Fluorescent Nanoparticles of Chitosan Complex for Real-Time


Jun 10, 2011 - Luminescence carbon dot-based nanofibers for a water-insoluble drug release system and their monitoring of drug release. Yue Zhai , Xue...
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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

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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

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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 (<4.83).27 It was observed that FNPs solution was bluish opalescence when the charge ratio of AN to CHC was fixed at 12% by mole, and no precipitate appeared in solution. After lyophilization treatment, solid FNPs were obtained. The FTIR spectra of AN, CHC, and FNPs are shown in Figure 1. The characteristic peaks at 1732 and 1624 cm1 of the FNPs are assigned to be the stretching vibration of the carbonyl band from the AN unit and amide band from the CHC unit, respectively. A significant shift from 1717 to 1732 cm1 of the carbonyl band of AN unit should be resulted from the complex formation between the carboxyl group of AN and the ammonium group of CHC in FNPs.28,29 Moreover, 1H NMR spectra further confirmed the formation of FNPs as shown in Figure 2. The 1H NMR spectrum of CHC dissolved in D2O is given in Figure 2b. The multiplet signals at 3.04.0 ppm correspond to the ring methenyl protons of CHC. Figure 2c is the 1H NMR spectrum of FNPs dispersed in D2O. The signals of CHC (3.04.0 ppm) were also observed, but the characteristic signals at 8.527.48 ppm for AN molecules are very weak by comparison with 1H NMR spectrum of AN in

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Figure 2. 1H NMR spectra of (a) AN dissolved in DMSO-d6, (b) CHC dissolved in D2O, (c) FNPs dispersed in D2O, and (d) FNPs dissolved in DMSO-d6.

Figure 2a. It is because that hydrophobic groups in FNPs are confined within coreshell structure, thus causing shielding of proton signal of AN molecules.30,31 This shielding effect is further evidenced by the fact that characteristic signals at 8.52 7.48 and 6.2 ppm for AN became much more pronounced when FNPs was dissolved in DMSO, as shown in Figure 2d. The assembling behavior of AN and CHC in aqueous media was investigated using scanning electron microscopy (SEM). As shown in Figure 3a, spherical FNPs are clearly observable in the SEM image, and the size is ca. 200 nm. The formation of FNPs could be attributed to the difference in hydrophobic properties between AN molecules and the CHC chains: AN molecules are highly hydrophobic and CHC chains possess plenty of hydrophilic groups. In order to minimize the system energy of the complex of AN and CHC, AN molecules lies in the inner part of the complex and spontaneously formed FNPs. Particularly, spherical FNPs exhibit a good dispersion in aqueous media. This is very important to the application of nanoparticles because undesired agglomeration within capillaries could result in their occlusion and serious organ damage.32 As shown in Figure 3b, DLS also shows FNPs with the same size (ca. 200 nm) and monodisperse distribution in aqueous media, which agrees well with the results of SEM observation. Figure 3c shows the changes of diameter of FNPs in solution with increasing the charge ratio. The charge ratio (Z value) is ratio of anionic charge to cationic charge. From 3% to 25%, the mean diameter of the FNPs decreased slightly because of the enhancement of the hydrophobic interactions.33 However, as the Z value increases to 80%, the size of FNPs increased from ca. 200 to 500 nm. It might be attributed to the overloading of AN molecules in the aqueous solution, resulting in the formation of FNPs aggregates.34 As shown in Figure 3d, the size distribution of FNPs (Z = 80%) displays bimodal distribution, suggesting that FNPs with several different sizes exist in solution. Therefore, the suitable charge molar ratio of AN to CHC is between 3% and 25% for obtaining FNPs which are applicable in drug delivery. In this work, the Z value of AN to CHC for FNPs was fixed at 12%. The assembly behavior of AN and CHC in aqueous media was further studied using the fluorescence probe technique. Nile red as fluorescence probe has very low fluorescence emission in an aqueous solution due to its hydrophobic nature, and the emission increases significantly once it is transferred into a hydrophobic environment.35 As a control experiment with pure chitosan, the fluorescence intensity of Nile red is constant with increasing CHC concentration, which indicated that pure CHC cannot 8386

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Figure 3. (a) SEM image, (b) size distribution of FNPs at Z = 12%, (c) diameter of FNPs with various Z values, and (d) the size distribution of FNPs at Z = 80%.

Figure 4. (a) Emission spectra of Nile red (1.0  106 M) as a function of various concentrations of the FNPs (λex = 550 nm). (b) Plot of the fluorescence intensity for Nile red from emission spectra (at λem = 660 nm) versus concentration of FNPs.

form nanoparticles (Figure S1). Figure 4 shows the change in the fluorescence emission intensity of Nile red as a function of the concentration of FNPs solutions. The fluorescence intensity of Nile red is almost constant at low concentrations of FNPs solutions, and above a certain concentration, the intensity increases dramatically, indicating the transfer of Nile red into a hydrophobic environment. Such a sudden change of the fluorescent intensity indicated the formation of FNPs from complex of AN and CHC at this concentration. This concentration can be defined as the critical aggregation concentration (CAC). The CAC value of the FNPs at the turning point is 6  102 mg mL1, indicating that such a low concentration of complex of AN and CHC can form FNPs and maintain the stability in dilute condition. Nicardipine as a model drug was loaded in FNPs. It is a medication widely used for the treatment of hypertension, which decrease blood pressure by blocking calcium channels in cardiac muscle and smooth muscles of blood vessels.36 So it is generally used in blood environment with neutral physiological conditions

(pH 7.4). Three samples of FNPs with different drug feedings were prepared and recorded as FNPs-1, FNPs-2, and FNPs-3. The UV spectrum indicates that Nicardipine was successfully loaded in FNPs for appearance of Nicardipine characteristic absorbance at 236 nm, as shown in Figure S2. FTIR characterization as another proof for drug loading is provided as shown in Figure S3. The characteristic peak at 1700 cm1 is assigned to carbonyl band vibration of ester group of Nicardipine. The peaks at 1527 and 1349 cm1 are due to the nitro group vibration of Nicardipine. Major properties of these three samples of Nicardipine loaded FNPs are summarized in Table 1. After drug being loaded, the diameters of FNPs decrease from ca. 200 to ca. 150 nm. It is because drug loading enhanced the hydrophobic interaction between drug molecules and AN in the inner of FNPs. The DLS results also show that size distributions of FNPs with loaded drug are monodisperse distribution, and spherical FNPs with loaded drug were also observed in SEM image (Figures S4 and S5). The drug loading efficiency (DL) in Table 1 is defined as the ratio of the loaded Nicardipine weight to total 8387

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weight of loaded Nicardipine and FNPs, and entrapment efficiency (EE) is defined as the ratio of the loaded Nicardipine weight to initial weight of Nicardipine feeding. In order to determine the DL and EE, the drug loaded FNPs solution was lyophilized, dissolved in methanol, and analyzed by UV absorbance of Nicardipine at 236 nm.7 As shown in Table 1, by varying drug feeding, DL can be conveniently adjusted from 13% to 35%. More importantly, satisfactory DL (up to 35%) as well as high EE (up to 90%) was obtained, suggesting the high effective drug encapsulation capability of FNPs.37 The high DL of FNPs may be attributed to the following explanation: during the formation of ANCHC complex by electrostatic interactions, hydrophobic drug molecules act as nucleation sites and are entrapped into the complex to form FNPs by hydrophilic/hydrophobic interactions, leading to high DL as well as high EE. In addition, the positive Table 1. Diameter, DL, and EE of Nicardipine Loaded FNPs Nicardipine feeding sample (FNPs based w/w)

a

diameter

zeta potential

(nm)

(mV)

DLa

EEb

(wt %) (wt %)

231.0 ( 2.0 þ35.4 ( 1.8

FNPs

0

FNPs-1

0.16

155.8 ( 1.1 þ36.6 ( 1.0

12.6

60.0

FNPs-2

0.32

153.1 ( 2.9 þ35.5 ( 1.7

28.0

89.6

FNPs-3

0.64

148.4 ( 1.4 þ32.5 ( 2.7

34.8

75.0

DL = drug loading efficiency. b EE = encapsulation efficiency.

Figure 5. Fluorescence emission spectra of (a) FNPs, (b) FNPs-1, (c) FNPs-2, and (d) FNPs-3 solutions excited at 365 nm. Inset: corresponding fluorescence photographic images of FNPs and FNPs-3 solutions.

zeta potential ranging from þ32.5 to þ36.6 mV comes from the ionization of the residue amino group of CHC distributed on the FNPs surface. DLS measurement was applied to investigate the storage stability of FNPs with loaded drug. FNPs-3 solution was tested under 25 °C after 2 days in darkness storage. The results show that the diameter of FNPs-3 significant increased from 148 to 185 nm after 2 days, indicating FNPs-3 is relatively stable. The luminescence properties of FNPs before and after drug loading were investigated by fluorescence spectroscopy. As shown in Figure 5a, with excitation wavelength of 365 nm, remarkable blue fluorescence was observed. However, after loading of drug Nicardipine, as shown in Figure 5bd, the fluorescence intensity of FNPs gradually decreased with increasing DL for FRET effect and electron transfer from anthracene derivative to Nicardipine.38 Compared with the fluorescence emission intensity (at λem = 415 nm) of FNPs without loaded drug, 40.1% of fluorescence intensity was quenched for FNPs-1. Moreover, with further increased drug loading, 64.0% and 95.6% of fluorescence intensity were quenched for FNPs-2 and FNPs-3, respectively. These results demonstrate that FNPs could serve as a drug carrier with real-time monitoring of the drug release. The drug release experiments were carried out by dialysis in vitro. Figure 6a shows the fluorescence changes of quenched FNPs-3 with different dialysis times at pH 7.4. The fluorescence intensity of FNPs-3 solution gradually recovered with increasing dialysis time. Moreover, the fluorescence recovery rate depends strongly on the pH value of dialysis solution, as shown in Figure 6b. For pH 7.4 of dialysis solution, ∼3.3-fold increase in the relative

Figure 7. Nonlinear curve fitting of accumulative release of Nicardipine vs accumulative fluorescence recovery of FNPs-3 at pH 7.4.

Figure 6. (a) Fluorescence recovery of the FNPs-3 with increasing dialysis time at pH 7.4. (b) Relative fluorescence intensity of FNPs-3 without dialysis (black line) and with dialysis at pH 5.0 (red line) and pH 7.4 (blue line) as a function of dialysis time. 8388

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Figure 8. Fluorescence microscopic images of (a) FNPs, (b) FNPs-3 with loading drug, and (c) fluorescence recovery of FNPs-3 after 540 min dialysis at pH 7.4. Inset: corresponding fluorescence photographic images.

fluorescence intensity was observed after 540 min dialysis. In comparison, when pH value of dialysis solution was reduced to 5.0, only 1.7-fold increase in relative intensity was observed at the same dialysis time, indicating that drug release from FNPs was easier at pH 7.4 than at pH 5.0. This is because most of the amino groups in chitosan are deprotonated at pH 7.4 (pKa of chitosan is about 6.5).25 AN molecules prefer to depart from complexes; as a result, NPs decomposed and drug molecules were released. Thus, drug release from FNPs was easier at pH 7.4 than at pH 5.0. These results indicated that the FNPs are more suitable for use in neutral physiological conditions (such as blood with pH 7.4), and Nicardipine loaded in FNPs is an antihypertensive drug which is used in blood environment. Drug loaded FNPs are relatively stable in weak acid environment, such as pH 5.06.0. However, it is found that the FNPs-3 would be dissociated and aggregate after 540 min dialysis. A control experiment for fluorescence changes of FNPs without loaded drug was also carried out. The fluorescence intensity of FNPs without loaded drug gradually decreased with increasing dialysis time. After 540 min dialysis, approximately 50% and 55% of fluorescence intensities were left at pH 7.4 and pH 5.0, respectively (Figure S6). It is probably due to fluorescence molecules suffered from leaking from FNPs in the process of dialysis. After dialysis, leakage of fluorescence molecules AN disturbed the hydrophobic/ hydrophilic balance of complexes and finally result in the dissociation of complexes. We further investigated the relationship between the accumulative release of Nicardipine drug and accumulative fluorescence recovery of FNPs. The accumulative fluorescence recovery can be calculated by following equation accumulative fluorescence recovery ¼

In  I0  100% Ia  I0

where Ia is fluorescence intensity of FNPs without drug loading. I0 is initial fluorescence intensity of FNPs-3, and In is fluorescence intensity of FNPs-3 at different inspection time. Fluorescence intensity at 415 nm was recorded. Simultaneously, the amount of Nicardipine released from FNPs-3 was measured using UV absorbance at 236 nm. The relationship between the accumulative releases of Nicardipine as a function of accumulative fluorescence recovery of FNPs-3 is also plotted in Figure 7. It can be described by the following equation which is given by software of Origin 8.0 with a good fitting coefficient of 0.992 (allometric relationship). y ¼ 17:10x0:55 where y is accumulative release of Nicardipine and x is accumulative fluorescence recovery of FNPs-3. As a result, the amount of

drug released could be calculated by observing the level of fluorescence recovery of FNPs-3. Fluorescence microscopy was also employed to investigate the fluorescence recovery of FNPs-3 during drug release. As shown in Figure 8a, blue light emitted from FNPs. However, the FNPs fluorescence is efficiently quenched after drug loading, as shown in Figure 8b. After 540 min dialysis for drug release at pH 7.4, fluorescence of FNPs-3 was recovered again as shown in Figure 8c, indicating that a portion of drug molecules had been released from FNPs. However, blue fluorescence emission of FNPs-3 is the relatively weak. It cannot return to original fluorescence intensity, possibly caused by leakage of fluorescent molecules in dialysis. Therefore, one can readily monitor the drug release from FNPs by measuring fluorescence recovery in real time. This provides a new, facile method for the investigation of drug release through readily measurable fluorescent image changes.

’ CONCLUSION In summary, we have prepared a new type of ionic selfassembly complex using cationic chitosan hydrochloride (CHC) as the hydrophilic backbone and an anionic compound 4-(anthracen-9-ylmethoxy)-4-oxobutanoic acid (AN) as the hydrophobic unit. The complex was further self-assembled to form FNPs and well dispersed in aqueous solution. Strong blue fluorescence of these FNPs was observed as originated from anthracene groups in AN. The fluorescence of FNPs was quenched and recovered when drug molecules (e.g., Nicardipine) were loaded into and released from FNPs. FNPs exhibit the different release behavior upon the pH value, which suitable for controlling and monitoring the release of drug molecules in neutral physiological conditions (such as blood with pH 7.4). Therefore, our study may provide useful information for design of diverse functional FNPs to monitor the controlled drug release. ’ ASSOCIATED CONTENT

bS

Supporting Information. Fluorescence emission spectra of Nile red as a function of various concentrations of the CHC without added AN as a control experiment; UVvis absorbance spectra of Nicardipine, AN, and FNPs-3 solutions; FTIR spectra of Nicardipine, FNPs, and FNPs-3; size distributions of FNPs-1, FNPs-2, and FNPs-3; SEM image of FNPs-3; fluorescence changes of FNPs without loaded drug in dialysis process as a control experiment; detailed explanation for fluorescence of FNPs quenched by Nicardipine drug. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (Q.L.), [email protected] (Y.W.); Tel: (021)54747535; Fax: (021)54747535.

’ ACKNOWLEDGMENT We gratefully acknowledge financial support from the National Natural Science Foundation of China (NSFC) for Distinguished Young Scholars Grant Award (50925310 to Q.L.), NSFC Grants (50902094 and 20874059), “973” Projects (2009CB930403 and 2011CB935700), Hi-Tech Research and Development Program of China (2009AA03Z329), the Shanghai Municipal Science and Technology Commission (08JC1412300), and the Shanghai Leading Academic Discipline Project (No. B202). ’ REFERENCES (1) Slowing, I.; Trewyn, B. G.; Lin, V. S.-Y. J. Am. Chem. Soc. 2006, 128, 14792–14793. (2) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487–492. (3) Park, H.; Yang, J.; Lee, J.; Haam, S.; Choi, I. H.; Yoo, K. H. ACS Nano 2009, 3, 2919–2926. (4) Wu, G. H.; Mikhailovsky, A.; Khant, H. A.; Fu, C.; Chiu, W.; Zasadzinski, J. A. J. Am. Chem. Soc. 2008, 130, 8175–8177. (5) Schroeder, A.; Avnir, Y.; Weisman, S.; Najajreh, Y.; Gabizon, A.; Talmon, Y.; Kost, J.; Barenholz, Y. Langmuir 2007, 23, 4019–4025. (6) Ryu, J.-H.; Roy, R.; Ventura, J.; Thayumanavan, S. Langmuir 2010, 26, 7086–7092. (7) Wu, D. Q.; Lu, B.; Chang, C.; Chen, C. S.; Wang, T.; Zhang, Y. Y.; Cheng, S. X.; Jiang, X. J.; Zhang, X. Z.; Zhuo, R. X. Biomaterials 2009, 30, 1363–1371. (8) Lo, C.-L.; Lin, K.-M.; Huang, C.-K.; Hsiue, G.-H. Adv. Funct. Mater. 2006, 16, 2309–2316. (9) Ma, D.; Tu, K.; Zhang, L. M. Biomacromolecules 2010, 11, 2204–2212. (10) Tang, H. W.; Duan, X. R.; Feng, X. L.; Liu, L. B.; Wang, S.; Li, Y. L.; Zhu, D. B. Chem. Commun. 2009, 641–643. (11) Zahr, A. S.; de Villiers, M.; Pishko, M. V. Langmuir 2005, 21, 403–410. (12) Gerelli, Y.; Barbieri, S.; Di Bari, M. T.; Deriu, A.; Cantu, L.; Brocca, P.; Sonvico, F.; Colombo, P.; May, R.; Motta, S. Langmuir 2008, 24, 11378–11384. (13) Mornet, S.; Vasseur, S.; Grasset, F.; Duguet, E. J. Mater. Chem. 2004, 14, 2161–2175. (14) Wang, D.; Sima, M.; Mosley, R. L.; Davda, J. P.; Tietze, N.; Miller, S. C.; Gwilt, P. R.; Kopeckova, P.; Kopecek, J. Mol. Pharmaceutics 2006, 3, 717–725. (15) van Schooneveld, M. M.; Vucic, E.; Koole, R.; Zhou, Y.; Stocks, J.; Cormode, D. P.; Tang, C. Y.; Gordon, R. E.; Nicolay, K.; Meijerink, A.; Fayad, Z. A.; Mulder, W. J. M. Nano Lett. 2008, 8, 2517–2525. (16) Zhang, X. K.; Meng, L. J.; Lu, Q. H.; Fei, Z. F.; Dyson, P. J. Biomaterials 2009, 30, 6041–6047. (17) Francis, M. F.; Cristea, M.; Winnik, F. M. Biomacromolecules 2005, 6, 2462–2467. (18) Weinstain, R.; Segal, E.; Satchi-Fainarob, R.; Shabat, D. Chem. Commun. 2010, 46, 553–555. (19) Feng, X. L.; Lv., F. T.; Liu, L. B.; Tang, H. W.; Xing, C. F.; Yang, Q.; Wang, S. ACS Appl. Mater. Interfaces 2010, 2, 2429–2435. (20) Wu, E. C.; Park, J.-H.; Park, J.; Segal, E.; Cunin, F.; Sailor, M. J. ACS Nano 2010, 2, 2401–2409. (21) Baky, S. H. Cardiovasc. Drug Rev. 1985, 3, 153–172. (22) Lei, X. G.; Porco, J. A., Jr. Org. Lett. 2004, 6, 795–798. (23) Jiang, G. B.; Quan, D. P.; Liao, K. R.; Wang, H. H. Mol. Pharmaceutics 2005, 3, 152–160.

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