Arch Pharm Res Vol 35, No 5, 839-850, 2012

DOI 10.1007/s12272-012-0509-9

839

Preparation and Characterization of a Novel pH-Sensitive Coated Microsphere for Duodenum-Specific Drug Delivery

Dan Zhou1, Xi Zhu1, Yang Wang2, Yun Jin1, Xuefan Xu1, Tingting Fan1, Yan Liu1, Zhirong Zhang1, andYuan Huang1

1Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy,Sichuan University, Chengdu 610041, China and 2Teaching & Research Unit of Hygienic Toxicology, School of PublicHealth, Chongqing Medical University, Chongqing 400016, China

(Received August 6, 2010/Revised October 22, 2010/Accepted January 31, 2011)

The aim of this study is to develop a duodenum-specific drug delivery system on the basis ofa pH-sensitive coating and a mucoadhesive inner core for eradication of Helicobacter pylori (H.pylori) in the ulcer duodenum. Hydroxypropyl methylcellulose acetate maleate (HPMCAM)was used as the pH-sensitive material, which dissolves around pH 3.0. The mucoadhesivemicrospheres loaded with furazolidone (FZD-ad-MS) were prepared by the emulsification-sol-vent evaporation method using Carbopol 971NP as the mucoadhesive polymer. The preparedpH-sensitive coated mucoadhesive microspheres (AM-coated-MS) were characterized inregards to particle size, drug loading efficiency, morphological change, drug stability, drugrelease and in vitro anti-H. pylori activity. The particle size was 160.97 ± 47.24 µm and 336.44± 129.34 µm, and the drug content was 42.33 ± 3.43% and 10.96 ± 1.29% for FZD-ad-MS andAM-coated-MS, respectively. The morphological changes in different pH media were charac-terized by scanning electron microscopy (SEM). HPMCAM coating improved the stability ofthe FZD-ad-MS and these particles were expected to remain intact until their arrival in theduodenum. The drug release was extremely suppressed at pH 1.2 for AM-coated-MS, butincreased at pH 4.0 after regeneration of FZD-ad-MS. In addition, FZD-ad-MS exhibited excel-lent anti-H. pylori activity in vitro. Thus, the HPMCAM-coated microspheres developed in thisstudy hold great promise for use as a duodenum-specific drug delivery system for H. pyloriclearance.

Key words: HPMCAM-coated mucoadhesive microspheres, Furazolidone-loaded mucoadhe-sive microspheres, Duodenum-specific, pH-Sensitive, Morphological changes, Drug release

INTRODUCTION

There are undoubtedly multiple pathogenic mecha-

nisms involved in the development of duodenal ulcer

(DU) disease (Olbe et al., 2000). Helicobacter pylori

(H. pylori) infection, however, is the most common

denominator. In fact, clinical treatments have demon-

strated that successful H. pylori eradication heals

ulcers and virtually prevents ulcer relapse (Majumdar

et al., 2007). Eradication of H. pylori using antibiotics,

such as amoxicillin, clarithromycin, metronidazole or

tetracycline (O’Connor et al., 2009), has been widely

shown to play a critical role in preventing gastroduo-

denal diseases. The duodenum, which is the initial

part of the intestines, is about 25 cm long in humans.

Drugs taken by oral administration are always de-

graded by digestive enzymes in the stomach and also

go quickly through the duodenum; therefore, these

drugs cannot reach their effective concentrations at

the pathological site for complete H. pylori clearance.

Thus, since therapeutic regimens for DU are not duo-

denum-specific, there is a need to develop a duodenum-

specific drug delivery system that can deliver the

drugs directly to the duodenum for their release to

effective concentrations. Considering that the pH of

the duodenal juice is lower (about 2.9-4.0) in DU

Correspondence to: Yuan Huang, Key Laboratory of Drug Tar-

geting and Drug Delivery System of Ministry of Education,

West China School of Pharmacy, Sichuan University, Chengdu

610041, China

Tel: 86-28-8550-1617, Fax: 86-28-8550-1617

E-mail: huangyuan0@yahoo.com.cn

840 D. Zhou et al.

patients than the normal subjects (about 4-5), our group

previously (Huang et al., 2005) developed a novel

duodenum-specific tablet, which was coated with the

pH-sensitive polymer hydroxypropyl methylcellulose

acetate maleate (HPMCAM). HPMCAM was obtained

from HPMC (hydroxypropyl methylcellulose) through

chemical modification using maleic anhydrides, and

found to show not only good film forming properties,

but also it was pH-sensitive from 3.0 to 3.7, which was

suitable for use as a duodenum-specific coating. After

‘the press-coated tablets’ were prepared, in vitro results

demonstrated that HPMCAM could completely suppress

drug release within 2 h in a simulated gastric fluid

(pH 1.2) and rapidly release of the drug was observed

in a simulated pathological duodenal fluid (pH 3.4).

These results indicated that HPMCAM might be a

useful material for the development of a duodenum-

specific drug delivery system.

However, when the duodenum-specific drug is admin-

istrated in tablet form, the drug may or may not accu-

mulate in the duodenum, due to its large size, which

results in a relative small effective surface area for

contact with the duodenal epithelia (Muramatsu and

Kondo, 1995). In addition, the large mass of the tablet

form and the vigorous movement of the gastrointest-

inal (GI) tract may result in a large variation in the

intestinal transit (Takishima et al., 2002; Chun et al.,

2005). Thus, a novel drug delivery system that can

increase the contact area, prolong the residence time

in the duodenum, and be less affected by the GI transi-

tion is needed to improve the duodenum-targeting

efficiency.

At present, numerous mucoadhesive drug delivery

systems have been developed for targeting antibiotics

to the gastric mucosa for an extended period of time to

improve their anti-H. pylori effect (Rajinikanth et al.,

2008). In this regard, mucoadhesive microspheres have

gained considerable attention. In general, mucoadhesive

microspheres offer obvious advantages over a single-

unit dosage form such as tablets and capsules. The

smaller size of the microspheres could increase the

effective contact area with the GI tract, while a smaller

mass and the ability to adhere to the mucus layer

could result in a controlled GI transit rate (Takishima

et al., 2002). Moreover, it has been shown that the

extended release of the drug from the mucoadhesive

microspheres can produce a higher antibiotic concen-

tration in the gastric region where H. pylori exists,

which ultimately improves therapeutic efficacy (Hejazi

and Amiji, 2002; Liu et al., 2005; Ishak et al., 2007;

Rajinikanth et al., 2008). Similarly, mucoadhesive

microspheres could also be used to improve the accu-

mulation of antibiotics in the duodenum for H. pylori

clearance.

Thus, the aim of this study was to develop a novel

duodenum-specific drug delivery system that com-

bines the advantages of mucoadhesion and pH-sensi-

tive controlled drug delivery. Due to the special envi-

ronmental pH in the ulcer duodenum, the pH-sensi-

tive polymer, HPMCAM, which dissolves around pH

3.0, was synthesized and used as a coating agent. Mu-

coadhesive microspheres were prepared using Carbopol

(Cb) as the mucoadhesive material and ethylcellulose

(Ec) as the matrix material, which has been widely

used in mucoadhesive delivery systems. Furazolidone

(FZD) was chosen as the model drug, and so far as we

know, no micro-/nano-particles of furazolidone have

been previously reported. We hypothesized that fura-

zolidone could be directed to the duodenum by using

HPMCAM-coated mucoadhesive microspheres to pre-

vent drug release in the stomach, which would allow

the released drug to accumulate at the surface of the

pathological ulcer of duodenum, thereby achieving

duodenum-specific therapy for eradication of H. pylori.

MATERIALS AND METHODS

MaterialsFurazolidone (FZD) (99.3% of purity) was purchased

from Fangxing Technology Development Co., Ltd.

Carbopol 971P NF was received as a gift from Lubrizol

Corporation. Ec and aluminum stearate were obtained

from Kelong Chemical Industry Corporation. HPMC

was kindly supplied as a gift from Colorcon Corpor-

ation. Pepsin and pancreatin were bought from Bio

Life Science & Technology Co., Ltd. and Huishi Bio-

chemistry Co., Ltd., respectively. Maleic anhydrides

and sodium acetate anhydrous were purchased from

Zhiyuan Chemical Reagent Co., Ltd. All other chemi-

cals were of reagent grade.

Preparation of mucoadhesive microspheres and

non-adhesive microspheresThe FZD-loaded mucoadhesive microspheres (FZD-

ad-MS) were prepared using an emulsion solvent

evaporation method similar to that described by Tao

et al. (2009). 1 g of Carbopol 971P NF and Ec with Cb-

Ec ratio (2/1, w/w) were dissolved in 25 mL of 90%

ethanol solution. 1 g of FZD was suspended in the

polymer solution. The polymer-FZD mixture was then

poured into 200 mL of light liquid paraffin containing

1% (w/v) Span80 and stirred until complete evapora-

tion of the solvents. The resultant microspheres were

collected by filtration using a sintered filter funnel

(#G5, pore size = 1.5-2.5 µm), then washed with n-

hexane, dried in a vacuum at 35oC. After drying, the

Development of Novel Microsphere for Duodenum Delivery 841

microspheres were sieved with a 50-mesh.

FZD-loaded non-adhesive microspheres with only Ec

as a matrix (FZD-non-MS) were prepared using a

similar method.

Synthesis of pH-sensitive HPMCAMThe method used to synthesize the pH-sensitive

HPMCAM was adapted from Huang et al. (2005). 5 g

of HPMC was dissolved in 31 g of acetic acid in a three-

necked flask at 85-90oC, followed by the addition of 2

g of maleic anhydrides, 3 g of acetic anhydrides and 2

g of sodium acetate anhydrous, which was used as a

catalyst. The reaction mixture was incubated for 5 h,

and then terminated by adding 10 g of purified water

and 3.5 g of concentrated hydrochloric acid. Finally,

the polymer was separated by pouring the mixture

into an excess amount of purified water. The polymer

was then washed and dried under vacuum at 50oC.

Preparation of HPMCAM-coated mucoadhesive

microspheres (AM-coated-MS)FZD-loaded mucoadhesive microspheres were coated

with HPMCAM using a previously reported oil-in-oil

solvent evaporation method (Maestrelli et al., 2008)

with slight modifications. The coating solution was

prepared by dispersing 200 mg of HPMCAM and 20

mg of aluminum stearate into 5 mL acetone. The

microspheres were immersed in the coating solution

and then emulsified into 75 mL light liquid paraffin

that had been pre-saturated with 5 mL acetone under

agitation. After the evaporation of acetone, the

HPMCAM-coated microspheres were collected in the

same way as described for the FZD-ad-MS in section

“Preparation of mucoadhesive microspheres and non-

adhesive microspheres”, except the samples were

sieved with a 20-mesh.

Particle characterizationThe shape and surface morphology of FZD-ad-MS

and AM-coated-MS were investigated using scanning

electron microscopy (SEM). The samples were coated

with gold to a thickness of about 200 and observed using

a JEOL JSM-5900LV scanning electron microscope.

The size distributions of FZD-ad-MS and AM-coated-

MS were examined by measuring the Green diameters

of 200 particles chosen at random using an Axiovert

40 inverted optical microscope (Carl Zeiss Shanghai

Co., Ltd.) equipped with a Pixera Penguin 150CL-

COOLED CCD digital camera systems (Pixera).

Determination of drug contentsAppropriate amounts of microspheres were dispersed

in 2 mL of 80% acetonitrile solution. The solution was

sequentially subjected to vortex, ultrasonication, and

centrifugation. This procedure was repeated three

times. All clear supernatants were then collected and

diluted to a certain volume. After filtration through a

0.45 µm membrane filter (Xinya), the filtrate was

analyzed at 368 nm using UV-vis spectroscopy (Varian

Cary 100 UV-VIS spectrophotomete). Cb, Ec and

HPMCAM did not interfere under this condition. Each

determination was made in triplicate.

Differential Scanning Calorimetry (DSC) and

X-ray Powder Diffraction (XRPD) In order to obtain qualitative information about the

physical state of FZD and any possible drug-polymer

interactions in the microspheres formulations (Türk et

al., 2009), FZD, FZD-ad-MS, AM-coated-MS and blank

mucoadhesive microspheres were analyzed by DSC

and XRPD. Samples were each placed in a close alu-

minum pan and then analyzed on a differential scann-

ing calorimeter (EXSTAR6000 DSC) as the tempera-

ture was increased from 25 to 280oC at a heating rate

of 10oC/min under a nitrogen purge at 50 mL/min.

XRPD patterns were obtained using a PHILIPS X’Pert

Pro MPD DY 1291 diffractometer. Samples were an-

alyzed in the range of 5-55o (2θ) at room temperature.

In vitro mucoadhesion test on porcine mucosaFZD-ad-MS was tested for mucoadhesion by modi-

fying the method designed by Ranga Rao and Buri

(1989). In this experiment, mucosa was isolated from

pig rather than mice, and gastric and duodenal mucosa

were employed. FZD-non-MS was used as the control

group. Different media were used to mimic the gastro-

intestinal environment. Briefly, the mucosa was cut

into pieces (2 cm × 1 cm) and rinsed with 2 mL of phy-

siological saline. 100 FZD-ad-MS or FZD-non-MS par-

ticles were scattered uniformly on the surface of the

mucosa, which was fixed on a glass slide. The mucosa

with the microspheres was then placed in a chamber

maintained at a relative humidity of 93% and room

temperature. After 30 min, the glass slide was taken

out and adjusted to the inclined position of 45o. The

mucosa was rinsed with the simulated gastric fluids

(SGF, pH 1.2, without enzyme) or the simulated

pathological duodenal fluids (SPDF, pH 4.0, without

enzyme) for 5 min at a rate of 22 mL/min (BT00-100

M, Baoding Longer Precision Pump Co., Ltd). The

number of remaining microspheres was counted and

the percentage of mucoadhesion was calculated. The

experiment was performed in triplicate.

Stability of furazolidone in SGF and SPDFThe stability of FZD was examined in SGF at pH 1.2

842 D. Zhou et al.

and SPDF pH 4.0. First, FZD was dissolved in buffer

solutions to prepare a stock solution with a concen-

tration of 100 µg/mL. The stock solutions were then

diluted 10-fold to produce a final concentration of 10

µg/mL. The final solutions (in triplicate) were incubat-

ed at 100 rpm and 37 ± 0.5oC using a thermostatic

shaker. At appropriate time intervals, 3 mL of media

were removed and filtered through 0.45 µm filters.

The concentration of parent drug remaining in the

media was then determined by RP-HPLC. Pepsin and

pancreatin were also added into SGF and SPDF, re-

spectively, to investigate their influences on drug

stability.

The HPLC system consisted of a G1310A pump, a

G1314B UV spectrophotometer detector and an Agilent

Chemstation for LC system (Rev B 04.01.sp1, Agilent

Technologies Inc.). An analytical Diamonsil® C18 (5

µm, 250 mm × 4.6 mm) reverse-phase column (Dikma

Technologies) was used. The mobile phase consisted of

a mixture of acetonitrile-0.033 M KH2PO4 (40:60, v/v)

at a flow rate of 1 mL/min. The detector was set at 368

nm. All analyses were performed at room tempera-

ture.

In vitro drug release in simulated gastrointes-

tinal fluids The drug release study was performed in SGF (pH

1.2) and SPDF (pH 4.0) at 37 ± 0.5oC. Appropriate

amounts of FZD-ad-MS or AM-coated-MS were dis-

persed in 75 mL of SGF or SPDF and incubated under

the same conditions described for the stability studies

(referred to section above). At predetermined intervals,

aliquots (3 mL) were removed and replaced with an

equal volume of fresh media to maintain the initial

volume of the dissolution fluid. The withdrawn sam-

ples were filtered through 0.45 µm filters and analyzed

immediately at 368 nm using UV-vis spectroscopy.

A release study was also carried out in media with

digestive enzymes. Pepsin and pancreatin were added

into SGF and SPDF, respectively, at a concentration

of 0.18% (w/w) (Zhang et al., 2004; Chinese Pharma-

copoeia Commission, 2005).

Morphological changes of microspheres in dif-

ferent pH mediaFZD-ad-MS and AM-coated-MS were dispersed in 3

mL enzyme-free SGF or SPDF, and incubated with

horizontal shaking at 100 rpm at 37 ± 0.5oC. After

different time intervals, suspensions were centrifuged

at 4000 rpm for 5 min. The resultant solid were then

washed, lyophilized, and observed by SEM using a

JEOL JSM-5900LV scanning electron microscope.

In vitro anti-H. pylori efficacy The antimicrobial activity of FZD, FZD-ad-MS, and

blank mucoadhesive microspheres was evaluated using

the diffusion method (Ferraz et al., 2007). Appropriate

amounts of drug or microspheres were suspended in

PBS (pH 6.8) to produce final concentrations of FZD

or microspheres of 10 µg/mL, 100 µg/mL, and 1000 µg/

mL. A standard strain (ATCC 11637) of H. pylori pre-

actived in seed culture was diluted in Brucella broth

to a final concentration of 1×108 CFU/mL. Molten agar

media was transferred to sterilized petridishes and

allowed to solidify. The plates were swabbed with 10

µL liquor of the microorganism. Wells were made in

the solidified agar medium, and then filled with sus-

pensions of the drug or microspheres. The plates were

then incubated at 37 ± 0.5oC for 72 h under micro-

aerophilic conditions. The diameters of the inhibition

zones were measured. All tests were performed in tri-

plicate.

RESULTS AND DISCUSSION

Preparation and characterization of Micro-

spheresPreparation, morphology, particle size and

drug loading capacity of microspheres

In the present study, FZD was chosen as a model

drug, since it has been used to treat peptic ulcer

disease in China for several decades (Cheng and Hu,

2009). Its low-level resistance and MICs make it a

good alternative for H. pylori therapy (Kwon et al.,

2001). However, compared with other antibiotics such

as amoxicillin, clarithromycin, metronidazole and tetra-

cycline (Hejazi and Amiji, 2002; Liu et al., 2005; Ishak

et al., 2007; Rajinikanth et al., 2008), FZD was formu-

lated into microspheres for the first time. The pre-

paration of furazolidone-loaded mucoadhesive micro-

spheres (FZD-ad-MS) was performed using the emul-

sion-solvent evaporation technique described in pre-

vious studies. An ethanol-water mixture (9:1, v/v) was

used as the dispersed phase instead of acetone, because

Cb and Ec are almost completely dispersed in the

former and obvious precipitates were observed in the

latter. Spherical microspheres were obtained using

this approach and scanning electron images of the

microspheres are shown in Fig. 1A. Several crystals

were also found to be scattered on the surface of the

microsphere (Fig. 1B), which might result in a burst

release and enhance the FZD concentration for effect-

ive H. pylori clearance (Liu et al., 2005).

The AM-coated-MS were prepared using modified

Eudragit-coated microsphere formulations (Onishi et

al., 2007; Paharia et al., 2007; Oosegi et al., 2008) and

Development of Novel Microsphere for Duodenum Delivery 843

were found to be almost spherical as observed by SEM

(Fig. 1C). The surface texture of the AM-coated-MS

was much smoother than the uncoated microparticles

and no crystals were present on the surface (Fig. 1D),

indicating that FZD-ad-MS was successfully enclosed

and the burst release might be reduced by HPMCAM.

Several solvents were used as the coating solution in

preliminary experiments, e.g. ethanol, acetone, di-

chloromethane, ethanol-acetone mixture (4:1, v/v),

ethanol-dichloromethane mixture (9:1, v/v). Finally,

acetone was chosen because it allowed complete dis-

solution of the enteric HPMCAM, while maintaining

the integrity of the inner microspheres. Numerous

polymer-coated microspheres have been developed in

previous studies including the Eudragit-coated chito-

san or pectin microspheres (Onishi et al., 2007; Paharia

et al., 2007; Oosegi et al., 2008), PLGA-coated chitosan

particles (Jeong et al., 2008) and Ec-coated chitosan

microcores (Remuñán-López et al., 1998). However,

this is the first study to report on the development of

HPMCAM-coated mucoadhesive microspheres com-

posed of Cb and Ec.

The pH-sensitive coating material, HPMCAM, which

dissolves at a pH of around 3.0, was synthesized ac-

cording to a previous study published by our group

(Huang et al., 2005). Taking into account the special

environmental pH of the ulcer duodenum (pH 2.9-4.0),

HPMCAM, which has a relative low pH-sensitive value

(pH 3.0), would dissolve quickly after been transferred

into the duodenum, resulting in the rapid release of

the drug to effective concentrations for anti-H. pylori

therapy.

The particle size and drug contents of FZD-ad-MS

and AM-coated-MS are listed in Table I. FZD-ad-MS

had a mean diameter of 160.97 µm, and the sizes

ranged from 72.49−313.22 µm. The size of the AM-

coated-MS ranged from 197.32 to 861.16 µm and were

bigger than FZD-ad-MS, suggesting that the micro-

particles were well coated (Onishi et al., 2007), which

was also demonstrated by the SEM (Fig. 1C and D).

The drug content of the FZD-ad-MS and AM-coated

MS was 42.33 ± 3.43% (w/w) (n = 3), and 10.96 ± 1.29%

(w/w) (n = 3), respectively. The addition of a fairly large

amount of HPMCAM and loss of FZD-ad-MS during

the preparation could explain the lower FZD content

in the AM-coated-MS. In summary, furazolidone-loaded

mucoadhesive microspheres were successfully formu-

lated and coated with pH-sensitive HPMCAM through

an emulsion-evaporation method.

DSC and XRPD

DSC and XRPD studies were performed to under-

stand the drug-polymer interactions and the crystal-

line or amorphous nature of the drug after encapsula-

tion into a polymeric microsphere formulation (Türk

et al., 2009). DSC scans of the FZD, FZD-ad-MS, AM-

coated-MS and blank mucoadhesive microspheres are

presented in Fig. 2. In the thermogram of FZD (Fig.

2A), a single sharp endothermic peak was observed at

258.2oC, which corresponded to the melting point of

the drug (255−259oC, Chinese Pharmacopoeia Com-

mission, 2010). The same fusion peak at 256.7 and

255.5o were also observed in the thermograms of FZD-

ad-MS and AM-coated-MS (Fig. 2B and C), indicating

the possible existence of crystalline drug in the micro-

spheres. The small decrease in the melting point of

drug, which was also observed in the immiscible or

partially miscible system, also demonstrated that the

FZD may have been in the crystalline form in the

microspheres (Marsac et al., 2009; Türk et al., 2009).

Fig. 1. Scanning electron photomicrographs of FZD-loadedmucoadhesive microspheres (A, ×150; B, surface character,×1000) and HPMCAM-coated mucoadhesive microspheres(C, ×70; D, surface character, ×1000).

Table I. Particle characteristics of FZD-ad-MS and AM-coated-MS

FormulationParticle sizea

(µm)Size distributiona

(min. - max., µm)Drug contentb

(%, w/w)

FZD-ad-MS 160.97 ± 047.24 72.49 - 313.22 42.33 ± 3.43

AM-coated-MS 336.44 ± 129.34 197.32 - 861.16 10.96 ± 1.29aThe results are expressed as the mean ± S.D. (n = 200); bThe results are expressed as the mean ± S.D. (n = 3).

844 D. Zhou et al.

Furthermore, SEM (Fig. 1B) images showed drug

crystals scattered on the surface of FZD-ad-MS, which

also suggests that the drug was in the crystalline

state in microspheres. In contrast, the blank mucoad-

hesive microspheres (Fig. 2D) did not show any fusion

peak, which further proved that the visible endother-

mic peak around 255−259oC (Fig. 2A, B and C) may be

attributed to the entrapment of crystalline drug in the

microspheres. In addition, few crystals were observed

on the surface of the AM-coated-MS, which may due

to the HPMCAM coating.

XRPD analysis was used to confirm the physical

state of FZD in the microspheres. The graphs depicted

in Fig. 3 show the XRPD patterns of FZD, FZD-ad-

MS, AM-coated-MS and blank mucoadhesive micro-

spheres. Using this analysis, the blank microspheres

(Fig. 3D) were found to be in the typical amorphous

state (Pignatello et al., 2002), while the drug powder

(Fig. 3A) exhibited sharp peaks indicative of the

crystalline state of FZD (Türk et al., 2009). The crystal

diffraction peaks of FZD were still visible for the FZD-

ad-MS and AM-coated-MS, suggesting that FZD was

present in a crystalline state in the microspheres,

which was consistent with the results of the DSC

analysis and SEM images.

Percent mucoadhesion

The in vitro mucoadhesive properties of microspheres

were tested according to the method reported by Ranga

Rao and Buri (1989). The prepared FZD-ad-MS and

FZD-loaded non-adhesive microspheres (FZD-non-MS)

sieved at 150−355 µm were used. The percentage of

FZD-ad-MS remaining in the gastric mucosa was

87.67 ± 2.52% (n = 3), which was higher than that of

FZD-non-MS (23.00 ± 10.54%, n = 3). On the other

hand, more than 90% of FZD-ad-MS was trapped in

the duodenal mucosa (99.67 ± 0.58%, n = 3), compared

to 54.67 ± 9.07% (n = 3) for the FZD-non-MS. Micro-

spheres containing Cb adhered to the mucosa more

strongly than FZD-non-MS, which indicated that Cb

had a strong ability to interact with mucus (Oosegi et

al., 2008). According to Park and Robinson (1985), the

mucoadhesion of Cb was directly related to the pH of

the medium. They reported a higher binding interac-

tion at pH values lower than the pKa of polyacrylic

acid (4.75) and suggested that strong mucoadhesion

occurred only when the carboxylic groups were in their

acid forms. Our result was in agreement with these

findings. Thus, it was thought that mucoadhesive micro-

Fig. 2. DSC thermograms of FZD (A), FZD-ad-MS (B), AM-coated-MS (C) and blank mucoadhesive microspheres (D).

Fig. 3. XRPD diffraction patterns of FZD (A), FZD-ad-MS(B), AM-coated-MS (C) and blank mucoadhesive micro-spheres (D).

Development of Novel Microsphere for Duodenum Delivery 845

spheres containing Cb were good candidates for use in

mucoadhesive drug delivery systems for targeting the

ulcer duodenum, which has a pH ranging from pH

2.9-4.0. This is the case because Cb exhibited strong

mucoadhesion in its protonated form to prolong the

residence time of the microspheres, resulting in an

effective accumulation of antibiotics for H. pylori

clearance.

Drug stability, in vitro drug release and mor-

phological change of microspheres in different

pH media Stability studies of furazolidone in simulated

gastric fluid (SGF) and simulated pathological

duodenal fluid (SPDF)

Many antibiotics, such as amoxicillin, clarithromy-

cin and erythromycin, were reported to produce a

strong in vitro H. pylori clearance effect; however, the

efficacy of these antibiotics was poor in vivo. One of

the reasons was due to their instability in acidic me-

dium at the local site of infection. It has been reported

that FZD degraded rapidly when exposed to light

(Lunestad et al., 1995), or incubated with muscle,

intestinal, renal, and hepatic tissue (White, 1989).

But, its stability in simulated gastrointestinal fluids

at different pH values has not yet been evaluated. As

shown in Fig. 4, FZD was stable in SPDF pH 4.0 for

24 h and addition of pancreatic enzymes into the me-

dium did not result in degradation of the drug, which

would be ascribed to the low activity of pancreatic

enzymes at this pH (Aloulou et al., 2008). However,

slight decomposition (nearly 7%) of FZD was observed

at pH 1.2 after 10h of incubation but remained almost

constant up to 24 h. At 24 h, about 93.60% of the parent

drug still remained in the enzyme-absent medium and

93.22% of the drug remained when in the pepsin

solution. Interestingly, in pepsin-free SGF, furazolidone

degraded slowly for the initial 8 h (about 3.88%),

followed by a rapidly decomposition during 8-10 h

(about 2.31%). Meanwhile, in pepsin containing SGF,

the degradation pattern consisted of a remarkable

decline during the initial 4 h (about 5.75%), followed

by a gradual decrease from 4 h to 10 h (about 0.87%)

and this difference was statistically significant (p <

0.05, t-test, SPSS16.0 software for Windows®), which

suggested that pepsin accelerated the degradation or

furazolidone was vulnerable to pepsin during the first

few hours. At longer times, the influence of the enzyme

on drug stability could be ignored. Products of degrad-

ation were not detected in the present HPLC assay,

since no new peaks were found in the chromatograms.

To act effectively against H. pylori in the ulcer duo-

denum, the antibiotics in the formulations had to be

tolerant of the harsh acidic environment of the lumen.

Furazolidone remained stable at pH 4.0, indicating

that it would be a good candidate for use in duodenum-

specific drug delivery systems for H. pylori eradication.

In vitro drug release and changes in particle

features in different pH media

In vitro FZD release of FZD-ad-MS and AM-coated-

MS was examined in SGF (pH 1.2) and SPDF (pH 4.0)

at 37 ± 0.5oC in the presence and absence of a commer-

cial digestive enzyme. The drug release profiles are

shown in Fig. 5. At pH 1.2 (Fig. 5A and B), the drug

release rate was suppressed greatly in AM-coated-MS.

At 0.5 h, the release of FZD from AM-coated-MS in the

dissolution medium was too low for detection, while

12.84% of the loaded drug was released from the FZD-

ad-MS due to the burst release of the surface asso-

ciated drug. After 24 h, the cumulative release was

98.02% for FZD-ad-MS, which was higher than the

release observed for the AM-coated-MS (69.44%) in

SGF without pepsin (Fig. 5A). When pepsin was added

to the SGF (Fig. 5B), FZD-ad-MS also exhibited a

higher release (87.99%) than AM-coated-MS (70.45%)

after 24 h incubation. It was possible that the drug

release was hindered when in the AM-coated-MS be-

cause of the insolubility of the HPMCAM coating at

pH 1.2, which was lower than its pH-sensitive point

(pH 3.0). This conclusion was supported by the ob-

served changes in morphology (Fig. 6). After 2 h of

incubation in SGF, the AM-coated-MS did not change

shape significantly (Fig. 6A-C); however, the FZD-ad-

MS became porous and contained many cavities on

the surface (Fig. 6D-F). These cavities formed by diffu-

sion of the drug from the surface, which might have

promoted outward migration of the drug (Perugini et

al., 2001), resulting in a faster drug release. Addition-

Fig. 4. Stability of furazolidone at 37oC in SGF (pH 1.2) (-◆-),SGF (pH 1.2) with pepsin (-◇-), SPDF( pH 4.0) (-▲-) and SPDF(pH 4.0) (-△-) with pancreatin

846 D. Zhou et al.

ally, a slight decrease in the cumulative release was

observed when pepsin was added, which would contri-

bute to the instability of FZD at pH 1.2 as mentioned

above. However, these differences were not significant

for FZD-ad-MS (p > 0.05) but were for AM-coated-MS

(p < 0.05). This may have occurred because the decom-

position of the drug was compensated by a more rapid

drug release from FZD-ad-MS relative to the sup-

pressed drug release from AM-coated-MS due to the

HPMCAM coating. Finally, the decreased cumulative

release could be neglected for FZD-ad-MS but not for

AM-coated-MS.

At pH 4.0, the release profiles of FZD-ad-MS and

AM-coated-MS were comparable (p > 0.05) (Fig. 5C),

which consisted of a burst release followed by a gradual

release phase. These results implied that the HPMCAM

coating had little influence on drug release. As the pH

was increased, the drug release rate of AM-coated-MS

was accelerated and the cumulative release at 24 h

was augmented dramatically from 69.44% (pH 1.2,

Fig. 5A) to 83.92% (pH 4.0, Fig. 5C), suggesting that

the dissolution of HPMCAM primarily governed the

drug release (Onishi et al., 2007). The SEM analysis

(Fig. 6G-I) further demonstrated the rapid dissolution

of the HPMCAM coating at pH 4.0. The core particles

of the AM-coated-MS (i.e. FZD-ad-MS) were partly ex-

posed at 0.5 h and completely exposed after 1 h of

incubation with obvious pores on the surface, indicat-

ing that the drug release was correlated with the

dissolution of the HPMCAM coating. This process was

further confirmed by quantifying the presence of fur-

azolidone in the dissolution media. At 0.5 h, the cumu-

lative release was 2.08% for AM-coated-MS. The rapid

drug release from the AM-coated-MS over a short lag

time would promote fast and prolonged drug action

after transportation into the ulcer duodenum.

In contrast, the rate and extent of drug release for

FZD-ad-MS was retarded when the pH was raised

from 1.2 to 4.0 (p < 0.05). After 24 h, drug release was

nearly complete at pH 1.2 (98.02%), compared to

84.46% at pH 4.0. Generally, an increase in pH would

promote the swelling and erosion of Cb, since the

content of carboxyl groups of this polymer was more

than 50% (Wang, 2007). As reported by Park and

Robinson (1985), the pKa of polyacrylic acid was 4.75;

thus Cb would mostly remain in its protonated form

at pH < pKa. Upon approach to the pKa, deprotonation

would occur to some extent causing the polymer

networks to decomplex, leading to increased swelling

(Brock Thomas et al., 2007). At the same time, erosion

would occur at a very slow rate due to the insolubility

of Cb at acidic pH (Wang, 2007). It was likely that the

outer layers of the microspheres were hydrated and

formed a thicker gel layer at pH 4.0 than at pH 1.2,

which impeded water penetration into the core of the

particles (Kockisch et al., 2005), and also increased

the drug diffusion path length from the inner region of

the matrix (Nagarwal et al., 2010). Consequently, the

drug release was reduced at pH 4.0 for FZD-ad-MS. In

parallel, morphological changes of FZD-ad-MS at pH

4.0 were quite different from that at pH 1.2. During

the initial 0.5 h, pores and cracks were found on the

surface of FZD-ad-MS (Fig. 6J), as was observed at pH

1.2. However, it appeared that the tough features

disappeared with time, and instead, a viscous gelat-

inous layer with only a few large holes was formed

around the microspheres (Fig. 6K and L). At higher

pH values, the polymer chains of Cb tended to unfold,

which allowed further swelling. Under these conditions,

the viscous gel layer around the matrix will increase

with time creating a longer path length for the drug to

Fig. 5. Percentage cumulative in vitro FZD release fromFZD-ad-MS (-●-) and AM-coated-MS (-○-) in SGF withoutenzyme (A), in SGF with pepsin (B), and in SPDF withoutenzyme (C)

Development of Novel Microsphere for Duodenum Delivery 847

diffuse into the dissolution media, resulting in a delayed

drug release. The release profiles of FZD-ad-MS and

AM-coated-MS in the SPDF containing pancreatic

enzymes were evaluated, too (Date not shown). No

remarkable effect of the enzyme on drug release was

observed (p > 0.05), indicating that HPMCAM, Cb and

Ec might not be sensitive to the pancreatin as was

observed for FZD. Controlled drug release is important

for efficiently delivering drugs to the diseased area.

When simple mucoadhesive microspheres were admin-

istered orally, they were first trapped by the gastric

mucosa, where they collapsed and/or dissolved; there-

fore, protection of microspheres from the effect of gas-

tric pH and mucosa is needed (Onishi et al., 2007). In

this study, the morphology of HPMCAM-coated muco-

adhesive microspheres, namely AM-coated-MS, were

not significantly affected at gastric pH, and the inner

mucoadhesive particle, that is FZD-ad-MS, was regen-

erated at ulcer duodenal pH within less than 1 h due

to the dissolution of the HPMCAM coating layer. It

was thought that FZD-ad-MS could be protected effec-

tively in the stomach by HPMCAM coating and the

drug would be released soon after being transited into

the duodenum. Thus, a more effective H. pylori eradi-

cation would be expected by the combination of pH-

sensitive controlled drug release and strong mucoad-

hesion to prolong the residence time of microspheres

at ulcer duodenal pH.

To better understand the kinetics and mechanism

that governed drug release from the microspheres, the

release data (Fig. 5A and C) were evaluated kinetically

by zero-order, first-order, Higuchi, Baker-Lonsdale and

Korsmeyer-Peppas models (Costa and Lobo, 2001). The

line regression analysis is summarized in Table II.

For FZD-ad-MS, the best correlation was found to be

the first-order model (R2 = 0.9887) at pH 1.2 and the

Baker-Lonsdale (R2 = 0.9865) at pH 4.0. Similarly, For

AM-coated-MS, a very high correlation was achieved

Fig. 6. The morphological changes of AM-coated-MS and FZD-ad-MS in SGF after 0.5 h (A, D), 1 h (B, E), 2 h (I, L), and inSPDF after 0.5 h (G, J), 1 h (H, K), 2 h (C, F).

848 D. Zhou et al.

for the first-order model (R2 = 0.9999) at pH 1.2, as

well as the Baker-Lonsdale model at pH 4.0 (R2 =

0.998). These fits proved that a diffusive mechanism

was mostly involved in the drug release from both

microparticles (Segale et al., 2008), and the drug

release was a diffusion-rate-limiting process (Kockisch

et al., 2005). This behaviour appeared to be consistent

with the cumulative release and morphological changes

described above. When hydrophilic polymer based

microspheres such as Cb and HPMCAM were immersed

in an aqueous medium, they swelled and formed a gel

diffusion layer that hampered the outward transport

of the drug within the matrix, hence producing a con-

trolled release effect (Esposito et al., 2005). Further-

more, the n value calculated from the Korsmeyer-Peppas

model was an empirical parameter that could be used

to distinguish the release mechanism. To determine

the exponent n, only the initial portion (<60%) of the

release curve was used (Mathew et al., 2007). For

spheres, the n values were 0.43 and 0.85 for diffusion

and “Case-II Transport” drug release, respectively (Arifin

et al., 2006). The n values ranged between 0.6678-

0.8635, which indicated that the drug release varied

from non-Fickian diffusion to “Case-II Transport”

(Agnihotri and Aminabhavi, 2006) depending upon

the formulation.

In short, the HPMCAM-coated mucoadhesive micro-

spheres were considered to be suitable for specific

delivery of furazolidone to the ulcer duodenum. The

drug release was suppressed in the stomach by the

HPMCAM coating layer and promoted in the duode-

num. In addition, the combination of the strong muco-

adhesion and pH-sensitive controlled drug release at

ulcer duodenal pH would result in improved treat-

ment of H. pylori infection.

In vitro antimicrobial efficacyThe bioactivities of the FZD and FZD-ad-MS were

examined using the agar diffusion method. The anti-

biotic-free mucoadhesive microspheres were used as

the negative control samples. A phosphate buffered

solution (pH 6.8) was employed to mimic the environ-

ment pH of the gastroduodenal epithelium where H.

pylori is localized, because the bacterial could not sur-

vive in SGF (pH 1.2) without urea. H. pylori urease

hydrolyses the urea present in the gastric juices to

generate ammonia and bicarbonate, which effectively

neutralize the acidic pH of its environment (Lin et al.,

2009). Microbiological test data are listed in Table III.

No inhibitory effect was detected for the correspond-

ing negative control groups, indicating that the inhibi-

tory effect was due to the antibiotics being released

from the microspheres during incubation (Cheng and

Hu, 2009). The diameters of the kill zone expanded at

higher sample concentrations, which suggested that

the antibacterial activity was concentration-dependent.

Taking into account the actual drug content (38.94%)

of the FZD-ad-MS, encapsulation of FZD in the micro-

spheres resulted in an increase in the apparent anti-

H. pylori activity with lower drug amounts at the

given concentrations. This result indicates that encap-

sulation improves drug activity (Giunchedi et al.,

Table II. Model fitting of in vitro drug release data in different pH media without enzymes

SamplesCorrelation coefficients (R2) Release exponent (n)

Zero-order First-order Higuchi Baker-Lonsdale Korsmeyer-Peppas

FZD-ad-MSpH 1.2 0.6988 0.9887 0.8876 0.9085 0.6678

pH 4.0 0.7399 0.915 0.9183 0.9865 0.7867

AM-coated-MSpH 1.2 0.9716 0.9999 0.9962 0.9721 0.8301

pH 4.0 0.8085 0.973 0.9555 0.998 0.8635

Table III. Inhibition of H. pylori growth in the presence of furazolidone and furazolidone-loaded mucoadhesivemicrosphere and drug-free microspheres

Concentration of furazolidone ormicrospheres (µg/mL)

Mean zone diameter (cm ± S.D.) (n = 3)

FurazolidoneFurazolidone-loaded

microspheres*drug-free

microspheres

1000 5.37 ± 0.12 5.0 ± 0.06 0

100 3.60 ± 0.17 3.9 ± 0.10 0

10 1.90 ± 0.10 1.3 ± 0.12 0

0: No zone of inhibition*The drug content was 38.94% (w/w).

Development of Novel Microsphere for Duodenum Delivery 849

1998). Although FZD has been shown to be highly

effective in controlling H. pylori infection, its produces

serious adverse effects at the therapeutic dose (400

mg/day), which limits its widespread use. Thus, de-

creasing the dose might help to decrease its side effects

(Hasan et al., 2010). Therefore, the application of FZD-

loaded microsphere formulation would be expected to

relieve drug adverse reactions with a lower risk of

drug dumping and improved patient compliance

(Akiyama et al., 1995; Nagahara et al., 1998).

In conclusion, a novel combination approach, i.e. pH-

sensitive and mucoadhesive delivery system, namely

HPMCAM-coated mucoadhesive microsphere (AM-

coated-MS), was developed to improve H. pylori eradi-

cation. Furazolidone was chosen as the model drug in

the mucoadhesive microsphere formulation (FZD-ad-

MS). The HPMCAM coating was shown to protect the

inner core of the microspheres (FZD-ad-MS) at gastric

pH and allowed almost complete regeneration of FZD-

ad-MS at pathological duodenal pH. The drug release

was also suppressed in the stomach by HPMCAM and

immediately increased in the ulcer duodenum. FZD-

ad-MS exhibited a very strong mucoadhesiveness to

the duodenal mucosa at ulcer duodenal pH and FZD

was stable in SPDF. The in vitro anti-H. pylori experi-

ments demonstrated that encapsulation improved the

efficacy of the drug. These properties demonstrated

that AM-coated-MS could be used as a specific delivery

system for duodenum-targeted treatment of H. pylori

infection.

ACKNOWLEDGEMENTS

This research was supported by National Natural

Science Foundation of the People’s Republic of China

(30772667) and the National S & T Major Project of

China (2009ZX09310-002).

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