ARTICLE pubs.acs.org/Langmuir
Fluorescent Nanoparticles of Chitosan Complex for Real-Time Monitoring Drug Release Wei Cui,† Xuemin Lu,† Kun Cui,† Jun Wu,† Yen Wei,*,‡ and Qinghua Lu*,† †
School of Chemistry and Chemical Engineering, the State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China ‡ Department of Chemistry, Tsinghua University, Beijing 100084, China
bS Supporting Information ABSTRACT: New types of fluorescent nanoparticles (FNPs) were prepared through ionic self-assembly of anthracene derivative and chitosan for applications as drug delivery carriers with real-time monitoring of the process of drug release. Because of the presence of the hydrophilic groups, these FNPs showed excellent dispersion and stability in aqueous solution. The structure and properties of the FNPs were investigated by using means of 1H NMR, FTIR, SEM, dynamic light scattering (DLS), and so on. The potential practical applications as drug delivery carriers for real-time detection of the drug release process were demonstrated using Nicardipine as a model drug. Upon loading the drug, the strong blue fluorescence of FNPs was quenched due to electron transfer and fluorescence resonance energy transfer (FRET). With release of drug in vitro, the fluorescence was recovered again. The relationship between the accumulative drug release of FNPs and the recovered fluorescence intensity has been established. Such FNPs may open up new perspectives for designing a new class of detection system for monitoring drug release.
’ INTRODUCTION Drug delivery systems (DDS) are intensively investigated in the recent years for their great potential to improve the therapeutic index of small molecules drugs.13 Various carriers for drug molecules have been reported, such as liposomes,4,5 micelles,68 gelatin,9,10 and nanoparticles.1113 To evaluate the DDS efficacy, it is necessary to obtain crucial data on exact time and location of drug release from the carriers. Despite the intensive study on the pharmacokinetic,14,15 cell permeation efficiency,16 and pathway17 of DDS, few reports focused on the real-time acquisition of these crucial data of the drug molecules released from the delivery carriers. Weinstain et al.18 reported a coumarin-based drug delivery system with real-time monitoring of drug release. In this system, drug molecules as end units could be detached from the DDS after undergoing a specific activation. At the same time, the fluorescence was generated and drug release process could be observed using the noninvasive fluorescence detection techniques. Feng et al.19 also prepared FNPs from conjugated polymer (PFO) and poly(L-glutamic acid) and used them for loading anticancer drug doxorubicin by covalent linkage. This complex system can be also used to monitor the doxorubicin release by using fluorescence “turn-on” signal of PFO. Wu et al.20 reported an oxidation-triggered release of fluorescence molecules based on mesoporous Si microparticles. However, most efforts of DDS based on FNPs for real-time monitoring drug release are concentrated on using covalent linkage for fluorescent tectonic units attached in the polymeric chains to construct FNPs, which often involves lengthy and multistep synthesis and delicate design of r 2011 American Chemical Society
the polymer architecture. Moreover, uncontrollable assembly structure of matrix and heavy aggregation of the obtained nanoparticles make it still a challenge to seek an applicable approach to detect the crucial data of drug molecules released from DDS. Here we report a facile method to prepare noncovalent FNPs which can be used to monitor the real-time release of drug molecules. The FNPs were prepared by forming complexes between chitosan and fluorescent molecules through ionic selfassembly and hydrophobic/hydrophilic interactions. Compared with previous report on FNPs preparation, this approach was mainly based on the ionic interaction between cationic chitosan and anionic fluorescent unit. So there is a wide range of choice to select functional molecules according to different requirement. The obtained FNPs exhibited excellent dispersive properties and stability in aqueous media. Nicardipine as model drug molecule was encapsulated inside the FNPs during self-assembly due to its hydrophobic property.21 The fluorescence of FNPs was quenched effectively by Nicardipine due to the electron transfer and fluorescence resonance energy transfer (FRET) between the fluorescent molecules and drug molecules. Upon releasing the drug molecules from the FNPs, the fluorescence was regenerated, and the extent of fluorescence recovery was dependent on the amount of drug molecules released. Received: February 11, 2011 Revised: May 5, 2011 Published: June 10, 2011 8384
dx.doi.org/10.1021/la200552k | Langmuir 2011, 27, 8384–8390
Langmuir
ARTICLE
Scheme 1. (a) Synthetic Pathway of 4-(Anthracen-9-ylmethoxy)-4-oxobutanoic Acid (AN) and FNPs of ANCHC and (b) Formation of Drug Loaded FNPs Where Drug Release from FNPs Results in Fluorescence Recovery
’ EXPERIMENTAL SECTION Materials. Chitosan hydrochloride (CHC) (water-soluble, deacetylation degree of 80.0%90.0%, and viscosity 10120 mPa 3 s) was purchased from Golden-shell Biochemical Co., Ltd. (China). 9-Anthracenemethanol and Nicardipine were purchased from Alfa Aesar and Sigma. Succinic anhydride (chemical pure), pyridine, sodium bicarbonate, hydrochloric acid, and organic solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received without further purification. The water used was doubly distilled. Preparation of FNPs of ANCHC. 4-(Anthracen-9-ylmethoxy)4-oxobutanoic acid (AN) was prepared according to previously reported procedures in the literature.22 As a typical procedure to generate ANCHC complex, 1.0 mL of various concentrations of AN tetrahydrofuran solutions (0.102.7 mg mL1) was added dropwise to 9.0 mL of chitosan hydrochloride aqueous solution (0.30 mg mL1). At the same time, ANCHC complex self-assembled to form FNPs due to hydrophobic interactions between AN molecules and CHC. After stirring for 2 h at room temperature, the solutions of FNPs were passed through a membrane filter (pore size: 0.8 μm, Millipore) and stored at room temperature. Unless otherwise noted, the FNPs presented in this work refer to the 12% of charge ratio. The charge ratio (Z value) is the ratio of anionic charge to cationic charge. The cationic charge amount of CHC was calculated based on a deacetylation degree of 80.0%. Drug Loading and in Vitro Drug Release. For drug loading, 0.10 mL of Nicardipine in methanol solution (520 mg mL1) and 1.0 mL of AN tetrahydrofuran solutions (0.40 mg mL1) were in sequence added dropwise to 9.0 mL of chitosan hydrochloride aqueous solution (0.30 mg mL1). After stirring for 2 h, the solution was exposed to the air atmosphere, allowing the organic solvent to evaporate. Excess of unloaded Nicardipine was removed by repeated centrifugation and filtration for three times.23,24 Three samples with drug feeding of 0.16, 0.32, and 0.64 w/w based on weight of complex were prepared and recorded as FNPs-1, FNPs-2, and FNPs-3.
For drug release experiments in vitro, 10 mL of Nicardipine loaded FNPs solution was placed into dialysis membrane (MW cutoff 3500) and dialyzed against 250 mL of distilled water at room temperature. Aliquots of 3.0 mL were withdrawn from the solution periodically. The volume of solution was maintained constant by adding 3.0 mL of distilled water after each sampling. The amount of Nicardipine released from FNPs was measured using UV absorbance at 236 nm.
Measurement of Critical Aggregation Concentration (CAC). In this experiment, the FNPs solution prepared as described above was lyophilized to obtain the solid sample. Then by diluting lyophilized solid sample with water, various concentrations of FNPs solution were obtained. 25 μL of 1.6 104 M Nile red in acetone was added into an empty vial and evaporated to dry. Then 4.0 mL of various concentrations of FNPs solution was added. The final concentration of Nile red in the solution was 1.0 106 M. The FNPs solutions containing Nile red were equilibrated at room temperature for 24 h before measurement. Fluorescence emission spectra of the solutions were excited at 550 nm and recorded in the range from 600 to 750 nm on a fluorescence spectrometer. Measurements of Fluorescence Recovery. The 10 mL of Nicardipine loaded FNPs solution was placed into dialysis membrane (MW cutoff 3500) and dialyzed against 250 mL of water at pH 7.4 or pH 5.0 (pH value was adjusted with 0.5 M HCl and 0.5 M NaOH at room temperature). At various time intervals, FNPs solution was taken out from dialysis membrane and transferred to 25 mL volumetric flask. The volume of solution was kept constant by adding distilled water into volumetric flask after each sampling for fluorescence measurement. Fluorescence emission spectra of these solutions were excited at 365 nm and recorded in the range from 380 to 500 nm on a fluorescence spectrometer. Instruments and Characterization. The samples for Fourier transform infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy were prepared by lyophilization of the FNPs solution. FTIR spectra were recorded on a Perkin-Elmer Paragon 1000 8385
dx.doi.org/10.1021/la200552k |Langmuir 2011, 27, 8384–8390
Langmuir
Figure 1. FTIR spectra of (a) AN, (b) CHC, and (c) FNPs. FTIR spectrometer using pressed thin transparent disks of the samples mixed with potassium bromide. Spectra were obtained by collecting and averaging 64 scans. 1H NMR studies were carried out on a Varian Mercury Plus 400 MHz spectrometer in D2O or DMSO-d6 solvent. UV absorption was recorded on a Perkin-Elmer lambda 20 UVvis spectrophotometer. Fluorescence emission spectra were measured on a Photon Technology International QM-4 luminescence spectrometer. The fluorescent morphologies of FNPs were observed with optical microscopy (Leica DM4500 B). The FNPs solution was dropped onto glass slide and dried under the vacuum for SEM observation. Emission scanning electron microscopic (SEM) characterization was carried out on a JEOL JSM-7401F electron microscope operating at 5 KV. DLS and zeta potential measurements were performed using a Malvern Instruments Zetasizer Nano ZS.
’ RESULTS AND DISCUSSION The preparation of FNPs is illustrated in Scheme 1. Chitosan with cationic amino groups in its backbone at low pH (pH < 6.5) provides a platform to construct various functional materials by ionic self-assembly with various opposite charged components.25,26 AN with blue fluorescence was chosen as anionic tectonic unit. FNPs of ANCHC were prepared through electrostatic interactions of cationic chitosan hydrochloride with anionic AN molecules at pH 6.0. AN in the solution with pH 6.0 should be deprotonated according to its pKa. Comparing with butanoic acid with pKa 4.83, the presence of an electron-withdrawing ester group in AN structure shifts electron density away from the proton and results in a lower pKa (
