CoreâShell Chitosan Microcapsules for Programmed Sequential Drug

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01277 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 11, 2016

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ACS Applied Materials & Interfaces

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, Sichuan University, Chengdu 610065, P. R. China

‡

State Key Laboratory of Polymer Materials Engineering, and Collaborative Innovation

Center for Biomaterials Science and Technology, Sichuan University, Chengdu 610065, P. R. China

1 ACS Paragon Plus Environment


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ABSTRACT A novel type of core-shell chitosan microcapsules with programmed sequential drug release is developed by 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 exposed to acid stimulus, the resultant microcapsules can keep structural integrity without leakage of encapsulated substances.

Upon acid-triggering, the microcapsules can achieve

the first burst-release due to the acid-induced decomposition of chitosan shell.

The

encapsulated free drug molecules and drug-loaded PLGA nanoparticles are rapidly released within 60 seconds.

Next, the drugs loaded in the PLGA nanoparticles are slowly released

for several days to achieve the second 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 Microcapsules, Chitosan, PLGA, Nanoparticles, Programmed sequential drug release


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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 the treatment level and drug could quickly take effect after administration. dose is more appropriate in this case.

Thus, burst-release mode with large drug

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, 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 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.



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Microcapsules, which can encapsulate various active substances to protect them from the surrounding environment, are of great interest for many applications, especially in drug delivery field.5,6

Recently, microcapsules with various structures and functions are

developed to achieve programmed sequential release, and are considered to be very applicable as drug delivery carriers. categories.

These functional microcapsules are mainly divided into two

One is the stimuli-responsive microcapsule with programmed pulsed-release

ability based on repeated “on-off” mechanism.7-16

However, triggered by such

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 one 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 firstly 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, there exists

drug leakage problem before these core-shell microcapsule carriers reach the targeted sites. To the best of our knowledge, such kind of drug-loaded microcapsules that can achieve burst release first and then sustained release has not been reported yet.

Previous studies bring us

inspirations that we can design a kind of microcapsules with special core-shell structure and stimuli-responsive property to achieve the programmed sequential drug release that we expect.


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Here, we report on a novel type of core-shell microcapsules with programmed sequential drug release, i.e., burst release in stomach first and then sustained release in 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 oil-water interface between inner oily core and hydrous chitosan shell, there will be no-leakage of 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 great acid-induced 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 chitosan shell under the unique acidic condition of stomach (Scheme 1A2-A3).

Simultaneously, the

co-encapsulated drug-loaded PLGA nanoparticles are also released out, which could provide 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 symptom 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 the complications.

That is, this kind of novel

core-shell microcapsules, which can achieve programmed sequential drug release, is of great potential to realize more rational drug administration for the treatment of acute stomach



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illness.

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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.

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.

(B) Second sustained-release of drugs from the PLGA

nanoparticles can be achieved via drug diffusion and PLGA degradation.

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 = 20000) is purchased from Sichuan Dikang Sci & Tech Pharmaceutical Co., Ltd.

Soybean oil (Kerry Oils & Grains) is used as the oil phase. 6 ACS Paragon Plus Environment

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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. other chemicals are of analytical grade and used as received.

All

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 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.

30 ml of PVA aqueous solution (1.0%, w/v) is used as the water phase.

The oil phase is dropwise added into the water phase under agitation (300 rpm) for 10 min, followed by homogeneous emulsification (19000 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



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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 for preparing 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 be observed,

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 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 disc technique.

The morphologies of the drug-loaded PLGA nanoparticles in dried state are


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observed by scanning electron microscopy (SEM, JSM-7500F, JEOL), and their morphologies in water and oil solutions are observed by confocal laser scanning microscope (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 efficiency of of PLGA nanoparticles are measured by UV-visible spectrophotometer (UV-Vis, UV-1700, Shimadzu).

A given

amount of freeze-dried nanoparticles (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. methanol solution is centrifuged at 12000 rpm and the supernatant is collected.

The After

dilution, the drug concentration in the supernatant is determined by UV-vis at 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: =

× 100%

(1)

= × 100%

2.4. Preparation of Core-shell Chitosan Microcapsules.

(2)

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 capillary microfluidic technique according to our published method.24 Microfluidic technique is an excellent method to prepare multiple emulsions with precisely controlled size.

To better achieve our proposed design purpose, firstly the formed O/W/O



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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 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 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 keep the inner cores still inside the microcapsules.


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2.5. Characterization of O/W/O Emulsions and Microcapsules.

The morphologies of

O/W/O emulsions are characterized by optical microscope (BX 61, Olympus).

The size and

size distribution are calculated based on the obtained optical micrographs using an 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 no-leakage of drug molecules from the microcapsules before they reach stomach, the stability of composite core-shell microcapsules in neutral environment is investigated by recording the variation of relative fluorescence intensity of 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).

Firstly, 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 surrounding solution.

That is,

the following sustained-release 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 firstly.

These release experiments are



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carried out in phosphate buffer 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 12000 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 method to calculate the release 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 method 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 12000 rpm for 10 min, and then dispersed into PBS solution (pH 7.4) for further investigating the second sustained-release by conducting experiments similar to the above-mentioned simple sustained-release experiment for drug-loaded PLGA nanoparticles.

The use of ethanol for the first burst-release is to ensure


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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 that 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 2 h to ensure the maximum dissolution of drug molecules in the ethanol solution.

After that, the ethanol solution is centrifuged (12000

rpm) for 10 min and the supernatant solution is collected. supernatant solution is measured by UV-vis method.

The drug concentration in the

To completely extract drugs from the

nanoparticles, the precipitant is re-dispersed 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



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drug-loaded PLGA nanoparticles are shown in Figure 1.

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Specifically, the characteristic

bands of curcumin molecule (Curve B), including a wide peak around 3400 cm-1 for O-H stretching vibration of phenol group, three peaks at 1625~1500 cm-1 for C=C skeletal stretching vibration in benzene ring, and two peaks at 860~800 cm-1 for C-H bending vibration of benzene ring, are all found in 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 O-H stretching vibration of phenol group, three peaks at 1625~1500 cm-1 for C=C skeletal stretching vibration in benzene ring, and two peak at 860~800 cm-1 for C-H bending vibration of benzene ring, are all found in FT-IR spectrum of C-PLGA-NPs (Curve E).

For

RhB-PLGA-NPs, the characteristic bands of RhB (Curve F), including the characteristic peak at 1700 cm-1 for C=O stretching vibration of carboxyl group and two peaks at 1650~1550 cm-1 for C=C skeletal stretching vibration in benzene ring, can also be found in FT-IR spectrum of RhB-PLGA-NPs (Curve G).

Furthermore, the characteristic peak at 1750 cm-1 for C=O

stretching vibration of ester bond in PLGA nanoparticles (Curve A) can be found in FT-IR 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.


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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).

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 do not exhibit fluorescence under CLSM observation



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(Figure 3A2).

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For better observation, RhB-PLGA-NPs are used as the substituted samples

of PLGA nanoparticles containing hydrophilic drugs, which display obvious red fluorescence from RhB dye (Figure 3A3).

It can be seen that these three kinds of nanoparticles are

well-dispersed in water without bulk aggregation, which benefit 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 nm, 478.4 nm 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 good monodispersity of these nanoparticles. Uniform size of 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, Cur-PLGA-NPs,

C-PLGA-NPs and RhB-PLGA-NPs also exhibit good dispersibility in soybean oil without bulk aggregation, which benefit the generation of O/W/O emulsion templates in microfluidic devices without clogging the microchannel. 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. Because hydrophilic drug molecules can easily diffuse from organic phase into 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


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2.94% and 60.23%.

Certainly, the loading capacity of hydrophilic drug in PLGA

nanoparticles can be improved by various methods through changing the preparation parameters.34

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.

Morphologies of Emulsion Templates and Microcapsules.

The purpose of this work is 17

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to develop a novel type of core-shell microcapsules 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

microcapsules with such core-shell structures provide more flexiblity 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 the microcapsules containing free

drug molecules and PLGA nanoparticles with the same drug molecules.

Such kind of

microcapsules are 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 microcapsules is used to prove that same drugs can be released

in the 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 microcapsules 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.

Figures 4A-4H show the optical micrographs of different kinds of O/W/O

emulsions prepared by microfluidic method. core-shell structures.

These emulsions all show clear and stable

Similarly, since catechin exhibits no color and no fluorescence, RhB is

used instead of catechin as the hydrophilic model drug for optical characterization.


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Curcumin has a bright-yellow color and RhB is a red dye. emulsions have different colors in the inner cores.

As a result, different O/W/O

Figures 4A-4D are the control groups

with inner cores contain only free drugs or only drug-loaded PLGA nanoparticles.

The

O/W/O emulsions containing only free oleophilic curcumin (Figure 4A) or only Cur-PLGA-NPs (Figure 4B) show obvious yellow color in inner cores.

Specially, due to

larger amount of curcumin, the emulsions containing only free curcumin show brighter yellow color in inner cores than those containing only Cur-PLGA-NPs.

In addition, there are lots of

black dots in 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 inner cores.

Figures 4E-4H show 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 inner cores, is nearly an overlay of Figures 4A and 4B. Similarly, the image of O/W/O emulsions containing both free RhB and RhB-PLGA-NPs (Figure 4G) seems to be an overlay of Figures 4C and 4D.

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 mixed-color of that in Figures 4A and 4D.

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 inner cores of O/W/O emulsions individually or together.

In



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addition, encapsulated free drug molecules and drug-loaded PLGA nanoparticles do not affect the structural integrity and stability of the emulsions. Figures 4E1-4H1 are 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 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.


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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.

Using the monodisperse O/W/O emulsions as templates, core-shell chitosan microcapsules with uniform size and structure are prepared via interfacial cross-linking reaction.

Figure 5



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shows the CLSM images of core-shell chitosan microcapsules loading with different substrates in 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 higher loading amount of free drug molecules, the microcapsules containing only free curcumin or only free RhB have brighter fluorescence in inner cores compared with the microcapsules containing only Cur-PLGA-NPs or only RhB-PLGA-NPs.

Compared with those in Figures 5A and 5C, 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 Figures 5B and 5D.

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


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(Figure 5F), which just show green fluorescence.

The reason is that the loading amount of

RhB in the PLGA nanoparticles is much smaller than that of free curcumin in 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 microcapsules. (A) Microcapsules 23 ACS Paragon Plus Environment


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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.

(A, C, E) are on green fluorescent channel and (B, D, F-H) are

the overlap of images on green and red fluorescent channels.

Stability of Drug-Loaded Microcapsules.

Scale bars are all 500 µm.

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 leakage.

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 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 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 loading with oleophilic curcumin (Figure 6A),

the relative fluorescence intensity remains nearly unchanged at ~1, indicating nearly


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no-leakage of curcumin from the microcapsules.

For hydrophilic RhB-loaded microcapsules

(Figure 6B), the fluorescence intensity of inner cores slightly decreases after 3 h.

After

dispersed in aqueous solution for 3 h, the chitosan shells of microcapsules swell completely, so that the pores of 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), 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 inner cores and hydrous chitosan shells.

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



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and RhB-PLGA-NPs.

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 stomach and second sustained-release in gastrointestinal tract.

The

controlled-release behaviors of two kinds of representative core-shell microcapsules containing both free drug molecules and drug-loaded nanoparticles are investigated. Firstly, 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 7 shows the CLSM microscope snapshots of

acid-triggered burst-release processes of these two kinds of 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 at first, and then a rapid and complete decomposition is achieved within 60 seconds.

Such decomposition of chitosan

shells in acidic solution is a result of acid-induced hydrolysis of Schiff base bonds between chitosan and terephthalaldehyde.24,25 With the break-up of chitosan shells, both free drug


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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 achieves the first 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 next drug sustained-release from the PLGA nanoparticles. The movies of the acid-triggered burst-release processes are also shown in the Supporting Information (Movie S1 and Movie S2).



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Figure 7.

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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 channel).

HCl solution with pH 1.5 is added at t = 0 s.

The scale bars are all 500 µm.

At the first acid-triggered burst-release stage, the co-encapsulated drug-loaded PLGA nanoparticles are also released from the microcapsules, which could provide 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 a simulating physiological condition (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 typical sustained-release “first-order kinetic model”.

A sustained and prolonged release of oleophilic curcumin in the

PBS up to 28 days is observed in Figure 8A.

In the initial period of 6 h, approximate 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


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upon the solubility and diffusion of drug, as well as the biodegradation of the matrix materials. Catechin is a hydrophilic molecule with larger solubility in aqueous environment, so that 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.

Figure 8.

Cumulative releases of curcumin (A) and catechin (B) from PLGA nanoparticles

in PBS (pH 7.4, 37 °C).

To display the entire programmed sequential drug release process, a continuous release experiment combining first burst-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 initial 10 min equilibrium time, there is nearly no-leakage of drug from the



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microcapsules.

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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 seconds, which directly shows the first burst-release performance.

The

following sustained-release experiments are carried out for two 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 two days. The curve shapes and change tendencies of sustained-release parts in Figure 9 are similar to that in Figure 8, which also present 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 in Figure 9 seem to be relatively gentle than that in Figure 8.

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.


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Figure 9.

Programmed sequential release behaviors of curcumin-loaded (A) and

catechin-loaded (B) composite core-shell microcapsules.

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

microcapsule 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 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 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 nanoparticles are successfully encapsulated inside the inner cores of the microcapsules.

The microcapsules can keep the

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 stomach first and then sustained release in gastrointestinal tract.

When the microcapsules are transferred to an acidic environment like



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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 seconds at the first burst-release stage.

Simultaneously, the co-encapsulated drug-loaded PLGA

nanoparticles are also released to provide the second sustained-release.

Respectively, about

19.3% of curcumin and 32.3% of catechin are slowly released from PLGA nanoparticles within two 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 versatile for loading different drugs, such as oleophilic drugs, hydrophilic drugs and multiple drugs with synergistic efficacy.

Moreover, the results in this

study also provide a versatile strategy for designing and developing novel functional microcapsules with various programmed sequential release properties for biomedical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Movies of the acid-triggered burst-release processes of microcapsules, CLSM images of Cur-PLGA-NPs, C-PLGA-NPs and RhB-PLGA-NPs in soybean oil, stabilities of the


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microcapsules containing only free drug molecules or containing only drug-loaded nanoparticle or containing both free drug molecules and drug-loaded nanoparticles (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] 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.

ACKNOWLEDGMENT 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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