Core−Shell chitosan microcapsules for programmed sequential

Research Article www.acsami.org Core−Shell Chitosan Microcapsules for Programmed Sequential Drug Release Xiu-Lan Yang,† Xiao-Jie Ju,*,†,‡ Xiao-Ting Mu,† Wei Wang,† Rui Xie,† Zhuang Liu,† and Liang-Yin Chu†,‡ † School of Chemical Engineering and ‡State Key Laboratory of Polymer Materials Engineering, and Collaborative Innovation Center for Biomaterials Science and Technology, Sichuan University, Chengdu 610065, P R China S Supporting Information * ABSTRACT: A novel type of core−shell chitosan microcapsule with programmed sequential drug release is developed by the microfluidic technique for acute gastrosis therapy The microcapsule is composed of a cross-linked chitosan hydrogel shell and an oily core containing both free drug molecules and drug-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles Before exposure to acid stimulus, the resultant microcapsules can keep their structural integrity without leakage of the encapsulated substances Upon acid-triggering, the microcapsules first achieve burst release due to the acidinduced decomposition of the chitosan shell The encapsulated free drug molecules and drug-loaded PLGA nanoparticles are rapidly released within 60 s Next, the drugs loaded in the PLGA nanoparticles are slowly released for several days to achieve sustained release based on the synergistic effect of drug diffusion and PLGA degradation Such core−shell chitosan microcapsules with programmed sequential drug release are promising for rational drug delivery and controlled-release for the treatment of acute gastritis In addition, the microcapsule systems with programmed sequential release provide more versatility for controlled release in biomedical applications KEYWORDS: microcapsulesc, chitosan, PLGA, nanoparticles, programmed sequential drug release INTRODUCTION The incidence of gastropathy increases every year because of unreasonable eating and living habits, the abuse of drugs, or inherited factors.1,2 Acute gastritis attacks rapidly, and often causes dehydration and acid−base disturbance Without treatment in time, acute gastritis can even bring a variety of complications, which will badly endanger the health of patients Traditional dosage forms, such as tablets, capsules, and granules, for gastroenteritis treatment have many disadvantages, such as frequent drug administration, large fluctuation of plasma drug concentration, untargeted action, and low bioavailability.3,4 Considering the characteristics of acute gastroenteritis and the clinical needs, controlled drug release systems are expected to obtain more effective treatment When gastroenteritis attacks, it is desired that the plasma drug concentration could immediately reach to the treatment level and that the drug could quickly take effect after administration Thus, the burst-release mode with large drug dose is more appropriate in this case After the first burst release, it is desired that the drug dose could be constantly supplied to keep the plasma drug concentration within safe and effective range for a long time, which can maintain therapeutic effect and restrain complications Thus, the sustained-release mode is more suitable in this case If burst-release and sustained-release modes are orderly combined into a single drug carrier to achieve sequential release behaviors, i.e., burst release first and © 2016 American Chemical Society then sustained release, it would be very beneficial to more rational and effective therapy for gastroenteritis Therefore, the design and preparation of drug delivery systems with programmed sequential release ability, which can reduce the frequency of administration and increase patient compliance, are of great scientific and technological importance Microcapsules, which can encapsulate various active substances to protect them from the surrounding environment, are of great interest for many applications, especially in the drug delivery field.5,6 Recently, microcapsules with various structures and functions are developed to achieve programmed sequential release, and they are considered to be very applicable as drug delivery carriers These functional microcapsules are mainly divided into two categories One is the stimuli-responsive microcapsule with a programmed pulsed-release ability based on a repeated “on−off” mechanism.7−16 However, triggered by such a programmable pulse-type external stimulus, the drug release from the microcapsules is either in an “on” state or in an “off” state, so the drug release mode is relative simplex.7,8 The other category is the core−shell-structured microcapsule with drugs loaded in different layers, so that the drugs can be released sequentially.17−23 The drugs loaded in outer shell are Received: January 31, 2016 Accepted: April 7, 2016 Published: April 7, 2016 10524 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces Scheme Schematic Illustration of the Programmed Sequential Drug Release from Core−Shell Chitosan Microcapsule: (A) First, Burst Release of Free Drug Molecules and Drug-Loaded PLGA Nanoparticles from the Microcapsule Can Be Achieved via the Rapid Decomposition of Chitosan Shell in Acidic Solution, and (B) Second, Sustained-Release of Drugs from the PLGA Nanoparticles Can Be Achieved via Drug Diffusion and PLGA Degradation first released when the shell layer is eroded, swelled, or decomposed, and then the drugs in the core layer diffuse out to achieve the second-stage release However, these sequential release manners are usually both sustained-release mode, so that the plasma drug concentration cannot immediately reach effective value after the first dosing.17 Furthermore, drug leakage problems exist before these core−shell microcapsule carriers reach the targeted sites To the best of our knowledge, drug-loaded microcapsules that can achieve burst release first and then sustained release have not been reported yet Previous studies inspired us to design a kind of microcapsule with special core−shell structure and a stimuli-responsive property to achieve the programmed sequential drug release that we expect Here, we report on a novel type of core−shell microcapsule with programmed sequential drug release, i.e., burst release in the stomach first and then sustained release in the gastrointestinal tract As illustrated in Scheme 1A1, the proposed microcapsule is composed of a cross-linked chitosan hydrogel shell and an oily core Particularly, the oily core contains both free drug molecules and drug-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles Because of the existence of an oil−water interface between the inner oily core and the hydrous chitosan shell, there will be no-leakage of the encapsulated drugs before these microcapsule carriers reach the stomach In our previous studies, we find that chitosan hydrogels prepared using terephthalaldehyde as the cross-linker exhibit a great acidinduced dissolution property.24,25 There is an obvious change in pH along the gastrointestinal tract, and the stomach is a special acidic environment with low pH value (pH 1−3).26 Therefore, the encapsulated free drug molecules with large dose can be suddenly released due to the decomposition of the chitosan shell under the unique acidic condition of the stomach (Scheme 1A2,A3) Simultaneously, the coencapsulated drugloaded PLGA nanoparticles are also released out, which could provide a second, sustained release based on the synergistic effect of drug diffusion and PLGA degradation,27,28 as shown in Scheme 1B1−B3 The first burst-release mode can make the plasma drug concentration rapidly reach the treatment level, which can relieve the symptoms of acute gastritis quickly The second sustained-release mode can constantly supply drug dosage to keep the plasma drug concentration within a safe and effective range for a long time, which can cure acute gastritis and suppress complications That is, this kind of novel core− shell microcapsule, which can achieve programmed sequential drug release, is of great potential to realize more rational drug administration for the treatment of acute stomach illness In addition, these microcapsules provide more flexibility for versatile loading of different drugs, such as oleophilic drugs, hydrophilic drugs, and multiple drugs with synergistic efficacy EXPERIMENTAL SECTION 2.1 Materials Water-soluble chitosan (CS, Mw = 5000, degree of deacetylation = 85%) is provided by Ji’nan Haidebei Marine Bioengineering Co., Ltd PLGA (≥99%, lactide/glycolide = 50/50, Mw = 20 000) is purchased from Sichuan Dikang Sci & Tech Pharmaceutical Co., Ltd Soybean oil (Kerry Oils & Grains) is used as the oil phase Oleophilic curcumin (HPLC ≥ 98%, Chengdu Herbpurify), hydrophilic catechin (HPLC ≥ 98%, Chengdu Herbpurify), and hydrophilic Rhodamine B (RhB, ≥ 99%, Chengdu Kelong Chemicals) are all used as model drugs Poly(vinyl alcohol) (PVA, ≥97%, Chengdu Kelong Chemicals) is used as emulsion stabilizer for preparation of drug-loaded PLGA nanoparticles Pluronic F127 (Bio-Reagent, Sigma-Aldrich) and polyglycerol polyricinoleate (PGPR, ≥99.8%, Danisco) are used as surfactants in aqueous phase and organic phase, respectively Hydroxyethylcellulose (HEC, ≥98%, Lingxianzi Cellulose) is used for viscosity adjustment Terephthalaldehyde (≥98%, Sinopharm Chemical Reagent) is used as cross-linker All other chemicals are of analytical grade and used as received Deionized water (18.2 MΩ, 25 °C) from a Millipore Milli-Q Plus water purification system is used throughout the experiments 2.2 Preparation of Drug-Loaded PLGA Nanoparticles In this work, oleophilic curcumin and hydrophilic catechin, which are both typical gastrointestinal drugs with good anti-inflammatory effect, are used as model drugs to prepare different drug-loaded PLGA nanoparticles Curcumin-loaded PLGA nanoparticles (Cur−PLGA-NPs) are prepared by a modified emulsion solvent evaporation method.29,30 Briefly, PLGA (300 mg) and curcumin (40 mg) are dissolved in a mixed organic solvent (10 mL) of dichloromethane and ethyl acetate (3:2, v/v) as the oil phase Thirty milliliters of PVA aqueous solution (1.0%, w/v) is used as the water phase The oil phase is dropwise 10525 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces added into the water phase under agitation (300 rpm) for 10 min, followed by homogeneous emulsification (19 000 rpm) for using a BRT homogenizer (B25, 10 mm head) to obtain oil-in-water (O/W) emulsions Next, the O/W emulsions are transferred into deionized water and stirred overnight at room temperature for complete evaporation of the organic solvent The solidified nanoparticles are purified by repeated centrifugation with deionized water Catechin-loaded PLGA nanoparticles (C−PLGA-NPs) are prepared by a similar emulsion solvent evaporation process as mentioned above, except water-in-oil-in-water (W1/O/W2) double emulsions are used as the synthesis templates.31 Briefly, ethanol (1 mL) containing catechin (40 mg) is dispersed in organic solution (10 mL) containing PLGA to obtain W1/O primary emulsions Then, the primary emulsions are dropwise added into aqueous solution (30 mL) containing PVA (2.0%, w/v) under agitation to prepare the double emulsion templates The increase of PVA concentration is to improve the loading capacity of catechin Next, after complete solvent evaporation and centrifugation-based purification, C−PLGA-NPs are obtained Because the color and fluorescence of catechin are difficult to observe, RhB with similar hydrophilicity property and molecular weight is used as the model hydrophilic drug instead of catechin for optical and fluorescent characterization Thus, RhB-loaded PLGA nanoparticles (RhB−PLGA-NPs) are also prepared using the same method as for C−PLGA-NPs To maintain the drug activity and avoid PLGA hydrolysis, the drugloaded PLGA nanoparticles are freeze-dried and then stored in a dry cabinet at °C 2.3 Characterization of Drug-Loaded PLGA Nanoparticles The chemical compositions of Cur−PLGA-NPs, C−PLGA-NPs, and RhB−PLGA-NPs are confirmed by Fourier transform infrared spectroscopy (FT-IR, IR Prestige-21, Shimadzu) using the KBr disk technique The morphologies of the drug-loaded PLGA nanoparticles in the dried state are observed by scanning electron microscopy (SEM) (JSM-7500F, JEOL), and their morphologies in water and oil solutions are observed by confocal laser scanning microscopy (CLSM) (SP5-II, Leica) Moreover, the size and size distribution of the drugloaded PLGA nanoparticles are measured by dynamic light scattering (DLS) (Zetasizer Nano ZS90-ZEN3690, Malvern) The drug-loading capacity and encapsulation efficiency of PLGA nanoparticles are measured by UV−visible spectrophotometry (UV− vis) (UV-1700, Shimadzu) A given amount of freeze-dried nanoparticles (2 mg for Cur−PLGA-NPs or mg for C−PLGA-NPs/ RhB−PLGA-NPs) is dissolved in mL of methanol, and then the solution is treated with ultrasonic oscillation for h to ensure the complete extraction of the loaded drugs The methanol solution is centrifuged at 12 000 rpm and the supernatant is collected After dilution, the drug concentration in the supernatant is determined by UV−vis at a specific wavelength (435 nm for curcumin and 278 nm for catechin) The drug-loading capacity (LCNP) and encapsulation efficiency (EENP) of PLGA nanoparticles are calculated as follows: LC NP = mass of drug in nanoparticles × 100% total mass of nanoparticles EE NP = mass of drug in nanoparticles total mass of drug used for nanoparticle preparation × 100% burst release Taking these factors into consideration and to have comparability, the flow rates of three-phase fluids and the size of the microfluidic device have been optimized and fixed to use in this work Briefly, a mixture of soybean oil and benzyl benzoate (1:1, v/v) containing free drug molecules (3 mg/mL), drug-loaded PLGA nanoparticles (3 mg/mL), terephthalaldehyde (2.4 wt %), and PGPR (8.0%, w/v) is used as the inner oil phase Soybean oil is used as the oily solvent, and benzyl benzoate is added to adjust the density and viscosity of the inner oil phase Deionized water containing chitosan (2.0%, w/v), F127 (1.5%, w/v), and HEC (2.0%, w/v) is used as the middle aqueous phase The outer oil phase is soybean oil containing PGPR (8.0%, w/v) The flow rates of the inner, middle, and outer fluids are QI = 400 μL/h, QM = 800 μL/h, and QO = 5000 μL/h, respectively The obtained O/W/O emulsions are collected in a glass container and left for 10 h at room temperature to ensure the complete cross-linking of the chitosan in the water phase Here, we present several kinds of composite core−shell microcapsules containing different free drug molecules and different drug-loaded PLGA nanoparticles Moreover, other kinds of chitosan microcapsules are also prepared by the same method except that the inner cores contain only free drug molecules or only drug-loaded PLGA nanoparticles Generally, the prepared microcapsules can be placed in a small amount of soybean oil for storage Before characterization, these prepared microcapsules are washed with a mixture of acetone and deionized water (1:1, v/v) to remove the outer oil and simultaneously to keep the inner cores still inside the microcapsules 2.5 Characterization of O/W/O Emulsions and Microcapsules The morphologies of O/W/O emulsions are characterized by optical microscopy (BX 61, Olympus) The size and size distribution are calculated on the basis of the obtained optical micrographs using analytic software (Tiger 3000, Chongqing Xinminfeng Instruments) The morphologies of resultant core−shell chitosan microcapsules are observed by CLSM (SP5-II, Leica) Furthermore, to confirm that there is no leakage of drug molecules from the microcapsules before they reach the stomach, the stability of composite core−shell microcapsules in neutral environment is investigated by recording the variation of the relative fluorescence intensity of the inner cores 2.6 Programmed Sequential Drug Release of Microcapsules The whole release behavior of the core−shell chitosan microcapsules is a programmed combination of, first, burst release of free drug molecules and, second, sustained release from drug-loaded PLGA nanoparticles The acid-triggered burst-release behavior of the microcapsules is monitored by CLSM (SP5-II, Leica) First, the composite core−shell microcapsules are equilibrated in a small amount of deionized water in a transparent glass container To change the ambient solution into an acidic medium, excess HCl solution (pH 1.5) is added into the container rapidly All experiments on burst-release behaviors of microcapsules are performed at room temperature After acid-triggered burst release, the free drug molecules and drugloaded PLGA nanoparticles in inner cores are both released and dispersed in the surrounding solution That is, the following sustainedrelease behavior is similar to the simple drug release from PLGA nanoparticles To verify our hypotheses, the sustained-release behaviors of curcumin and catechin directly from PLGA nanoparticles are studied first These release experiments are carried out in phosphate-buffered saline (PBS, pH 7.4) at 37 °C using a waterbathing shaker at 100 rpm Because of the poor water solubility of curcumin, PBS containing ethanol (5%, v/v) is employed for Cur− PLGA-NPs to increase the solubility of curcumin Each sustainedrelease experiment is performed in triplicate at the same time and under sink condition In detail, 12 mL of PBS containing 18 mg of drug-loaded nanoparticles is divided equally into three parts and then separately placed into three centrifuge tubes At predetermined time intervals, the nanoparticle suspensions are centrifuged at 12 000 rpm for 10 min, and then mL of supernatant is removed and replaced with fresh PBS Drug concentrations of these supernatants are determined by UV−vis to calculate the released amount of drug at different time intervals (1) (2) 2.4 Preparation of Core−Shell Chitosan Microcapsules The core−shell chitosan microcapsules containing both free drug molecules and drug-loaded PLGA nanoparticles are prepared with oil-in-water-in-oil (O/W/O) emulsions as templates, which are fabricated by the capillary microfluidic technique according to our published method.24 The microfluidic technique is an excellent method to prepare multiple emulsions with precisely controlled size To better achieve our proposed design purpose, first the formed O/W/O emulsion templates and as-prepared microcapsules should be stable; moreover, the prepared microcapsules with large inner volume and proper membrane thickness are good for loading more drugs and for rapid 10526 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces To display the entire programmed sequential drug release process, a continuous release experiment combining, first, burst release and, second, sustained release is also studied Before adding acid, the prepared composite core−shell microcapsules (1 mL) are immersed into mL of ethanol for ∼10 Then, the pH value of the ethanol solution is adjusted to 1.5 by immediately adding hydrochloric acid The drug concentrations in ethanol solution before and after adding acid are measured by UV−vis to determine the amount of released drugs After complete decomposition of the chitosan shell for burst release, the drug-loaded PLGA nanoparticles are also released into the ethanol solution These drug-loaded PLGA nanoparticles are collected by centrifugation at 12 000 rpm for 10 and then dispersed into PBS solution (pH 7.4) to investigate further the second, sustained release by conducting experiments similar to the above-mentioned simple sustained-release experiment of drug-loaded PLGA nanoparticles The use of ethanol for the first burst release is to ensure that, the free drug molecules including both oleophilic curcumin and hydrophilic catechin can be rapidly dispersed into the surrounding solution from oily cores This process is similar to the actual situation in which the free drug molecules can be immediately dispersed in gastric fluid when composite core−shell microcapsules reach stomach The drug-loading capacity of the composite microcapsules (LCMC) is defined as the ratio of the total drug-loading amount to the mass of microcapsules The total drug-loading amount in the microcapsules is the sum of the amounts of the encapsulated free drugs and the drugs contained in the encapsulated PLGA nanoparticles To calculate the drug-loading capacity of the microcapsules, a certain amount of microcapsules are immersed into a given volume of ethanol solution (pH 1.5) Due to the acid-induced decomposition of chitosan shell, free drug molecules and drug-loaded nanoparticles are all dispersed in ethanol solution Then, the ethanol solution is ultrasonically treated for h to ensure the maximum dissolution of drug molecules in the ethanol solution After that, the ethanol solution is centrifuged (12 000 rpm) for 10 and the supernatant solution is collected The drug concentration in the supernatant solution is measured by UV−vis To completely extract drugs from the nanoparticles, the precipitant is redispersed in a given volume of ethanol solution, followed with repeated ultrasonic treatment and centrifugation until the drug concentration in the supernatant solution cannot be detected The sum of the drug amounts in all supernatant solutions is the total drugloading amount in the microcapsules Figure FT-IR spectra of blank PLGA nanoparticles (A), curcumin drug (B), Cur−PLGA-NPs (C), catechin drug (D), C−PLGA-NPs (E), RhB model drug (F), and RhB−PLGA-NPs (G) spectra of Cur−PLGA-NPs (curve C), C−PLGA-NPs (curve E), and RhB−PLGA-NPs (curve G) All the results confirm that curcumin, catechin, and RhB are successfully encapsulated in PLGA nanoparticles SEM images of Cur−PLGA-NPs, C−PLGA-NPs, and RhB− PLGA-NPs clearly show that all drug-loaded nanoparticles show good spherical shape and uniform size (Figure 2) CLSM is used to observe the dispersibility and morphology of the drug-loaded PLGA nanoparticles in water As shown in Figure 3A1, PLGA nanoparticles containing oleophilic curcumin exhibit obvious green fluorescence due to the autofluorescence of curcumin Because catechin has nearly no fluorescence, C− PLGA-NPs not exhibit fluorescence under CLSM observation (Figure 3A2) For better observation, RhB− PLGA-NPs are used as a substitute sample for PLGA nanoparticles containing hydrophilic drugs, because they display obvious red fluorescence from the RhB dye (Figure 3A3) It can be seen that these three kinds of nanoparticles are well-dispersed in water without bulk aggregation, which benefits the drug release from the nanoparticles The DLS results show that the average sizes of Cur−PLGA-NPs, C− PLGA-NPs, and RhB−PLGA-NPs are 551.6, 478.4 and 466.6 nm, respectively The polydispersity index (PDI) values of Cur−PLGA-NPs, C−PLGA-NPs, and RhB−PLGA-NPs are 0.122, 0.114, and 0.091, respectively, indicating the good monodispersity of these nanoparticles A uniform size for polymer particles is crucial for their use as drug delivery carriers, since it allows precise manipulation of the drug-loading amount, optimization of the release kinetics, and repeatability of the release profiles.32 The dispersibility of drug-loaded PLGA nanoparticles in oil solution is also studied As shown in Figure S1 (Supporting Information), Cur−PLGA-NPs, C−PLGANPs, and RhB−PLGA-NPs also exhibit good dispersibility in soybean oil without bulk aggregation, which benefits the generation of O/W/O emulsion templates in microfluidic devices without clogging the microchannel RESULTS AND DISCUSSION 3.1 Composition and Morphology of Nanoparticles FT-IR spectra of different drug-loaded PLGA nanoparticles are shown in Figure Specifically, the characteristic bands of curcumin molecule (curve B), including a wide peak around 3400 cm−1 for the O−H stretching vibration of the phenol group, three peaks at 1625−1500 cm−1 for the CC skeletal stretching vibration in the benzene ring, and two peaks at 860− 800 cm−1 for the C−H bending vibration of the benzene ring, are all found in the FT-IR spectrum of Cur−PLGA-NPs (curve C) Similarly, the characteristic bands of catechin (curve D), including a wide peak around 3380 cm−1 for the O−H stretching vibration of the phenol group, three peaks at 1625− 1500 cm−1 for the CC skeletal stretching vibration of the benzene ring, and two peak at 860−800 cm−1 for the C−H bending vibration of the benzene ring, are all found in the FTIR spectrum of C−PLGA-NPs (curve E) For RhB−PLGANPs, the characteristic bands of RhB (curve F), including the characteristic peak at 1700 cm−1 for the CO stretching vibration of the carboxyl group and two peaks at 1650−1550 cm−1 for the CC skeletal stretching vibration of the benzene ring, can also be found in the FT-IR spectrum of RhB−PLGANPs (curve G) Furthermore, the characteristic peak at 1750 cm−1 for the CO stretching vibration of the ester bond in PLGA nanoparticles (curve A) can be found in the FT-IR 10527 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces Figure SEM images of Cur−PLGA-NPs (A), C−PLGA-NPs (B), and RhB−PLGA-NPs (C) Figure CLSM images (A) and size distributions (B) of Cur−PLGA-NPs (A1, B1), C−PLGA-NPs (A2, B2), and RhB−PLGA-NPs (A3, B3) in water drug molecules and PLGA nanoparticles with the same drug molecules Such kind of microcapsule is demonstrated by preparing microcapsules containing free oleophilic curcumin and Cur−PLGA-NPs and microcapsules containing free hydrophilic catechin and C−PLGA-NPs This kind of microcapsule is used to prove that the same drugs can be released in a programmed sequential release manner to reduce the frequency of drug administration The other kind of the microcapsules contain free drug molecules and PLGA nanoparticles with different drug molecules This is demonstrated by preparing microcapsules containing free curcumin and C−PLGA-NPs and microcapsules containing free catechin and Cur−PLGANPs Such type of microcapsule is used to verify that multiple drugs with synergistic efficacy35,36 can be sequentially released to enhance the therapeutic effect O/W/O emulsions are used as templates to prepare the designed core−shell chitosan microcapsules Figure 4A−H shows the optical micrographs of different kinds of O/W/O emulsions prepared by the microfluidic method These emulsions all show clear and stable core−shell structures Similarly, since catechin exhibits no color and no fluorescence, RhB is used instead of catechin as the hydrophilic model drug for optical characterization Curcumin has a bright-yellow color and RhB is a red dye As a result, different O/W/O emulsions have different colors in the inner cores Figure 4A−D shows the control groups with inner cores containing only free drugs or The drug-loading capacities of Cur−PLGA-NPs and C− PLGA-NPs are 12.87% and 3.23%, respectively, and their encapsulation efficiencies are 64.76% and 66.85%, respectively The loading capacity of hydrophilic catechin is smaller than that of oleophilic curcumin The reason is that a large number of catechin is lost during the nanoparticle preparation process Hydrophilic drug molecules can easily diffuse from the organic phase into the outer aqueous phase during the solvent evaporation process, which results in small drug-loading capacity.33 Similarly, the drug-loading capacity and encapsulation efficiency of RhB−PLGA-NPs is 2.94% and 60.23% Certainly, the loading capacity of hydrophilic drugs in PLGA nanoparticles can be improved by various methods through changing the preparation parameters.34 3.2 Morphologies of Emulsion Templates and Microcapsules The purpose of this work is to develop a novel type of core−shell microcapsule for programmed sequential drug release The proposed microcapsule is composed of a crosslinked chitosan shell and an oily core containing both free drug molecules and drug-loaded PLGA nanoparticles The microcapsules with such core−shell structures provide more flexibility for versatile loading of different drugs, such as oleophilic drugs, hydrophilic drugs, and multiple drugs with synergistic efficacy To demonstrate the feasibility of our technique, two kinds of core−shell chitosan microcapsules are designed in this work One is microcapsules containing free 10528 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces Figure Optical micrographs (A−H) and size distributions (E1−H1) of different O/W/O emulsions: (A) emulsions containing only free curcumin, (B) emulsions containing only Cur−PLGA-NPs, (C) emulsions containing only free RhB, (D) emulsions containing only RhB−PLGANPs, (E, E1) emulsions containing both free curcumin and Cur−PLGA-NPs, (F, F1) emulsions containing both free curcumin and RhB−PLGANPs, (G, G1) emulsions containing both free RhB and RhB−PLGA-NPs, and (H, H1) emulsions containing both free RhB and Cur−PLGA-NPs Scale bars are 200 μm Figure 4E−H shows the optical micrographs of the final O/W/ O emulsions, which serve as templates to prepare core−shell microcapsules containing both free drug molecules and drugloaded PLGA nanoparticles The image of O/W/O emulsions containing both free curcumin and Cur−PLGA-NPs (Figure 4E), which shows both bright yellow color and lots of black dots in the inner cores, is nearly an overlay of parts A and B of Figure Similarly, the image of O/W/O emulsions containing both free RhB and RhB−PLGA-NPs (Figure 4G) seems to be an overlay of parts C and D of Figures On the other hand, the core color of O/W/O emulsions containing both free curcumin and RhB−PLGA-NPs (Figure 4F) is almost a mixture only PLGA nanoparticles with same drug molecules The O/ W/O emulsions containing only free oleophilic curcumin (Figure 4A) or only Cur−PLGA-NPs (Figure 4B) show obvious yellow color in their inner cores Specially, due to larger amount of curcumin, the emulsions containing only free curcumin show brighter yellow color in their inner cores than those containing only Cur−PLGA-NPs In addition, there are lots of black dots in the inner cores of emulsions containing only Cur−PLGA-NPs, which are slightly aggregated PLGA nanoparticles Similarly, the O/W/O emulsions containing only free hydrophilic RhB (Figure 4C) or only RhB−PLGA-NPs (Figure 4D) exhibit red color from RhB dye in the inner cores 10529 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces of the colors in Figure 4A,D A similar situation can also be found in O/W/O emulsions containing both free RhB and Cur−PLGA-NPs (Figure 4H) These results indicate that different free drug molecules and drug-loaded PLGA nanoparticles are successfully encapsulated into the inner cores of O/W/O emulsions individually or together In addition, encapsulated free drug molecules and drug-loaded PLGA nanoparticles not affect the structural integrity and stability of the emulsions Figure 4E1−H1 is the corresponding size distributions of these final O/W/O emulsions All emulsion templates show uniform size with a narrow size distribution A parameter called the coefficient of variation (CV), which is defined as the ratio of the standard deviation of the size distribution to its arithmetic mean, is used to evaluate the size monodispersity of the particles and emulsions The calculated CV values for the inner diameters (ID) and outer diameters (OD) of O/W/O emulsions shown in Figure 4E are 1.48% and 1.64%, respectively, indicating the high monodispersity of these emulsion templates The O/W/O emulsions shown in Figure 4F also show good monodispersity, and the CV values for ID and OD are 2.01% and 1.99%, respectively Similarly, CV values for ID and OD shown in Figure 4G are 1.85% and 1.2%, respectively, and CV values for ID and OD shown in Figure 4H are 1.67% and 1.2%, respectively Using the monodisperse O/W/O emulsions as templates, core−shell chitosan microcapsules with uniform size and structure are prepared via an interfacial cross-linking reaction Figure shows the CLSM images of core−shell chitosan microcapsules loading with different substrates in their inner cores Due to the formation of Schiff base bonds, chitosan hydrogels cross-linked by terephthalaldehyde can exhibit autofluorescence.24,25 Therefore, the chitosan shell layers in these different kinds of microcapsules all display obvious green fluorescence Due to loading with different substrates, there exist distinct differences in their inner cores Because curcumin and RhB are naturally fluorescent in the visible green and red spectra respectively, the inner cores of microcapsules containing only free curcumin (Figure 5A) or only free RhB (Figure 5B) show clear green fluorescence or red fluorescence Similarly, microcapsules containing only Cur−PLGA-NPs (Figure 5C) or only RhB−PLGA-NPs (Figure 5D) also show green fluorescence or red fluorescence Because of the higher loading amount of free drug molecules, the microcapsules containing only free curcumin or only free RhB have brighter fluorescence in their inner cores compared with the microcapsules containing only Cur−PLGA-NPs or only RhB− PLGA-NPs Compared with those in Figure 5A,C, the fluorescence intensity of the inner cores in microcapsules containing both free curcumin and Cur−PLGA-NPs (Figure 5E) obviously increases The composite core−shell microcapsules containing both free RhB and RhB−PLGA-NPs (Figure 5G) show the same phenomenon, that the red fluorescence intensity in the inner cores also increases compared with that in Figure 5B,D In addition, core−shell microcapsules containing both free RhB and Cur−PLGA-NPs (Figure 5H) display a mixed fluorescence color of red fluorescence from RhB and green fluorescence from Cur− PLGA-NPs However, in the overlap of CLSM images on green and red fluorescent channels, the phenomenon of mixed fluorescence color does not appear for the microcapsules containing both free curcumin and RhB−PLGA-NPs (Figure 5F), which just show green fluorescence The reason is that the Figure CLSM images of different core−shell chitosan microcapsules: (A) microcapsules containing only free curcumin, (B) microcapsules containing only free RhB, (C) microcapsules containing only Cur−PLGA-NPs, (D) microcapsules containing only RhB− PLGA-NPs, (E) microcapsules containing both free curcumin and Cur−PLGA-NPs, (F) microcapsules containing both free curcumin and RhB−PLGA-NPs, (G) microcapsules containing both free RhB and RhB−PLGA-NPs, and (H) microcapsules containing both free RhB and Cur−PLGA-NPs parts A, C, and E are on a green fluorescent channel and parts B, D, and F−H are the overlap of images on green and red fluorescent channels Scale bars are all 500 μm loading amount of RhB in the PLGA nanoparticles is much smaller than that of free curcumin in the inner cores, so the red fluorescence of RhB is shielded by the green fluorescence of curcumin All the results demonstrate that different free drug molecules and drug-loaded nanoparticles can be successfully encapsulated in the composite core−shell microcapsules 10530 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces Figure Relative fluorescence intensity of the inner cores at hourly intervals in neutral aqueous solution (pH 6.8, 37 °C): (A) microcapsules containing both free oleophilic curcumin and Cur−PLGA-NPs and (B) microcapsules containing both free hydrophilic RhB and RhB−PLGA-NPs 3.3 Stability of Drug-Loaded Microcapsules Before the proposed core−shell chitosan microcapsules reach the targeted stomach site, it is vital that microcapsules can maintain their structural integrity and prevent loaded drugs from leaking Therefore, no leakage of drugs from the microcapsules in neutral medium is confirmed before the controlled-release experiments The core−shell chitosan microcapsules containing both free drug molecules and drug-loaded PLGA nanoparticles are used as the typical examples to investigate the stability of different composite core−shell microcapsules In order to facilitate real-time monitoring by the CLSM method, the drug amounts of curcumin and RhB are represented by the fluorescence intensities of the inner cores After the microcapsules are placed into the neutral aqueous solution (pH 6.8, 37 °C), the fluorescence intensities of the inner cores are recorded at hourly intervals within h Relative fluorescence intensity, which is defined as the ratio of fluorescence intensity at a desired time to that at the initial time, is used to evaluate the drug leakage For microcapsules loaded with oleophilic curcumin (Figure 6A), the relative fluorescence intensity remains nearly unchanged at ∼1, indicating nearly no leakage of curcumin from the microcapsules For hydrophilic RhBloaded microcapsules (Figure 6B), the fluorescence intensity of inner cores slightly decreases after h After being dispersed in aqueous solution for h, the chitosan shells of microcapsules swell completely, so the pores of the cross-linked network become larger At this time, compared with oleophilic curcumin, hydrophilic RhB is easier to pass through the hydrous chitosan shell However, such slight leakage does not affect the actual clinical performance, because the delivery time of the microcapsules from oral administration to stomach site is usually less than h So, there is nearly no leakage of RhB before the drug-loaded microcapsules reach stomach We also test the stabilities of the microcapsules containing only free drug molecules, only drug-loaded nanoparticles, or other kinds of composite core−shell microcapsules (Figure S2, Supporting Information), which also show the similar results These results indicate that the loaded drug molecules scarcely escape from microcapsules within the required time due to the oil−water interface between the inner cores and the hydrous chitosan shells 3.4 Programmed Sequential Release Characteristics of Microcapsules The programmed sequential drug release of our proposed core−shell chitosan microcapsules is designed as, first, burst release in the stomach and, second, sustained release in the gastrointestinal tract The controlled-release behaviors of two kinds of representative core−shell microcapsules containing both free drug molecules and PLGA nanoparticles with same drug molecules are investigated First, the acid-triggered burst-release behaviors of proposed microcapsules are studied under CLSM Prior to tests, microcapsule samples are immersed in deionized water Then, we introduce a sudden change to the pH value of their environmental solution by quickly adding excess HCl solution (pH 1.5) Figure shows the CLSM microscope snapshots of acid-triggered burst-release processes of these two kinds of Figure CLSM microscope snapshots of acid-triggered burst-release processes of microcapsules containing both free curcumin and Cur− PLGA-NPs (A, green fluorescent channel) and microcapsules containing both free RhB and RhB−PLGA-NPs (B, overlap of images on green and red fluorescent channels) HCl solution with pH 1.5 is added at t = s The scale bars are all 500 μm 10531 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces Figure Cumulative releases of curcumin (A) and catechin (B) from PLGA nanoparticles in PBS (pH 7.4, 37 °C) Figure Programmed sequential release behaviors of curcumin-loaded (A) and catechin-loaded (B) composite core−shell microcapsules representative microcapsules One is the microcapsules containing both free oleophilic curcumin and curcumin-loaded PLGA nanoparticles (Figure 7A), and another one is the microcapsules containing both free hydrophilic RhB and RhBloaded PLGA nanoparticles (Figure 7B) The acid-induced decomposition phenomena of chitosan shell layers for these two kinds of microcapsules are almost the same That is, chitosan microcapsules maintain good spherical shape and structural integrity in neutral medium (pH 6.8, 37 °C) Once HCl solutions are added into the microcapsule suspensions, the chitosan shells swell immediately at first and then a rapid and complete decomposition is achieved within 60 s Such decomposition of chitosan shells in acidic solution is a result of acid-induced hydrolysis of the Schiff base bonds between chitosan and terephthalaldehyde.24,25 With the breakup of chitosan shells, both free drug molecules and drug-loaded PLGA nanoparticles are released into the surrounding medium, along with the dispersion of inner cores For our proposed microcapsules, the release of free drug molecules with large loading amount successfully first achieves the stomach-targeted burst release that can make the plasma drug concentration rapidly reach the treatment level Meanwhile, the quickly released drug-loaded nanoparticles disperse well in the aqueous medium, which is beneficial to the following sustained drug release from the PLGA nanoparticles Movies of the acidtriggered burst-release processes are also shown in the Supporting Information (Movie S1 and Movie S2) At the first acid-triggered burst-release stage, the coencapsulated drug-loaded PLGA nanoparticles are also released from the microcapsules, which could provide a second, sustained release based on the synergistic effect of drug diffusion and PLGA degradation The sustained-release behaviors of curcumin and catechin from PLGA nanoparticles are evaluated under simulated physiological conditions (PBS, pH 7.4, 37 °C) The in vitro release profiles of drugs are obtained by graphing the accumulated release percentage of drug from PLGA nanoparticles as a function of the time The cumulative release curves of oleophilic curcumin and hydrophilic catechin both present a typical sustained-release “first-order kinetic model” A sustained and prolonged release of oleophilic curcumin in the PBS for up to 28 days is observed in Figure 8A In the initial period of h, approximately 10% of curcumin is released, followed by a sustained drug release Within 28 days, 63.6% of the encapsulated curcumin is released from the nanoparticles Figure 8B shows the release profile of hydrophilic catechin, which also represents a good sustained-release behavior There is also a relative fast release of catechin within the initial h, and 60.86% of drug is slowly released within days It is noteworthy that the release rate and mechanism are different between curcumin and catechin In general, the drug release mechanisms depend upon the solubility and diffusion of drug, as well as the biodegradation of the matrix materials Catechin is a hydrophilic molecule with a greater solubility in aqueous environment, so its drug diffusion rate through the polymeric matrix is much faster than that of oleophilic curcumin In summary, the drug-loaded PLGA nanoparticles show good second, sustained release, which can bring constant and effective therapeutic action To display the entire programmed sequential drug release process, a continuous release experiment combining, first, burst 10532 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces release stage Simultaneously, the coencapsulated drug-loaded PLGA nanoparticles are also released to provide the second sustained-release stage Respectively, about 19.3% of curcumin and 32.3% of catechin are slowly released from PLGA nanoparticles within days Such well-designed core−shell chitosan microcapsules with programmed sequential drug release are promising to achieve a more rational drug delivery and controlled release for the treatment of acute stomach illness In addition, these microcapsules provide more versatility for loading different drugs, such as oleophilic drugs, hydrophilic drugs, and multiple drugs with synergistic efficacy Moreover, the results of this study also provide a versatile strategy for designing and developing novel functional microcapsules with various programmed sequential release properties for biomedical applications release and, second, sustained release is also studied Figure shows the drug release curves of two kinds of representative core−shell microcapsules One is for the microcapsules containing both free oleophilic curcumin and curcumin-loaded PLGA nanoparticles (Figure 9A), and the other one is for the microcapsules containing both free hydrophilic catechin and catechin-loaded PLGA nanoparticles (Figure 9B) During the initial 10 equilibrium time, there is nearly no leakage of drug from the microcapsules These data match well with the results of stability experiments of microcapsules in Figure After adding ethanol solution containing HCl (pH 1.5), free drug molecules are released immediately from the composite core−shell microcapsules Respectively, about 56.2% of curcumin (Figure 9A) and 59.6% of catechin (Figure 9B) are released within 60 s, which directly shows the first burst-release performance The following sustained-release experiments are carried out for days Relative to the total drug-loading amount in whole microcapsules, about 19.3% of curcumin (Figure 9A) and 32.3% of catechin (Figure 9B) are slowly released from PLGA nanoparticles within days The curve shapes and tendencies for change of sustained-release parts in Figure are similar to that in Figure 8, which also present a typical sustained-release “first-order kinetic model” However, since the accumulated release percentage of drug is relative to the total drug-loading amount in the whole microcapsules, the sustainedrelease curves in Figure seem to be relatively gentle compared to those in Figure The entire controlled release behaviors fully confirm that our proposed composite core−shell microcapsules possess the programmed sequential drug release properties for both oleophilic drugs and hydrophilic drugs ■ ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b01277 CLSM images of Cur−PLGA-NPs, C−PLGA-NPs, and RhB−PLGA-NPs in soybean oil and stabilities of the microcapsules containing only free drug molecules or containing only drug-loaded nanoparticle or containing both free drug molecules and drug-loaded nanoparticles (PDF) The acid-triggered burst-release process of chitosan core−shell microcapsules containing both free curcumin and Cur−PLGA-NPs (Supplementary Movie S1) (AVI) The acid-triggered burst-release process of chitosan core−shell microcapsules containing both free RhB and RhB−PLGA-NPs (Supplementary Movie S2) (AVI) CONCLUSIONS A novel type of core−shell microcapsules with programmed sequential drug release for acute gastrosis therapy has been successfully developed in this work The proposed microcapsule is composed of a cross-linked chitosan hydrogel shell and an oily core containing both free drug molecules and drugloaded PLGA nanoparticles Oleophilic curcumin and hydrophilic catechin are used as anti-inflammatory model drugs in this work, which also have synergistic efficacy in clinic We have designed and prepared several kinds of representative microcapsules For example, the core−shell microcapsules encapsulate the same drugs (free oleophilic curcumin and curcuminloaded PLGA nanoparticles, or free hydrophilic catechin and catechin-loaded PLGA nanoparticles), and the core−shell microcapsules contain different drugs (free curcumin and catechin-loaded PLGA nanoparticles, or free catechin and curcumin-loaded nanoparticles) CLSM results confirm that various free drug molecules and drug-loaded PLGA nanoparticles are successfully encapsulated inside the inner cores of the microcapsules The microcapsules can keep their structural integrity without leakage of drugs in neutral aqueous medium before they reach the acidic stomach environment Controlledrelease results indicate that the proposed microcapsules with this unique core−shell structure can successfully achieve programmed sequential drug release, i.e., burst release in the stomach first and then sustained release in the gastrointestinal tract When the microcapsules are transferred to an acidic environment like the stomach, the encapsulated free drug molecules are rapidly released as the first burst-release stage due to the acid-triggered decomposition of chitosan shell About 56.2% of curcumin and 59.6% of catechin are respectively released from the microcapsules within 60 s at the first burst- ■ AUTHOR INFORMATION Corresponding Author *E-mail: juxiaojie@scu.edu.cn Author Contributions The manuscript was written through contributions of all authors All authors have given approval to the final version of the manuscript Notes The authors declare no competing financial interest ■ ACKNOWLEDGMENTS The authors gratefully acknowledge support from the National Natural Science Foundation of China (21322605, 21276002, 81321002), the Training Program of Sichuan Province Distinguished Youth Academic and Technology Leaders (2013JQ0035), and the State Key Laboratory of Polymer Materials Engineering (sklpme2014-3-02) ■ REFERENCES (1) Siegel, R.; Ma, J M.; Zou, Z H.; Jemal, A Cancer Statistics, 2014 Ca-Cancer J Clin 2014, 64, 9−29 (2) Sinha, M.; Gautam, L.; Shukla, P K.; Kaur, P.; Sharma, S.; Singh, T P Current Perspectives in NSAID-Induced Gastropathy Mediators Inflammation 2013, 2013, 258209 (3) den Hollander, W J.; Kuipers, E J Current Pharmacotherapy Options for Gastritis Expert Opin Pharmacother 2012, 13, 2625− 2636 10533 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 Research Article ACS Applied Materials & Interfaces (25) Wang, X X.; Ju, X J.; Sun, S X.; Xie, R.; Wang, W.; Liu, Z.; Chu, L Y Monodisperse Erythrocyte-Sized and Acid-soluble Chitosan Microspheres Prepared via Electrospraying RSC Adv 2015, 5, 34243− 34250 (26) Evans, D F.; Pye, G.; Bramley, R.; Clark, A G.; Dyson, T J.; Hardcastle, J D Measurement of Gastrointestinal pH Profiles in Normal Ambulant Human Subjects Gut 1988, 29, 1035−1041 (27) Danhier, F.; Ansorena, E.; Silva, J M.; Coco, R.; Le Breton, A.; Preat, V PLGA-Based Nanoparticles: An Overview of Biomedical Applications J Controlled Release 2012, 161, 505−522 (28) Panyam, J.; Labhasetwar, V Biodegradable Nanoparticles for Drug and Gene Delivery to Cells and Tissue Adv Drug Delivery Rev 2012, 64, 61−71 (29) Yallapu, M M.; Gupta, B K.; Jaggi, M.; Chauhan, S C Fabrication of Curcumin Encapsulated PLGA Nanoparticles for Improved Therapeutic Effects in Metastatic Cancer Cells J Colloid Interface Sci 2010, 351, 19−29 (30) Pillai, J J.; Thulasidasan, A K T.; Anto, R J.; Devika, N C.; Ashwanikumar, N.; Kumar, G S V Curcumin Entrapped Folic Acid Conjugated PLGA-PEG Nanoparticles Exhibit Enhanced Anticancer Activity by Site Specific Delivery RSC Adv 2015, 5, 25518−25524 (31) Pool, H.; Quintanar, D.; Figueroa, J D.; Marinho Mano, C.; Bechara, J E H.; Godinez, L A.; Mendoza, S Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles J Nanomater 2012, 2012, 145380 (32) De La Vega, J C.; Elischer, P.; Schneider, T.; Hafeli, U O Uniform Polymer Microspheres: Monodispersity Criteria, Methods of Formation and Applications Nanomedicine 2013, 8, 265−285 (33) Jain, R A The Manufacturing Techniques of Various Drug Loaded Biodegradable Poly(lactide-co-glycolide) (PLGA) Devices Biomaterials 2000, 21, 2475−2490 (34) Astete, C E.; Sabliov, C M Synthesis and Characterization of PLGA Nanoparticles J Biomater Sci., Polym Ed 2006, 17, 247−289 (35) Aditya, N P.; Aditya, S.; Yang, H.; Kim, H W.; Park, S O.; Ko, S Co-Delivery of Hydrophobic Curcumin and Hydrophilic Catechin by a Water-in-Oil-in-Water Double Emulsion Food Chem 2015, 173, 7−13 (36) Manikandan, R.; Beulaja, M.; Arulvasu, C.; Sellamuthu, S.; Dinesh, D.; Prabhu, D.; Babu, G.; Vaseeharan, B.; Prabhu, N M Synergistic Anticancer Activity of Curcumin and Catechin: An In Vitro Study Using Human Cancer Cell Lines Microsc Res Tech 2012, 75, 112−116 (4) Rakatansky, H Review article: Gastroenterology and the Pharmaceutical Industry Aliment Pharmacol Ther 2002, 16, 1859− 1866 (5) Bysell, H.; Mansson, R.; Hansson, P.; Malmsten, M Microgels and Microcapsules in Peptide and Protein Drug Delivery Adv Drug Delivery Rev 2011, 63, 1172−1185 (6) Lensen, D.; Vriezema, D M.; van Hest, J C M Polymeric Microcapsules for Synthetic Applications Macromol Biosci 2008, 8, 991−1005 (7) Sukhorukov, G.; Fery, A.; Mohwald, H Intelligent Micro- and Nanocapsules Prog Polym Sci 2005, 30, 885−897 (8) Kost, J.; Langer, R Responsive Polymeric Delivery Systems Adv Drug Delivery Rev 2012, 64, 327−341 (9) Wang, C Y.; Yang, C H.; Lin, Y S.; Chen, C H.; Huang, K S Anti- Inflammatory Effect with High Intensity Focused UltrasoundMediated Pulsatile Delivery of Diclofenac Biomaterials 2012, 33, 1547−1553 (10) Long, Y.; Liu, C Y.; Zhao, B.; Song, K.; Yang, G Q.; Tung, C H Bio-Inspired Controlled Release through Compression-Relaxation Cycles of Microcapsules NPG Asia Mater 2015, 7, e148 (11) Chu, L Y.; Yamaguchi, T.; Nakao, S A Molecular-Recognition Microcapsule for Environmental Stimuli-Responsive Controlled Release Adv Mater 2002, 14, 386−389 (12) Chu, L Y.; Liang, Y J.; Chen, W M.; Ju, X J.; Wang, H D Preparation of Glucose-Sensitive Microcapsules with a Porous Membrane and Functional Gates Colloids Surf., B 2004, 37, 9−14 (13) Yang, W C.; Xie, R.; Pang, X Q.; Ju, X J.; Chu, L Y Preparation and Characterization of Dual Stimuli-Responsive Microcapsules with a Superparamagnetic Porous Membrane and ThermoResponsive Gates J Membr Sci 2008, 321, 324−330 (14) Masoud, H.; Alexeev, A Controlled Release of Nanoparticles and Macromolecules from Responsive Microgel Capsules ACS Nano 2012, 6, 212−219 (15) Zheng, C L.; Ding, Y F.; Liu, X Q.; Wu, Y K.; Ge, L Highly Magneto-Responsive Multilayer Microcapsules for Controlled Release of Insulin Int J Pharm 2014, 475, 17−24 (16) Jeong, W C.; Kim, S H.; Yang, S M Photothermal Control of Membrane Permeability of Microcapsules for On-Demand Release ACS Appl Mater Interfaces 2014, 6, 826−832 (17) Stubbe, B G.; De Smedt, S C.; Demeester, J Programmed Polymeric Devices” for Pulsed Drug Delivery Pharm Res 2004, 21, 1732−1740 (18) De Cock, L J.; De Koker, S.; De Geest, B G.; Grooten, J.; Vervaet, C.; Remon, J P.; Sukhorukov, G B.; Antipina, M N Polymeric Multilayer Capsules in Drug Delivery Angew Chem., Int Ed 2010, 49, 6954−6973 (19) Li, D.; Zhang, Y T.; Jin, S.; Guo, J.; Gao, H F.; Wang, C C Development of a Redox/pH Dual Stimuli-Responsive MSP@ P(MAA-Cy) Drug Delivery System for Programmed Release of Anticancer Drugs in Tumour Cells J Mater Chem B 2014, 2, 5187− 5194 (20) Windbergs, M.; Zhao, Y.; Heyman, J.; Weitz, D A Biodegradable Core−Shell Carriers for Simultaneous Encapsulation of Synergistic Actives J Am Chem Soc 2013, 135, 7933−7937 (21) Koppolu, B.; Rahimi, M.; Nattama, S.; Wadajkar, A.; Nguyen, K T Development of Multiple-Layer Polymeric Particles for Targeted and Controlled Drug Delivery Nanomedicine 2010, 6, 355−361 (22) Wang, Y Z.; Zhang, Y Q.; Wang, B C.; Cao, Y.; Yu, Q S.; Yin, T Y Fabrication of Core-Shell Micro/Nanoparticles for Programmable Dual Drug Release by Emulsion Electrospraying J Nanopart Res 2013, 15, 1726 (23) Zhang, Y Q.; Wang, B C.; Wang, Y Z.; Qiao, W L.; Shao, P Y A Novel Hypurgia for Cancer Chemotherapy: Programmable Release of Antineoplastics and Cytothesis Agents from Core-Shell Micro-/ Nano-particles Med Hypotheses 2011, 76, 201−203 (24) Liu, L.; Yang, J P.; Ju, X J.; Xie, R.; Liu, Y M.; Wang, W.; Zhang, J J.; Niu, C H.; Chu, L Y Monodisperse Core-Shell Chitosan Microcapsules for pH-Responsive Burst Release of Hydrophobic Drugs Soft Matter 2011, 7, 4821−4827 10534 DOI: 10.1021/acsami.6b01277 ACS Appl Mater Interfaces 2016, 8, 10524−10534 ... templates, core−shell chitosan microcapsules with uniform size and structure are prepared via an interfacial cross-linking reaction Figure shows the CLSM images of core−shell chitosan microcapsules. .. and Microcapsules The purpose of this work is to develop a novel type of core−shell microcapsule for programmed sequential drug release The proposed microcapsule is composed of a crosslinked chitosan. .. amount of drug at different time intervals (1) (2) 2.4 Preparation of Core−Shell Chitosan Microcapsules The core−shell chitosan microcapsules containing both free drug molecules and drug-loaded PLGA