pH-Sensitive Fluorescent Hepatocyte-Targeting Multilayer

Article pubs.acs.org/molecularpharmaceutics

pH-Sensitive Fluorescent Hepatocyte-Targeting Multilayer Polyelectrolyte Hollow Microspheres as a Smart Drug Delivery System Xubo Zhao and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Institute of Polymer Science and Engineering, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China S Supporting Information *

ABSTRACT: Novel multilayer polyelectrolyte hollow microspheres with pH-sensitive fluorescence and hepatocytetargeting functions were successfully fabricated via a layer-bylayer (LbL) assembly of fluorescein isothiocyanate (FITC)modified chitosan (CSFITC) and sodium hyaluronate (HA) (as the polycation and polyanion, respectively) on polystyrene sulfonate (PSS) templates with galactosylated chitosan (GC) as the outermost layer; after etching the templates by dialysis, the aim was to use the microspheres to target hepatocytes specifically. TEM analysis revealed that they have a hollow structure with a particle size of about 260 nm, and DLS analysis demonstrated that they have pH and ionic strength dual-responsive characteristics. The hollow microspheres showed pH-sensitive fluorescence at a very low concentration by fluorescent emission spectra. MTT assays revealed that doxorubicin (a water-insoluble anticancer drug)-loaded (CSFITC/HA)4/GC hollow microspheres can specifically target hepatocytes and exhibit favorable cytocompatibility. Three typical model drugs were loaded into the (CSFITC/HA)4/GC hollow microspheres, and their drug-release kinetics in simulated body fluid (SBF) were estimated with different mathematical models. The results demonstrated that the drugloading mechanism is chemosorption and the primary governing force for drug release is diffusion. Thus, the designed hollow microspheres are expected to be used for the diagnosis and therapy of hepatic cancer. KEYWORDS: drug delivery system, hollow microspheres, layer-by-layer assembly, pH-sensitive fluorescence, hepatocyte targeting, controlled release

1. INTRODUCTION

chemotherapeutic drugs deep into tumors by intravenous injection.9−11 In solid tumors, abnormal vascular structures are generated in newly formed vessels, causing leakage, tortuousness, dilation, and interconnection.12−14 Their discontinuous endothelium results in pores of 200 nm to 2 μm (mean size: 400 nm) in diameter on the vascular walls, so submicrometersized drug carriers are beneficial for their extravasation from the bloodstream into the tumor via the EPR effect.6,15 Fluorescent molecules have been widely used as bioimaging probes.16,17 Fluorescein isothiocyanate (FITC), a commonly used fluorescent probe, has been conjugated with all kinds of

Because of their well-defined, layered three-dimensional architecture with low polydispersity and abundant functional groups, polymer microspheres show promising potential for use as excellent carriers and convenient tools for biomedical applications,1−3 especially for the delivery and controlled release of bioactive substances.4,5 Recently, a layer-by-layer (LbL) assembly technique has been developed for use on colloidal spherical templates via electrostatic interactions between oppositely charged polyelectrolytes to obtain hollow microspheres after removing the template.6−8 This versatile approach allows the desired hollow microspheres to be obtained via regulating the size of the templates for use in controlled drug release. The particle size of the drug carriers is very important for overcoming the reticuloendothelial system (RES) to deliver © 2014 American Chemical Society

Received: Revised: Accepted: Published: 1599

December March 22, March 25, March 25,

23, 2013 2014 2014 2014

dx.doi.org/10.1021/mp400774v | Mol. Pharmaceutics 2014, 11, 1599−1610

Molecular Pharmaceutics

Article

Scheme 1. Fabrication and Drug Loading and Release Properties of pH-Sensitive Fluorescent Hepatocyte-Targeting Polyelectrolyte Multilayer Hollow Microspheres (CSFITC/HA)4/GC

technique for use as a drug delivery system (Scheme 1). After four bilayers of CSFITC/HA were assembled onto the PSS templates, the resultant core−shell PSS@(CSFITC/HA)4 microspheres were coated with a single layer of GC to produce the PSS@(CSFITC/HA)4/GC microspheres. Then, the (CSFITC/ HA)4/GC polyelectrolyte multilayer hollow microspheres were obtained by etching the templates. Here, CSFITC was selected as the functional polycation with pH-sensitive fluorescence, whereas GC was selected as the outermost layer to introduce the hepatocyte-targeting function. The stability of (CSFITC/HA)4/GC hollow microspheres in solutions with a higher salt concentration was investigated with dynamic light scattering (DLS). CSFITC endowed the hollow microspheres excellent pH-sensitive fluorescence, which was proved by their fluorescent emission spectra. MTT assays of the (CSFITC/HA)4/GC hollow microspheres in HepG2 cells revealed their excellent nontoxicity. Meanwhile, doxorubicin (DOX), a highly efficient anticancer drug, was loaded into the (CSFITC/HA)4/GC hollow microspheres, and the hepatocyte specificity of the (CSFITC/HA)4/GC hollow microspheres was examined using HepG2 cells. Three model drugs with different structures (ibuprofen (IBU), dipyridamole (DIP), and DOX) were used to investigate the cumulative release performance of the (CSFITC/HA)4/GC hollow microspheres. Furthermore, the drug-release kinetics from the model-drug-loaded (CSFITC/ HA)4/GC hollow microspheres are discussed with the versatile Higuchi and Korsmeyer−Peppas equations.

polymer macromolecules for bioimaging. Bae and co-workers synthesized FITC-conjugated poly( L -histidiene)-b-poly(ethylene glycol) to visualize the interaction between these polymeric micelles and cells.18 They also used FITC-TATconjugated poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(Lhistidine)-TAT to observe cellular uptake via flourescence.19 He et al. reported the generation of FITC-labeled nanocapsules to further understand the cellular uptake behavior of the Pluronic F127−chitosan nanocapsules.20 Most recently, the targeting function of drug carriers has attracted considerable attention in controlled drug release because of the serious side effects on healthy cells caused by commonly used anticancer drugs. Drug delivery systems using antibodies or a specific ligand can selectively bind to target cells and be internalized via receptor-mediated endocytosis.21 Among various targeting ligands, the galactose moiety, which has a very high affinity for galactose group receptors (ASGP-R (asialoglycoprotein)) on the surface of hepatocytes, is one of the most promising candidates for cancer cell targeting.22−24 Chitosan (CS) and hyaluronic acid (HA) have been used extensively in the biomedical field because of their biocompatibility, biodegradability, and ease of commercial accessibility.25−28 They have been widely used as drug delivery systems, including use in hydrogels,29 nanoparticles,30 and thin films.31 CS is a natural cationic polyelectrolyte with a pKa of about 6.5,29 which enables two- and three-dimensional surface modification.29,32 For its application as the polycation in the fabrication of multilayer polyelectrolyte hollow microspheres, CS has been mostly used directly without any modification. To avoid aggregation or fusion in higher saline media,33−37 to produce additional stimuli-responsive characteristics,34 to improve biocompatibility,35−37 and/or to introduce targeting function,37 only the outermost CS layer has been modified by grafting with functional molecules or polymers. After fabrication of the single-component multilayer polyelectrolyte hollow microspheres with anionized chitosan as the polyanion,38,39 the modified chitosan has been used for LbLengineered polyelectrolyte hollow microspheres via hydrogen bonding40 or click chemistry.31 In the present article, well-defined pH-sensitive fluorescent hepatocyte-targeting multilayer polyelectrolyte hollow microspheres were designed via a simple and efficient LbL assembly

2. EXPERIMENTAL SECTION 2.1. Materials. Chitosan (CS; viscosity-average molecular weight, 6.0 × 105; degree of deacetylation, 90%) was obtained from the Zhejiang Yuhuan Marine Biotechnique Company. Sodium hyaluronate (HA; MW ∼400 000) was purchased from Shandong Freda Biopharm. Co., Ltd. Doxorubicin hydrochloride (DOX·HCl) was obtained from Beijing Huafeng United Technology Co., Ltd. Ibuprofen (IBU) (purity > 99.7%) was provided by Shandong Xinhua Pharmaceutical Co., Ltd. Dipyridamole (DIP) was analytical reagent grade and was from J&K Chemical Ltd. Fluorescein isothiocyanate was purchased from Aldrich (WI, USA). Lactobionic acid (LA) was provided by Gracia Chemical Technology Co., LTD (Chengdu, China). 1-Ethyl-3-(31600



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with water to achieve the PSS templates encapsulated with one polyelectrolyte bilayer (PSS@(CSFITC/HA)1). Thus, CSFITC and HA were alternately adsorbed three more times onto the core−shell PSS@(CSFITC/HA)1 microspheres to achieve the PSS templates encapsulated with four polyelectrolyte bilayers (PSS@(CSFITC/HA)4). Finally, GC was deposited onto the surface of PSS@(CSFITC/HA)4 to obtain core−shell microspheres with an outermost GC layer (PSS@(CSFITC/HA)4/GC). The resulting product was centrifuged, washed with water, lyophilized, and stored at 4 °C. For comparison, (CSFITC/HA)4 hollow microspheres and shell-cross-linked (CSFITC/HA)4/GC hollow microspheres were also synthesized: 0.051 g of core−shell PSS@(CSFITC/ HA)4/GC microspheres were dispersed into 20 mL of water, and 2 mL of 1.0% glutaraldehyde was added to cross-link the CSFITC constituent in the core−shell microspheres to form the cross-linked shells. Lastly, the three core−shell microspheres (PSS@(CSFITC/HA)4, PSS@(CSFITC/HA)4/GC, and the covalently cross-linked PSS@(CSFITC/HA)4/GC) were dialyzed in DMF to etch the templates. Therefore, three polyelectrolyte multilayer hollow microspheres ((CSFITC/HA)4, (CSFITC/ HA)4/GC, and the covalently cross-linked (CSFITC/HA)4/ GC) were obtained, respectively. 2.6. Cell Toxicity and Hepatocyte-Specificity Assays. The cytocompatibility and hepatocyte specificity of the polyelectrolyte multilayer hollow microspheres were evaluated with HepG2 cells by the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, and DOX was used as a model drug to evaluate the hepatocyte specificity of the (CSFITC/HA)4/GC hollow microspheres. For the MTT assay, cells seeded in 96-well cell culture plates (1 × 105 cells/well) were cultured in 5% CO2 at 37 °C for 24 h. (CSFITC/HA)4/GC hollow microspheres, DOX-loaded (CSFITC/HA)4/GC and DOX-loaded (CSFITC/HA)4 hollow microspheres, or free DOX were added into the cells at different concentrations, and the cells were incubated for 48 h. The cells were washed with PBS, and the cell viability was determined by MTT assay: 100 μL of PBS, pH 7.4, containing 20 μL of MTT (5 mg/mL) solution was added to the wells, and the plates were incubated for another 4 h, after which the medium was removed from each well. Cell-bound dye was dissolved in 100 μL of DMSO in each well of the cell culture plate, which was then swung by a table concentrator for 20 min. The absorbance of the wells was measured with a microplate reader at 490 nm. 2.7. Drug Loading. The drug solution (1.0 mg/mL) was prepared by dissolving the drugs in water at pH 9.0 for IBU or pH 4.0 for DIP. (CSFITC/HA)4/GC hollow microspheres (30.3 mg) were dispersed in 5.0 mL of drug solution (IBU or DIP) for drug loading. After being swung by a table concentrator for 24 h, the IBU- or DIP-loaded hollow microspheres were centrifuged to remove the excess IBU or DIP. The drug concentration in the supernatant solution was monitored using an ultraviolet (UV) spectrophotometer (at 264.00 nm for IBU or 284.80 nm for DIP) to assess drug-loading capacity and loading efficiency. The drug-loading capacity and loading efficiency of the (CSFITC/HA)4/GC hollow microspheres were calculated from the drug concentrations before and after loading. (CSFITC/HA)4/GC hollow microspheres (20.2 mg) were dispersed into 5 mL of a 1.0 mg/mL aqueous DOX solution with the aid of ultrasound, and then the solution was adjusted to pH 7.4. After being stirred for 24 h and swung subsequently by table concentrator for 24 h in the dark, the DOX-loaded

dimethyl aminopropyl) carbodiimide hydrochloride (EDC· HCl) was purchased from Fluorochem. N-Hydroxylsuccinimide (NHS) was supplied by Aladdin Chemistry Co., Ltd. N,N,N′,N′-Tetramethylethylenediamine (TEMED) was purchased from Aldrich (WI, USA). Styrene (St), methacrylic acid (MAA), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetic anhydride, ether, trichloromethane (CHCl3), dichloromethane (CH2Cl2), ammonium persulfate (APS), sulfuric acid (H2SO4, 98%), and sodium carbonate (Na2CO3) were analytical reagents obtained from Tianjin Chemical Company, Tianjin, China. Deionized water was used throughout the experiments. 2.2. Polystyrene Sulfonate (PSS) Nanosphere Templates. The polystyrene (PS) nanospheres were synthesized via the emulsifier-free emulsion polymerization of styrene (St) and the surfmer, methacrylic acid (MAA).41 For a typical procedure, 10.0 mL of St, 2.0 mL of MAA, and 95 mL of water were charged in a three-necked flask equipped with a condenser and a magnetic stirrer in N2. Five milliliters of an aqueous solution containing 0.054 g of APS was added into the mixture under vigorous stirring. After being heated at 72 °C for 24 h, the product was purified by washing with ethanol, and the PS nanospheres were obtained as a white powder after being dried in a vacuum oven at 30 °C. Two grams of PS nanospheres was dispersed in 60 mL of 98% sulfuric acid with ultrasonic irradiation. The sulfonation reaction was allowed to take place at 45 °C for 8 h with stirring. After cooling to room temperature, the product (PSS templates) was separated, washed with water until neutral, dispersed, and stored in 100 mL of water for use.42 2.3. Isothiocyanate-Modified Chitosan (CSFITC). One gram of chitosan was added into 60 mL of water and was dissolved by adding 40 mL of 1 M acetic acid. Following the addition of 50 mL of dried methanol, 50 mL of methanol solution containing 82 mg of FITC was added. After stirring for 3 h, the mixture was diluted with deionized water to 500 mL. Then, the solution was neutralized with a 0.1 M NaOH solution to pH 8, and the precipitate was filtered and washed with a 70:30 (v/v) mixture of ethanol/deionized water until the filtrate was clear. The obtained CSFITC was lyophilized and stored at 4 °C.43 2.4. Galactosylated Chitosan (GC). LA was conjugated onto chitosan via EDC chemistry. Typically, 0.1911 g (0.5333 mmol) of LA was dissolved in 50 mL of TEMED/HCl buffer solution (pH 4.7), and its carboxyl groups were activated with NHS/EDC (0.0307/0.1022 g). Then, 1.0003 g of chitosan (5.5901 mmol, equivalent molar ratio to LA) was added, and the reaction was conducted for 72 h at room temperature. Lastly, the product was separated through a dialysis tube (12 000 MWCO) against water for 4 days, lyophilized, and stored at 4 °C .23 2.5. CSFITC/HA Multilayer Encapsulated PSS Templates. The polyelectrolyte multilayer-coated PSS templates were fabricated by the layer-by-layer assembly of polycation (CSFITC) and polyanion (HA) via the electrostatic interaction between CSFITC/HA, with the first layer being CSFITC. The assembly of CSFITC was completed by stirring 0.3 g of CSFITC in 150 mL of an aqueous dispersion of the PSS templates (0.3 g) at pH 4.8 for 8 h; the PSS@CSFITC microspheres were centrifuged and washed with water. Then, the PSS@CSFITC microspheres were dispersed into 150 mL of water followed by the addition of 0.3 g of HA at a pH of around 4.8 for 8 h with stirring, and the microspheres were centrifuged and washed 1601



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Figure 1. TEM images of the PSS nanospheres (a), core−shell PSS@(CSFITC/HA)4/GC) microspheres (b), covalently cross-linked PSS@(CSFITC/ HA)4/GC) core−shell microspheres (c), (CSFITC/HA)4/GC hollow microspheres (d), and covalently cross-linked (CSFITC/HA)4/GC hollow microspheres (e).

The linear form of the intraparticle diffusion equation can be given as

(CSFITC/HA)4/GC hollow microspheres were centrifuged to remove the free excess DOX. The drug-loading capacity and loading efficiency of (CSFITC/HA)4/GC were calculated from the drug concentrations in the solutions before and after loading by using UV−vis spectrometry at a wavelength of maximum absorbance (233 nm). The experimental data were analyzed with the pseudo-first order, pseudo-second-order, and intraparticle diffusion models.44 The pseudo-first-order model can be expressed as

ln qt = ln k i +

1 ln t 2

where ki (mmol/(g min1/2)) is the intraparticle diffusion rate constant. 2.8. Controlled Release. The drug-loaded (CSFITC/HA)4/ GC hollow microspheres were dispersed into 10 mL of water, and the dispersion was added into a 120 mL of PBS solution in a dialysis tube (MWCO of 10 000) at pH 7.4 and 1.8 for IBU and DIP, or pH 7.4 and 5.0 for DOX. Five milliliters of solution was taken out at certain intervals. The drug concentrations in the dialysates were measured by UV spectrophotometer to calculate the cumulative release from the drug-loaded (CSFITC/ HA)4/GC hollow microspheres. Five milliliters of fresh PBS was added after each sampling to keep the total solution volume constant. The cumulative release (%) can be calculated as the mass ratio of the cumulative amount of drug released and the total mass of loaded drug over time. The in vitro drug-release data was estimated for the release rate with the Higuchi and Korsmeyer−Peppas equations

k 1 1 = 1 + qt qet qe

where k1 (min−1) is the pseudo-first-order adsorption rate constant, qt (mmol/g) is the amount adsorbed at time t (min), and qe (mmol/g) denotes the amount adsorbed at equilibrium. The pseudo-second-order model is represented as t 1 t = + qt qe k 2qe 2

where k2 (g/(mmol min)) is the adsorption rate constant of the pseudo-second-order model. 1602



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Higuchi equation: M t = kt 1/2

were about 380 nm. Furthermore, the diameter of the covalently cross-linked PSS@(CSFITC/HA)4/GC microspheres was approximately 330 nm (Figure 1c). The well-defined (CSFITC/HA)4/GC hollow microspheres were nearly spherical in shape after the PSS templates had been etched with DMF. It can be seen that the diameter of the (CSFITC/HA)4/GC hollow microspheres was 260 nm, whereas the diameter of the covalently cross-linked (CSFITC/HA)4/GC hollow microspheres was 220 nm. This may be due to collapse of the (CSFITC/HA)4/GC shells, resulting from the removal of the PSS templates, compared to the covalently cross-linked (CSFITC/HA)4/GC hollow microspheres. However, obvious aggregation was found for the covalently cross-linked (CSFITC/ HA)4/GC hollow microspheres, which may be attributed to the side reaction of interparticle cross-linking during the crosslinking of the chitosan constituent in the core−shell PSS@(CSFITC/HA)4/GC microspheres. The change in the zeta potentials during the LbL assembly was evaluated to track the growth of the polyelectrolyte multilayer (Figure 2). The negative sign of the PSS templates

where Mt is the amount of drug release at time t and k is the rate constant.45 Korsmeyer−Peppas equation: M t /M∞ = kt n

where Mt/M∞ is the drug-release fraction at time t, k is a constant comprising the structural and geometric characteristics of the controlled release system, and n, the release exponent, is an important parameter that depends on the release mechanism.46,47 2.9. Characterizations. The morphology of the aqueous dispersion of the final (CSFITC/HA)4/GC multilayer polyelectrolyte hollow microspheres and other samples was analyzed with a JEM1200 EX/S transmission electron microscope (TEM). Fourier transform infrared (FT-IR) spectra were recorded with a Bruker IFS 66 v/s infrared spectrometer (Bruker, Karlsruhe, Germany) in the range of 400−4000 cm−1 with a resolution of 4 cm−1 as KBr pellets. The elemental analyses of the samples were performed with an Elementar Vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). The hydrodynamic diameter (Dh) and distribution of the obtained hollow microspheres were determined by the dynamic light scattering (DLS) technique with a light scattering system BI-200SM (Brookhaven Instruments) equipped with a BI200SM goniometer, a BI-9000AT correlator, a temperature controller, and a Coherent INOVA 70C argon ion laser at 20 °C. The measurements were performed using 135 mW laser excitation at 514.5 nm at a detection angle of 90° using their dispersion directly at 25 °C. The mean values of three measurements are given (SD < 5%). The zeta potentials of the PSS@(CSFITC/HA)4/GC coreshell microspheres after adsorption of each polyelectrolyte layer were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK) in media at pH 4.8. All fluorescence spectra were obtained with a PerkinElmer LS 55 luminescence spectrophotometer equipped with a 1.0 cm quartz cell. The fluorescence spectrum of CSFITC was conducted in the wavelength range of 480−680 nm upon excitation at 350 nm at different pH values (2, 3, 4, 5, 6, 7, 8, and 9). Typically, 1.0 mL of a 10 μg/mL (CSFITC/HA)4/GC aqueous dispersion was added into the color comparison tubes, and the synchronous fluorescence spectra were recorded in the wavelength range of 480−680 with Δλ of 50 nm. The drug-loading and controlled release behaviors of the (CSFITC/HA)4/GC hollow microspheres were measured with a PerkinElmer Lambda 35 UV−vis spectrometer (PerkinElmer Instruments, USA) at room temperature.

Figure 2. Zeta potentials of the core−shell microspheres after adsorption of each polyelectrolyte layer at pH 4.8.

because of the sulfonic acid groups made them suitable templates for the adsorption of the CSFITC polycation. After deposition of the positively charged chitosan derivatives, the zeta potential became positive. The zeta potential value was +10.3 mV after the first layer of chitosan derivative was assembled onto the PSS templates. Successive adsorption of the HA polyanion led to a negatively charged surface with zeta a potential value of −16.5 mV. The LbL encapsulation showed a full reversal of the surface charge sign four times, corresponding to the eight layers of polyelectrolyte (Scheme 1). Lastly, the zeta potential became positive with a zeta potential value of +6.2 mV after the outermost GC layer was deposited. This demonstrates that the LA coupled onto the backbone of chitosan partially shielded the positive sign of the CSFITC. To render the polyelectrolyte multilayer hollow microspheres fluorescence, FITC was conjugated onto the CS backbone. As a result, the product became orange−yellow compared to the original white one. The yield of FITC conjugation was measured to be 2.5−2.8% from the fluorescence intensity of the CSFITC. Then, fluorescent hepatocyte-targeting polyelectrolyte multilayer hollow microspheres were obtained after treating the core−shell microspheres with DMF to etch the inner PSS templates. The CSFITC and (CSFITC/HA)4/GC exhibited maximum fluorescent emission at 518 nm with 350 nm excitation (Figure 3). These results implied the presence of

3. RESULTS AND DISCUSSION 3.1. Fabrication of Hollow Microspheres. PS nanospheres were synthesized via facile surfactant-free emulsion polymerization and were then sulfonated to obtain a negatively charged surface. The (CSFITC/HA)4/GC hollow microspheres were fabricated by a simple and mild noncovalent LbL assembly (Scheme 1). The morphology and size of the (CSFITC/HA)4/ GC hollow microspheres were investigated by TEM. The PSS nanospheres were monodisperse with a particle size of about 350 nm (Figure 1a). The TEM image of the core−shell PSS@(CSFITC/HA)4/GC microspheres with the outermost GC layer is shown in Figure 1b. It was found that their diameters 1603



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fluorescein moieties in the modified chitosan (CSFITC) and the final hollow microspheres.

templates had been completely etched. Additionally, after 1.00 g of core−shell PSS@(CSTITC/HA)4/GC microspheres was dialyzed in DMF, 0.361 g of (CSTITC/HA)4/GC hollow microspheres were obtained. This indicated that a total mass of 0.56 g of the polyelectrolytes was coated onto 1.00 g of PSS templates in the LbL assemby. The elemental components of the samples were examined with an Elementar Vario EL instrument, and the results are summarized in Table 1. The FITC content (FITC%) in CSFITC was calculated from its N elemental content as 2.80%. The same conclusion was obtained from fluorescence analysis of CSFITC. Furthermore, the substitution degrees of chitosan coupled with LA in GC was estimated to be 5.61% from its N elemental content. Taken together, the molar concentration of the -NH2 groups of the chitosan derivatives and the -COOH groups of HA in every gram of the (CSFITC/HA)4/GC hollow microspheres was calculated to be 2.466 and 1.322 mmol/g, respectively. 3.2. pH and Ionic Strength Dual-Responsive and pHSensitive Fluorescent Properties. The hydrodynamic diameter distributions of the core−shell PSS@(CSFITC/HA)4/ GC microspheres and (CSFITC/HA)4/GC hollow microspheres at pH 7.4 were tracked using DLS. The core−shell PSS@(CSFITC/HA)4/GC microspheres displayed a narrow unimodal size distribution with an average hydrodynamic diameter (Dh) of 384 ± 17 nm (Figure 5). After removal of the

Figure 3. Fluorescent emission spectra of the fluorescent CSFITC (a) and (CSFITC/HA)4/GC hollow microspheres (b) in neutral media.

The IR spectra of chitosan, galactosylated chitosan (GC), core−shell PSS@(CS FITC /HA) 4 /GC microspheres, and (CSFITC/HA)4/GC hollow microspheres are shown in Figure 4. The characteristic peaks of the antisymmetric stretching of

Figure 4. FT-IR spectra of CS (a), GC (b), core−shell PSS@(CSFITC/ HA)4/GC microspheres (c), and (CSFITC/HA)4/GC hollow microspheres (d). Figure 5. Typical hydrodynamic diameter distributions of the PSS@(CSFITC/HA)4/GC core−shell microspheres and (CSFITC/ HA)4/GC hollow microspheres.

the C−O−C groups in the saccharide structure appeared at 1161, and the skeletal vibrations involving the CO stretching appeared at 1095 and 1022 cm−1.48 In the IR spectrum of GC, a distinctive absorbance peak appeared at 1716 cm−1 of the C O stretching of carboxylic groups compared to CS, which implied that LA was successfully coupled with chitosan. The characteristic absorbance bands of polystyrene at 3060, 3022, 2920, and 2845 cm−1 (stretching vibration of C−H); 1495 and 1457 cm−1 (stretching vibration of CC); and 700 cm−1 (outof-plane blending vibration δring) presented in the FT-IR spectrum of the PSS@(CSFITC/HA)4/GC disappeared in the IR spectrum of the (CSFITC/HA)4/GC hollow microspheres after being dialyzed with DMF, suggesting that the PSS

PSS templates, the (CSFITC/HA)4/GC hollow microspheres exhibited a Dh of 336 ± 11 nm, which is lower than that of the PSS@(CSFITC/HA)4/GC core−shell microspheres as a result of the collapse of the polyelectrolyte shell after the PSS templates were etched. The effect of the media’s pH value on the Dh of the (CSFITC/ HA)4/GC hollow microspheres was studied with DLS after they were dispersed into aqueous solutions with various pH values for 12 h, and the results are presented in Figure 6. Their Dh decreased from 556 to 319 nm with the pH value of the

Table 1. Chemical Components of the Samples Calculated from Elemental Analysis samples

CS

GC

CSFITC

(CSFITC/HA)4

(CSFITC/HA)4/GC

N (%) CS (%) -NH2 (mmol/g) -COOH (mmol/g)

8.950 93.10 5.675 0

7.660 87.49 4.773 0

8.250 90.23 5.295 0

3.235 38.36 2.188 1.457

3.655 61.23 2.466 1.322

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Figure 6. pH strength dependence of the average hydrodynamic diameter (Dh) and scattering intensity (Kcps) of the (CSFITC/HA)4/ GC hollow microspheres.

Figure 7. Fluorescent emission spectra of the aqueous dispersion of the (CSFITC/HA)4/GC hollow microspheres with pH values from 2.0 to 9.0.

medium increasing from 2.0 to 7.0, and it then increased, reaching 470 nm at pH 11.0. Increasing of the media’s pH value probably weakens the electrostatic attraction between HA and CSFITC, so the ionic cross-linking bonds within the internal structure of the (CSFITC/HA)4/GC hollow microspheres decrease. The multilayer polyelectrolyte hollow microspheres shrank as the media’s pH value increased from 2.0 to 7.0, and the size-changing trend showed a parabolic shape with pH variation and a small increase from 8.0 to 11.0. In lower pH media, the deprotonation of the -COOH groups of HA is decreased. At pH 2.0, CSFITC is adequately protonated into the -NH3+ groups,49 and the protonation decreases greatly with the increase of the media’s pH value. Thus, their Dh decreased with the increase of the media’s pH value because of the shrinkage of the polyelectrolyte multilayer shells. However, the Dh increased obviously in media with a pH range of 8.0−11.0. This may be due to the HA moiety in the (CSFITC/HA)4/GC hollow microspheres becoming more hydrated and extended because of its deprotonation in basic media. Furthermore, the scattering intensity only changed slightly until a steady state value of 77.23 was reached when the media’s pH value increased from 2.0 to 11.0, demonstrating that the (CSFITC/HA)4/GC hollow microspheres had a good stability and resistance against the changing pH value of the media except for the aggregation formed during the measurements.50 However, the fluorescence spectra of the (CSFITC/HA)4/GC hollow microspheres were recorded at seven different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) in the range of 480−680 nm upon excitation at 350 nm using a PerkinElmer LS 55 luminescence spectrophotometer equipped with a 1.0 cm quartz cell. It is worth mentioning that the fluorescence intensity of the hollow microspheres increased with an increase in the media’s pH value from 2.0 to 9.0 (Figure 7) because of the pH-sensitive fluorescence of FITC.51 This is a direct indication of the pH-sensitive fluorescence of the (CSFITC/ HA)4/GC hollow microspheres. The effect of the media’s ionic strength on the Dh of the (CSFITC/HA)4/GC hollow microspheres was also investigated using DLS (Figure 8). The Dh increased from 337 to 537 nm as the NaCl concentration of the media increased from 0 to 0.20 M. This was the result of the shielding effect of the small molecule electrolytes on the electrostatic interactions among the polyelectrolyte stretches in the hollow microspheres.52 Furthermore, the scattering intensity of the hollow microspheres decreased gradually from 78.84 to 69.51 as the media’s

Figure 8. Ionic strength dependence of the average hydrodynamic diameter (Dh) and scattering intensity (Kcps) of the (CSFITC/HA)4/ GC hollow microspheres.

ionic strength increased, indicating the aggregation of the hollow microspheres.50 3.3. Cell Toxicity and Hepatocyte Specificity Assays. The cytocompatibility of the pH-sensitive fluorescent (CSFITC/ HA)4/GC hollow microspheres was evaluated in HepG2 cells using the MTT assay. The viability of the HepG2 cells was near 100% (96.57−104.70%) at the tested concentrations (0−100 μg/mL) after 48 h of incubation (Figure 9), indicating that the pH-sensitive fluorescent (CSFITC/HA)4/GC hollow microspheres have excellent nontoxicity toward HepG2 cells. Then, the hepatocyte specificity of the DOX-loaded (CSFITC/HA)4/GC hollow microspheres was investigated in HepG2 cells with the MTT assay. The results are given in Figure 10 and suggest that the DOX-loaded hollow microspheres showed the pronounced cytotoxic effects. To evaluate the galactose group-mediated targeting performance of the hollow microspheres, the DOX-loaded (CSFITC/HA)4/GC hollow microspheres and the DOX-loaded (CSFITC/HA)4 hollow microspheres without the outermost GC layer were used for in vitro study with HepG2 cells. A significant reduction in cell viability was observed by introducing DOX, especially as the DOX concentration increased. The order of the killing efficacy is free DOX, DOX-loaded hepatocyte-specific (CSFITC/HA)4/GC hollow microspheres, and DOX-loaded non-hepatocyte-specific (CSFITC/HA)4 hollow microspheres. Furthermore, the DOX-loaded hepatocytespecific (CSFITC/HA)4/GC hollow microspheres showed obvious cell inhibition compared to the DOX-loaded non1605



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a), DOX (Figure 11, curve b), and DIP (Figure 11, curve c) into the (CSFITC/HA)4/GC hollow microspheres were examined at different pH values at 25 °C under magnetic stirring for 24 h. The drug-loading capacities were 61.8 ± 1.4, 87.1 ± 1.5, and 42.9 ± 0.1 mg/g, with drug-loading efficiencies of 37.4, 35.0, and 33.4% for IBU, DOX, and DIP, respectively. The drug-loading capacity for IBU was much higher than that for the other model drugs because of the higher content of the amino groups compared to the carboxyl groups in the (CSFITC/ HA)4/GC hollow microspheres, as shown in Table 1. The loading of IBU was derived from the electrostatic interaction between the -NH3+ groups of chitosan and the -COOH groups of IBU.53 The DIP molecules were loaded into the hollow microspheres through the electrostatic interaction between the -NR2 groups of DIP and the -COO− groups of HA.54 In addition, the DOX molecules were loaded into the hollow microspheres because of the electrostatic interaction or hydrogen bond between the -NH2 and -OH groups of DOX and the -COOH groups of HA and the -NH2 groups of CS. Compared with the formation of electrostatic interactions, the influence of hydrogen-bond interactions was merely a side effect.55 The bigger molecular structure of DOX and its poorer solubility in the selected loading medium might also affect the drug-loading capacity and rate. Its loading capacity increased continuously after 360 min, whereas those for IBU and DIP increased slightly after 360 min. At 360 min, the three model drugs, IBU, DOX, and DIP, reached 97.7, 85.8, and 89.1% of their equilibrium drug-loading capacity, respectively. The drug-loading process was also estimated via adsorption kinetics models, and the kinetic parameters are summarized in Table 2. On the basis of the obtained correlation coefficients (r2), the loading of the three model drugs (IBU, DOX, and DIP) into the (CSFITC/HA)4/GC hollow microspheres fit the pseudo-second-order model, not than the pseudo-first-order model, perfectly (Figure S1). In addition, the three r2 values for the pseudo-second-order kinetic model of the three model drugs were higher than 0.999. Moreover, the calculated equilibrium loading capacities, qe, cal, were very close to their experimental values, qe, exp. Thus, the rate-limiting step during drug-loading into the (CSFITC/HA)4/GC hollow microspheres is chemisorption.44 The multilinearity correlation was obtained from the intraparticle diffusion plots, a first initial linear portion followed by a plateau, indicating that two steps occurred during the drug-loading process (Figure S1c). This was deemed as an external surface adsorption at the first linear portion, and the second portion indicates a gradual adsorption stage where intraparticle diffusion is the rate-determined factor.44 3.5. Controlled Release and Kinetics. As pH and ionic dual-responsive drug carriers, the (CSFITC/HA)4/GC hollow microspheres exhibited not only a relatively stable structure but also a certain higher drug-loading capacity. Therefore, the drugrelease performance of the drug-loaded (CSFITC/HA)4/GC hollow microspheres in different PBS solutions (pH 7.4 as in the small intestine and colon and pH 1.8 as in gastric juice) at 37 °C was investigated with hydrophobic model drugs (IBU and DIP). Figure 12 shows the time dependence of the cumulative IBU and DIP release from the drug-loaded (CSFITC/HA)4/GC hollow microspheres in PBS at pH 7.4 and 1.8 at 37 °C, respectively. The cumulative release of IBU (Figure 12, curves a and c) and DIP (Figure 12, curves d and b) in PBS at pH 7.4 and 1.8 at 37 °C were calculated to be about 90.81 and 55.91% and 45.41 and 87.64%, respectively. The cumulative release

Figure 9. Cell viability assay in HepG2 cells. The cells were treated with the (CSFITC /HA) 4/GC hollow microspheres at various concentrations (0, 20, 40, 50, and 100 μg/mL) at 37 °C for 48 h. Cell viability (%) was determined by MTT assay.

Figure 10. Cell viability assay in HepG2 cells. The cells were treated with the DOX-loaded (CSFITC/HA)4/GC, DOX-loaded (CSFITC/ HA)4, and free DOX at various concentrations (0, 2, 4, 7, and 10 μg/ mL) at 37 °C for 48 h. Cell viability (%) was determined by MTT assay.

hepatocyte-specific (CSFITC/HA)4 hollow microspheres. This was mainly attributed to the ASGP-R receptors on the cell surface of the hepatocytes.22 Therefore, it can be concluded that the (CSFITC/HA) 4 /GC hollow microspheres have receptor-mediated hepatocyte specificity because of the galactose-specific recognition between GC molecules and the ASGR of hepatocytes. 3.4. Drug Loading and Kinetics. As shown in Figure 11, the drug-loading capacities and rates for IBU (Figure 11, curve

Figure 11. Drug loading of IBU (pH 4.0) (a), DOX (pH 7.4) (b), and DIP (pH 9.0) (c) into (CSFITC/HA)4/GC hollow microspheres. 1606



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Table 2. Kinetic Parameters for Drug Loading into the (CSFITC/HA)4/GC Hollow Microspheres qe, exp (mmol/g) Pseudo-First-Order qe, cal (mmol/g) r2 Pseudo-Second-Order qe, cal (mmol/g) r2 Intraparticle kinetic r2

IBU

DOX

DIP

0.2823

0.1624

0.1060

0.3213 0.9859

0.1541 0.9934

0.1126 0.9861

0.2880 0.9999 t ≤ 60 0.9727

0.1711 0.9992 t ≤ 60 0.9987

0.1088 0.9997 t ≤ 60 0.9494

t > 60 0.8724

t > 60 0.9174

t > 60 0.9343

diffusion. In addition, the positive axis intercept of 26.730 of the Higuchi equation demonstrated that the DIP release from DIPloaded (CSFITC/HA)4/GC had an obvious burst effect. This may be due to deprotonation of the CSFITC at this pH value, causing the collapse of HA, which forms pyknotic shells with HA as the shaggy shells. The special structure led to the enrichment of DIP molecules in the shaggy shells of the hollow microspheres. For a comparison with DIP, the model drug IBU containing a carboxyl group was used to study the release mechanism from the (CSFITC/HA)4/GC hollow microspheres. The plots of the iguchi and Korsmeyer−Peppas equations were used to describe the IBU release mechanism in PBS (pH 1.8, 37 °C) (Figure S2e,f). The plots for the Higuchi and Korsmeyer−Peppas models for the IBU-loaded (CSFITC/HA)4/GC resulted in linearity, with r2 and k values of 0.979 and 1.863 and r2 and the n values of 0.983 and 0.480, respectively. The corresponding plots (log cumulative percent drug release vs time (t) and cumulative release percent vs root square of time (t)) for the Korsmeyer−Peppas and Higuchi equations yielded comparatively good linearity (r2 of 0.983 and 0.979, respectively). These results suggested that drug release was controlled by Fickian diffusion and revealed that the release mechanism from IBUloaded (CSFITC/HA)4/GC is diffusion-controlled drug release in pH 1.8 PBS at 37 °C.47,57 However, the Korsmeyer−Peppas equation could not be used to explore the release behavior from IBU-loaded (CSFITC/HA)4/GC in pH 7.4 PBS at 37 °C because of the cumulative release of >60%. Therefore, only the Higuchi equation was used to analyze the IBU release mechanism of IBU-loaded (CSFITC/HA)4/GC in pH 7.4 PBS at 37 °C. As shown in Figure S2, the Higuchi equation plots were linear,with r2 and k values of 0.879 and 1.532, respectively. This suggests that the drug release was also controlled by Fickian diffusion. The positive axis intercept of 16.377 in the Higuchi equation plots also indicated the burst effect of the IBU release from IBU-loaded (CSFITC/HA)4/GC. This may be due to that IBU is an acidic molecule and could only be dissolved in alkaline media (pH 7.4). The increase of solubility of IBU has destroyed the electrostatic interaction between IBU molecule and CSFITC, which results in the burst effect.41 Therefore, the drug release from the (CSFITC/HA)4/GC hollow microspheres was mainly driven by the dissolution of the model drugs, and the diffusion was the primary governing force for the model drugs IBU and DIP. On the basis of the loading and release performance of IBU and DIP, the in vitro anticancer drug release from the DOXloaded pH-sensitive fluorescent hepatocyte-specific (CSFITC/ HA)4/GC hollow microspheres with a DOX-loading capacity of 87.1 ± 1.5 mg/g was investigated at 37 °C under simulated body fluid (SBF) conditions of pH 7.4 or 5.0. The sustained

Figure 12. Cumulative IBU release from the IBU-loaded (CSFITC/ HA)4/GC hollow microspheres in pH (a) 7.4 and (c) 1.8 PBS and cumulative DIP release from the DIP-loaded (CSFITC/HA)4/GC hollow microspheres in pH (b) 1.8 and (d) 7.4 PBS at 37 °C.

percent of DIP (Figure 12, curve d) was lower than that of IBU (Figure 12, curve a) in PBS (pH 7.4, 37 °C). However, the cumulative release of IBU (Figure 12, curve c) was lower than that of DIP (Figure 12, curve b) in PBS (pH 1.8, 37 °C). The lower cumulative release resulted from the different solubility of the two model drugs in saline at different pH values and the intrinsic polyelectrolyte nature of the assembling materials. IBU is easy to dissolve in alkaline media (pH 7.4), whereas DIP can not be dissolved in alkaline media.41 Furthermore, CS is a weak polybase with a pKa value of about 6.5.29 In medium with a pH value >6.5, it deprotonates, resulting in a decrease of the electrostatic interaction between the carboxyl groups of IBU and the amino groups of CS. HA is a weak polyacid with a pKa value of about 3.2.56 In medium with a pH value

pH-Sensitive Fluorescent Hepatocyte-Targeting Multilayer

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