. A novel, nondestructive purification protocol involving transient sodium dodecyl sulfate (SDS) adsorption to nanoparticles followed by centrifugation and dialysis was developed to yield purified, surfactant-free, CoQ10-loaded nanoparticles. This protocol permitted removal of unencapsulated CoQ10, prevented centrifugation-induced nanoparticle aggregation and preserved the surfactant-free and drug encapsulation properties of the nanoparticles. These CoQ10-loaded nanoparticles are promising as sustained drug delivery devices due to their extended CoQ10 release. Importantly, a surfactant-free nanoprecipitation procedure is presented that in combination with a novel purification step enables the synthesis of individual and purified CoQ10-loaded nanoparticles.

Introduction

Coenzyme Q10 (CoQ10), also called ubiquinone, is an extremely lipophilic antioxidant found in lipid membranes, especially the inner mitochondrial membrane. It is a cofactor in the electron transport chain, where it transfers free electrons from complexes I and II to complex III during oxidative phosphorylation and ATP synthesis (Ebadi et al., 2000). In addition to its bioenergetic and free radical scavenging functions, CoQ10 regenerates alpha-tocopherol, another critical antioxidant (Lass and Sohal, 1998). Experimental evidence established CoQ10's effectiveness at preventing lipid peroxidation (Forsmark-Andree et al., 1997) and decreasing apoptosis (Papucci et al., 2003). Additionally, early clinical trials showed minor improvements in neurological disorders due to CoQ10 therapy (Shults et al., 2002, Shults and Haas, 2005). These promising characteristics have spurred great interest in CoQ10 as a supplemental therapy for cardiovascular and mitochondrial disorders (Mortensen, 1993). Unlike symptomatic treatment, CoQ10 antioxidant therapy may slow the progression and exacerbation of diseases. However, the long isoprenoid side chain imparts extreme hydrophobicity to the CoQ10 molecule and thus renders it poorly bioavailable as a therapeutic. Therefore, there has been much interest in designing new technologies for efficient delivery of this promising lipophilic antioxidant.

Nanoparticles formulated as drug delivery devices are an innovative approach for CoQ10 therapeutics. Biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles are particularly interesting due to PLGA's clinical biocompatibility, tunable biodegradation and resorbable by-products (Hans and Lowman, 2002). PLGA nanoparticles loaded with drugs are preferable to traditional oral, intramuscular or intravenous therapies because they flow through capillaries, protect molecules from enzymatic degradation, enable delivery of non-traditional molecules, are modifiable for receptor targeting and can be internalized by cells for cytoplasmic drug delivery (Soppimath et al., 2001, Sahoo et al., 2002). Furthermore, they solubilize hydrophobic drug molecules, such as CoQ10, by encapsulating and separating drugs from aqueous physiological environments. Motivated by these advantages, several groups have employed emulsion-based techniques to encapsulate CoQ10 within nondegradable micro- and nanoparticles for improved oral bioavailability (Kommuru et al., 2001, Hsu et al., 2003). As an alternative to the aforementioned techniques, we investigated the feasibility of CoQ10 encapsulation within biodegradable nanoparticles using nanoprecipitation, a much simpler synthesis protocol. Since its introduction (Fessi et al., 1989), nanoprecipitation has been applied to formulate polymeric nanoparticles loaded with diverse drug molecules. In this simple and fast technique, a water-miscible organic solvent containing the dissolved polymer (with or without drug molecules) is injected into an aqueous nonsolvent; nanoparticles spontaneously precipitate. A major advantage of nanoprecipitation is that the stabilizing surfactants commonly added during nanoparticle synthesis are not required to prevent nanoparticle aggregation. Therefore, putative disadvantages of surfactants, like questionable biocompatibility, irreversible incorporation in the nanoparticles and alteration of nanoparticle surface properties (Sahoo et al., 2002), are eliminated. Furthermore, the nanoparticles are free for surface chemistry modifications and targeting applications. Here, it was demonstrated that CoQ10-loaded nanoparticles can be synthesized rapidly and simply via nanoprecipitation and that surfactants are not required to prevent nanoparticle aggregation in solution.

In this study, nanoprecipitation was used to produce novel CoQ10-loaded, surfactant-free, biodegradable PLGA nanoparticles, where CoQ10, a promising antioxidant, was controllably incorporated within the nanoparticles. These CoQ10-loaded drug delivery devices may significantly enhance CoQ10's therapeutic efficacy. The physical properties and drug encapsulation characteristics of the nanoparticles were controlled and optimized by varying the nanoprecipitation parameters. Drug release studies demonstrated steady release of CoQ10 for 2 weeks, an exciting result relevant for sustained drug delivery applications. A novel purification protocol is presented which did not alter the initial physicochemical properties of the surfactant-free nanoparticles. Specifically, transient SDS adsorption to synthesized nanoparticles combined with centrifugation and dialysis allowed efficient separation of unencapsulated CoQ10 from CoQ10-loaded nanoparticles without aggregation, which is normally induced by centrifugation in the absence of SDS. These purified CoQ10-loaded biodegradable nanoparticles are promising and novel drug delivery devices.

Section snippets

Materials

Poly(dl-lactide-co-glycolide)–COOH (referred to as PLGA) with 50:50 lactide:glycolide copolymer ratio was purchased from Lactel Absorbable Polymers (Pelham, AL, USA). Coenzyme Q10 was purchased from Spectrum Chemicals and Laboratory Products (Gardena, CA, USA). Omnipur phosphate buffered saline consisting of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4/KH2PO4 at pH 7.4 was referred to as 1× PBS (Fisher Scientific, Pittsburgh, PA, USA). Sodium dodecyl sulfate (SDS) was from Shelton Scientific-IBI

Surfactant-free nanoparticle synthesis and physicochemical characterization

Nanoprecipitation was used to synthesize surfactant-free empty and CoQ10-loaded PLGA nanoparticles. The parameters were first optimized for appropriately small and monodisperse nanoparticles. Diameters under 200 nm were desired because smaller nanoparticles are more easily engulfed by cells (Desai et al., 1997, Win and Feng, 2005) for intracellular drug delivery (Panyam et al., 2002, Elamanchili et al., 2004), and narrowly distributed nanoparticles were desired in order to minimize complications

Conclusions

Nanoprecipitation was a robust and versatile protocol for synthesizing CoQ10-loaded nanoparticles without the interference of surfactants. Control over the physicochemical and drug encapsulation properties was achieved simply by varying the nanoprecipitation formulation parameters, which may motivate other researchers to customize drug-loaded nanoparticles by nanoprecipitation. The extremely hydrophobic nature of CoQ10 facilitated steady CoQ10 release for 2 weeks along with long-term

Acknowledgements

The authors thank Dr. Xin Brown at Boston University for guidance with DLS. Anlee Krupp from Boston University was instrumental in SEM imaging. This work made use of STC shared experimental facilities supported by the National Science Foundation under Agreement Number ECS-9876771. B. Nehilla was supported by a grant from the American Foundation for Aging Research.

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