Core−Shell chitosan microcapsules for programmed sequential

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 f

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

micro-capsule 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)

nanopar-ticles 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

acid-induced 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

1 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 thefirst 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 releasefirst and

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 deliveryfield.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.17−23The drugs loaded in outer shell are

Received: January 31, 2016

Accepted: April 7, 2016

Published: April 7, 2016

www.acsami.org

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

gastro-intestinal 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, wefind that chitosan hydrogels prepared

using terephthalaldehyde as the cross-linker exhibit a great

acid-induced dissolution property.24,25There 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

drug-loaded 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

2 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 Terephthalalde-hyde ( ≥98%, Sinopharm Chemical Reagent) is used as cross-linker All other chemicals are of analytical grade and used as received Deionized water (18.2 M Ω, 25 °C) from a Millipore Milli-Q Plus water puri fication 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

Scheme 1 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

added into the water phase under agitation (300 rpm) for 10 min,

followed by homogeneous emulsification (19 000 rpm) for 2 min

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

nano-particles 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 Brie fly, 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 puri fication, 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

drug-loaded PLGA nanoparticles are freeze-dried and then stored in a dry

cabinet at 4 °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

drug-loaded PLGA nanoparticles are measured by dynamic light scattering

(DLS) (Zetasizer Nano ZS90-ZEN3690, Malvern).

The drug-loading capacity and encapsulation e fficiency of PLGA

nanoparticles are measured by UV−visible spectrophotometry (UV−

vis) (UV-1700, Shimadzu) A given amount of freeze-dried

nano-particles (2 mg for Cur−PLGA-NPs or 5 mg for C−PLGA-NPs/

RhB−PLGA-NPs) is dissolved in 2 mL of methanol, and then the

solution is treated with ultrasonic oscillation for 4 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 (LC NP ) and encapsulation

efficiency (EE NP ) of PLGA nanoparticles are calculated as follows:

LC mass of drug in nanoparticles

total mass of nanoparticles 100%

NP

(1)

=

×

EE mass of drug in nanoparticles

total mass of drug used for nanoparticle preparation

100%

NP

(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

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 Q I = 400 μL/h, Q M = 800 μL/h, and Q O = 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 di fferent 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 Micro-capsules 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 Micro-capsules 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 drug-loaded PLGA nanoparticles in inner cores are both released and dispersed in the surrounding solution That is, the following sustained-release behavior is similar to the simple drug sustained-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-bu ffered saline (PBS, pH 7.4) at 37 °C using a water-bathing 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 sustained-release 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 3 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.

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 4 mL of ethanol for ∼10 min 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 min 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

nano-particles 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 (LC MC )

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 2 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 min 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

drug-loading amount in the microcapsules.

3 RESULTS AND DISCUSSION

3.1 Composition and Morphology of Nanoparticles

FT-IR spectra of different drug-loaded PLGA nanoparticles are

shown in Figure 1 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−1for the CC skeletal

stretching vibration in the benzene ring, and two peaks at 860−

800 cm−1for 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),

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

RhB−PLGA-NPs, 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−1for the CC skeletal stretching vibration of the benzene

ring, can also be found in the FT-IR spectrum of RhB

−PLGA-NPs (curve G) Furthermore, the characteristic peak at 1750

cm−1for the CO stretching vibration of the ester bond in

PLGA nanoparticles (curve A) can be found in the FT-IR

E), and RhB−PLGA-NPs (curve G) All the results confirm that curcumin, catechin, and RhB are successfully encapsulated

in PLGA nanoparticles

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 inFigure

3A1, PLGA nanoparticles containing oleophilic curcumin exhibit obvious greenfluorescence due to the autofluorescence

of curcumin Because catechin has nearly nofluorescence, C−

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

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 inFigure

C−PLGA-NPs, 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

Figure 1 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).

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.33Similarly, the drug-loading capacity and

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

Micro-capsules 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

cross-linked chitosan shell and an oily core containing both free drug

molecules and drug-loaded PLGA nanoparticles The

micro-capsules 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

drug molecules and PLGA nanoparticles with the same drug molecules Such kind of microcapsule is demonstrated by preparing microcapsules containing free oleophilic curcumin

hydrophilic catechin and C−PLGA-NPs This kind of micro-capsule 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 −PLGA-NPs 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 all show clear and stable core−shell structures Similarly, since catechin exhibits no color and nofluorescence, 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

Figure 2 SEM images of Cur −PLGA-NPs (A), C−PLGA-NPs (B), and RhB−PLGA-NPs (C).

Figure 3 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.

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

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 drug-loaded 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 4 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 ofFigures 4 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

Figure 4 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−PLGA-NPs, (E, E1) emulsions containing both free curcumin and Cur −PLGA-NPs, (F, F1) emulsions containing both free curcumin and RhB−PLGA-NPs, (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.

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

nano-particles 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 do 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

respectively, indicating the high monodispersity of these

emulsion templates The O/W/O emulsions shown inFigure

4F also show good monodispersity, and the CV values for ID

and OD are 2.01% and 1.99%, respectively Similarly, CV values

respectively, and CV values for ID and OD shown inFigure 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 5 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 naturallyfluorescent 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 greenfluorescence 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

fluorescence intensity of the inner cores in microcapsules

containing both free curcumin and Cur−PLGA-NPs (Figure

5E) obviously increases The composite core−shell

micro-capsules 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

fluorescence color does not appear for the microcapsules

containing both free curcumin and RhB−PLGA-NPs (Figure

5F), which just show greenfluorescence The reason is that the

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

Figure 5 CLSM images of different core−shell chitosan micro-capsules: (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.

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

micro-capsules 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 6 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

RhB-loaded microcapsules (Figure 6B), thefluorescence intensity of

inner cores slightly decreases after 3 h After being dispersed in

aqueous solution for 3 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 2 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 micro-capsules 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 7shows the CLSM microscope snapshots of acid-triggered burst-release processes of these two kinds of

Figure 6 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.

Figure 7 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 = 0 s The scale bars are all 500 μm.

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

RhB-loaded 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 atfirst 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 successfullyfirst 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

acid-triggered burst-release processes are also shown in the

Supporting Information (Movie S1andMovie S2)

At thefirst acid-triggered burst-release stage, the

coencapsu-lated 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 inFigure 8A In the initial period of 6 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 6 h, and 60.86% of drug is slowly released within 6 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 Figure 8 Cumulative releases of curcumin (A) and catechin (B) from PLGA nanoparticles in PBS (pH 7.4, 37 °C).

Figure 9 Programmed sequential release behaviors of curcumin-loaded (A) and catechin-loaded (B) composite core−shell microcapsules.

release and, second, sustained release is also studied.Figure 9

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 min 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 7

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 thefirst burst-release

performance The following sustained-release experiments are

carried out for 2 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 2 days The curve shapes and

tendencies for change of sustained-release parts inFigure 9are

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

sustained-release curves inFigure 9seem to be relatively gentle compared

to those in Figure 8 The entire controlled release behaviors

fully confirm that our proposed composite core−shell

micro-capsules possess the programmed sequential drug release

properties for both oleophilic drugs and hydrophilic drugs

4 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

micro-capsule is composed of a cross-linked chitosan hydrogel shell

and an oily core containing both free drug molecules and

drug-loaded PLGA nanoparticles Oleophilic curcumin and

hydro-philic 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

micro-capsules For example, the core−shell microcapsules

encapsu-late the same drugs (free oleophilic curcumin and

curcumin-loaded 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

nano-particles 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

Controlled-release 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

stomachfirst 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 thefirst 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 thefirst

burst-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 2 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 bio-medical applications

■ ASSOCIATED CONTENT

*S Supporting Information

The Supporting Information is available free of charge on the

ACS Publications websiteat DOI:10.1021/acsami.6b01277

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)

■ 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 thefinal version of the manuscript

Notes

The authors declare no competingfinancial 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)

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