Paclitaxel distribution in poly(ethylene glycol)/poly(lactide-co-glycolic acid) blends and its release visualized by coherent anti-Stokes Raman scattering microscopy

Abstract

Mechanisms underlying the release of paclitaxel (PTX) from poly(ethylene glycol)/poly(lactic-co-glycolic acid) (PEG/PLGA) blends were investigated by coherent anti-Stokes Raman scattering (CARS) microscopy. PLGA, PEG, and PTX were selectively imaged by using the resonant CARS signal from the CH₃, CH₂, and aromatic CH stretch vibrations, respectively. Phase segregation was observed in PLGA films containing 10 to 40 wt.% PEG in the absence of PTX loading. The PEG phase existed in the form of crystalline fibers in the (8:2, weight ratio) and (7:3) PLGA/PEG films. CARS observation indicated that PTX preferentially partitioned into the PEG domains in the (9:1) and (8:2) PLGA/PTX films, but exhibited a uniform mixing with both PLGA and PEG in the (7:3) PLGA/PEG film. The solid dispersion of PTX into PEG domains was attributed to a strong interaction between PTX and PEG, supported by the disappearance of PEG crystallization in the PTX-loaded PLGA/PEG film evidenced through X-ray diffraction analysis. PTX release was induced by exposing the film to an aqueous solution and monitored in real time by CARS and two-photon fluorescence microscopy. Fast dissolution of both PEG and PTX was observed at the film surface. Upon infiltration of water into the film, the PEG domains were rearranged into ring structures enriched by both PTX and PEG. The CARS data provided visual evidence explaining the accelerated burst release followed by more sustained release of PTX from the PLGA/PEG films as measured by HPLC.

Introduction

PTX has been administered with a great success in the treatment of in-stent restenosis [1], [2], [3]. Being biodegradable and biocompatible, PLGA is widely used for delivery of PTX and other drugs coated on stents [1], [4], [5], [6]. To compromise the brittle mechanical properties of PLGA, PEG of low glass transition temperature was often added as a plastizer to the PLGA matrix [6], [7]. The PEG addition also resulted in accelerated or decelerated drug release in a controlled manner [7], [8], [9], [10]. For instance, adding 2% PEG to PLGA microparticles increased the in vitro release rate of PTX, while higher portion of PEG slowed down the PTX release [10].

In most studies, the drug release profiles were characterized by chemical analysis methods such as HPLC. While convenient and widely used, such chemical analysis does not tell the redistribution of drugs inside a delivery system that may occur during the release process. As a consequence, although the two basic pathways (diffusion and degradation) for drug release from a polymer matrix have been established [11], the detailed mechanisms which may be related to phase segregation according to polymer-drug interactions remain elusive.

Molecular imaging recently emerged as a new tool for drug release studies. FT-IR imaging of PEG dissolution in an aqueous environment has been implemented to investigate the drug release mechanism [12], [13], [14], [15]. While this method provides chemical selectivity, the long excitation wavelength used in FT-IR microscopy offers the spatial resolution of several to tens micrometer, which does not allow the visualization of 3D distribution of drug molecules in thin polymer films or microparticles. Raman microscopy [16], [17], [18] provides better spatial resolution by using a shorter excitation wavelength, but the weak Raman scattering necessitates a long image acquisition time from minutes to hours. A recently developed nonlinear optical imaging technique that is based on coherent anti-Stokes Raman scattering (CARS) circumvents these difficulties. In CARS microscopy [19], a pump laser beam at frequency ω_p and a Stokes laser beam at frequency ω_s are collinearly overlapped and tightly focused into a sample to generate a signal at the anti-Stokes frequency ω_as = ω_p − ω_s + ω_p. CARS microscopy offers the following advantages. First, the CARS signal can be significantly enhanced when ω_p − ω_s is tuned to a Raman-active molecular vibration, which provides the molecular selectivity. Second, the coherent addition produces a large CARS signal, which allows high-speed imaging of dynamic systems. Third, the nonlinear dependence of signal generation on the excitation laser intensity ensures that the CARS signal is only produced at the focal center, providing 3D spatial resolution. Recently, Kang et al. demonstrated CARS imaging of PTX dispersion and coagulation in films of different polymers [20]. In this work, CARS microscopy was used to map the distribution of PTX in spray-coated films of PLGA/PEG blend and to monitor the reorganization of PEG and PTX during the release process. The sample used in our study mimics the film coated on a drug-eluting stent. The CARS imaging results interpret the PTX release profiles measured by HPLC.