Real-time monitoring of sustained drug release using the optical properties of porous silicon photonic crystal particles

Abstract

A controlled and observable drug delivery system that enables long-term local drug administration is reported. Biodegradable and biocompatible drug-loaded porous Si microparticles were prepared from silicon wafers, resulting in a porous 1-dimensional photonic crystal (rugate filter) approx. 12 μm thick and 35 μm across. An organic linker, 1-undecylenic acid, was attached to the Si-H terminated inner surface of the particles by hydrosilylation and the anthracycline drug daunorubicin was bound to the carboxy terminus of the linker. Degradation of the porous Si matrix in vitro was found to release the drug in a linear and sustained fashion for 30 d. The bioactivity of the released daunorubicin was verified on retinal pigment epithelial (RPE) cells. The degradation/drug delivery process was monitored in situ by digital imaging or spectroscopic measurement of the photonic resonance reflected from the nanostructured particles, and a simple linear correlation between observed wavelength and drug release was observed. Changes in the optical reflectance spectrum were sufficiently large to be visible as a distinctive red to green color change.

Figure 1

Representative white light reflectance spectrum from a single porous Si particle immersed in buffer solution.

Figure 2

In vitro release of daunorubicin covalently attached to porous Si microparticles. (a) Control experiment, showing the rapid clearance (t_1/2 = 5 h) of an aliquot of free daunorubicin injected into the flow chamber at time t = 0 and then eluted with a continuous flow of 450 µL/h of pure PBS solution. The solid line shown is the calculated % cleared as a function of time in hours, based on the elution rate and chamber volume. (b) Percent of daunorubicin (solid circles, “drug”) and silicon (open circles, “Si”) released from porous Si microparticles into the effluent stream under the same flow conditions as in (a). Note the time axis here is in units of days. The effective t_1/2 for clearance of the covalently attached drug from the chamber is increased from 5 h (for free drug) to 15 d. Soluble silicon species (orthosilicate ions) and soluble daunorubicin quantified by ICP-OES and fluorimetry measurements, respectively. (c) Steady-state concentration of daunorubicin in the chamber as a function of time for the experiment described in (b). A therapeutic level of drug is maintained for > 30 d.

Figure 3

Optical microscope images showing the color change of particles as they degrade in pH = 5, 7, or 9 buffer solutions. The degree of color change correlates with the amount of drug released (see Figure 4). The scale bar is 100 µm.

Figure 4

Quantification of drug release, silicon dissolution, and spectral peak shift as a function of time for drug-loaded porous Si photonic crystal microparticles in aqueous buffer solutions at pH 5, 7, and 9. Degradation of the microparticles releases soluble silicon in the form of orthosilicate (a) and daunorubicin (b) at a rate that increases with increasing pH. The average value of the spectral maximum measured from the particles shifts to shorter wavelengths with time (c), and the rate of this shift also increases with increasing pH. The shift correlates with percent of daunorubicin cumulatively released from the particles in an approximately linear fashion over the range 0–60% drug released (d). All particles were prepared and loaded with drug using the same protocol, and results are presented for the three different solution pH values indicated. The data represent measurements made over a period of 7 days. Peak shifts in (c) and (d) are calculated as λ_t − λ₀, where λ_t is the average wavelength of the spectral reflectivity peaks determined at time t and λ₀ is the average wavelength at time t = 0 (measured on > 100 particles in 5 separate scans). The negative values of peak shift indicate a blue shift in the spectral peak; typical initial peak position was 660 nm.

Figure 5

Distribution of values of λ_max in an ensemble of particles, monitored during degradation in aqueous solution. Data for sample A were obtained on the first day after immersion. Data for samples B, C, and D were obtained on day 7, and they correspond to pH 5, 7, and 9, respectively. Each bar represents a 4 nm wavelength range about the value indicated on the x-axis. Photographic color images of the representative ensembles are shown at the bottom. The scale bar is 100 µm.

Figure 6

Viability of ARPE-19 cells (MTT assay) after exposure to as-received daunorubicin, silicon released (as soluble orthosilicate) from porous Si microparticles (containing the undecylenic acid linker chemistry but not containing any drug), and daunorubicin that had previously been attached to porous Si microparticles and then released by hydrolysis into aqueous solution. The x-axis displays the daunorubicin concentration in terms of the total amount of daunorubicin added per milliliter of media. The “Released Si” trace represents the soluble fraction of material released from a mass of porous Si microparticles that is equal to the mass of porous Si microparticles used in the “Released Daunorubicin” trace. Cell proliferation assessed 48 h after sample introduction.

Figure 7

Fluorescence microscope images of ARPE-19 cells comparing the toxicity of as-received daunorubicin (“Free Drug”), daunorubicin that had previously been attached to porous Si microparticles (“Released Drug”), and the soluble by-products of porous Si dissolution from non drug loaded porous Si (“Released Si”). A control experiment using PBS buffer only is shown for comparison (“Control-PBS”). The concentration of daunorubicin in the “Free Drug” and “Released Drug” images is 0.53 µg/mL. For the “Released Si” image, the soluble fraction of material released from a mass of porous Si microparticles equal to the mass of porous Si microparticles used in the “Released Drug” image was used. Green and red in images corresponds to calcein AM (live cells) and ethidium homodimer-1 (dead or damaged cells), respectively. The scale bar is 100 µm. Toxicity assessed 48 h after sample introduction.

Scheme 1

Loading of daunorubicin into porous Si microparticles. The process involves functionalization of the hydrogen-terminated porous Si surface (Si-H) by microwave-assisted hydrosilylation of undecylenic acid (a), followed by EDC-mediated coupling of daunorubicin via the pendant amino group (b).