Synthesis, characterization and evaluation of titanium carbonitride surface layers with varying concentrations of carbon and nitrogen

POLYMERS FOR ADVANCED TECHNOLOGIES

Polym. Adv. Technol. 2008; 19: 371–376

science.wiley.com) DOI: 10.1002/pat.1018

Synthesis, characterization and evaluation of semi-IPN

hydrogels consisted of poly(methacrylic acid) and guar

gum for colon-specific drug delivery

Shengfang Li* and Xianli LiuSchool of Chemical and Material Engineering, Huangshi Institute of Technology, Huangshi 435003, Hubei, PR China

Received 29 July 2007; Revised 24 September 2007; Accepted 26 September 2007

*Correspoeering, HHubei, PE-mail: li

The semi-IPN hydrogels consisting of poly(methacrylic acid) and guar gum (GG) are prepared at

room temperature using water as solvent. 5-aminosalicylic acid (5-ASA) is entrapped in the hydrogel

in the synthesis of hydrogel and all entrapment efficiencies are found above 85%. The hydrogel

shows excellent pH-sensitivity. It exhibited minimum swelling in an acidic pHmedium through the

formation of a complex hydrogen-bonded structure and maximal swelling due to the electrostatic

repulsion due to the ionization of the carboxylic groups in pH 7.4 medium. The degradation in vitro

shows that the degree of degradation (R%) depended on the concentration of cross-linking agent and

content of GG. The hydrogel shows a minimum release of 5-ASA due to the complex hydrogen

bonded structure of the hydrogels in the medium of pH 2.2. The enzymatic degradation of hydrogels

by cecal bacteria can accelerate the release of 5-ASA entrapped in the hydrogel in pH 7.4 medium.

Copyright # 2007 John Wiley & Sons, Ltd.

KEYWORDS: guar gum; Semi-IPN hydrogels; enzymatic degradation in vitro; release

INTRODUCTION

Colon-specific drug delivery has gained increased import-

ance not only for its potential for the delivery of proteins and

peptides but also for the delivery of the drugs for the

treatment special diseases such as ulcerative colitis, Crohn’s

diseases, inflammatory bowel diseases (IBD), infectious

diseases, and colon cancer.1,2 To achieve successful colonic

delivery, a drug needs to be prevented from absorption of the

environment of upper gastrointestinal tract (GIT) and then be

released into the colon, which is considered the optimum site

for colon-specific drug delivery. The various approaches for

colon-specific drug delivery mainly include time-dependent

release systems, pH-dependent systems and enzymatically

controlled delivery systems. pH-sensitive hydrogel could be

potentially used for the delivery of drugs to the colon.

However, site-specific drug delivery to the colon cannot be

achieved by the only pH-sensitive hydrogels, because the pH

of the small intestine and the large intestine are almost same.3

Guar gum (GG), a naturally occurring glactomannan

polysaccharide, has been well studied as a carrier for

colon-specific drug delivery due to its drug release retarding

property and susceptibility to microbial degradation in the

colon.4,5 From the literatures, GG used for colon-specific

ndence to: S. Li, School of Chemical and Material Engin-uangshi Institute of Technology, Huangshi 435003,R [email protected]

drug delivery includes mainly coating and hydrogels.6

However, the studies on hydrogels of GG mostly emphasize

on enzymatic degradation of GG and its release of drugs in

the environment of colon. Few researchers focus on the

release behavior in the pH environment of stomach.7–10

5-Aminosalicylic acid (5-ASA) is widely accepted in the

treatment of IBD, including ulcerative colitis and Crohn’s

disease. When orally administered, 5-ASA is unstable in the

gastric conditions and prone to be absorbed or degraded in

the stomach and small intestine before reaching the colon

sites, which causes low drug bioavailability and low

efficiency for inflammatory colon disease. In addition,

5-ASA is easily oxidated at high temperature. In the present

study, we synthesized a new pH sensitive and enzymatic

degradable semi-IPN hydrogel containing both pH-sensitive

acidic monomers and enzymatically degradable GG for

colon-specific drug delivery. The hydrogels are consisted of

poly(methacrylic acid) and GG. Swelling of such hydrogels

in the stomach (lower pH value) is minimal due to the

presence of carboxylic groups. The extent of swelling

increases as the hydrogel passes down the intestinal tract

because of increase in pH leading to ionization of the

carboxylic groups. Once inside the colon, highly swollen

hydrogels become accessible to the enzymes produced by

microflora in the colon. The enzymatic degradation of GG

will occur. 5-ASA, as model drug, was entrapped in the

hydrogel in the synthesis of hydrogel at room temperature

using water as a solvent. The semi-IPN hydrogels were

characterized by FTIR, SEM, etc. The swelling and degra-

Copyright # 2007 John Wiley & Sons, Ltd.

372 S. Li and X. Liu

dation properties of semi-IPN hydrogels were also studied.

The release behavior of 5-ASA in vitro from semi-IPN

hydrogel was evaluated.

EXPERIMENTAL

MaterialsMethacrylic acid (MAA) were purchased from Tianjin

Chemical Group, China and were distilled before use. GG,

N,N0-methylenebisacrylamide (MBA), K2S2O8, and NaHSO3

(purchased from Shanghai Chemical Group, China) were of

analytical reagent grade and used without any further

purification. 5-ASA was a gift from Yuancheng Co. (Wuhan,

China), and recrystallized with distilled water.

Preparation of semi-IPN hydrogelsThe hydrogels were prepared through the free-radical

copolymerization of MAA, MBA in the present of GG in

an aqueous medium. Briefly, the mixture solutions were

made up of varied amounts of MAA,MBA in distilled water.

Then various amounts of GG, K2S2O8, and NaHSO3 were

added into the mixture solutions. This homogeneous

solution was bubbled with nitrogen to discharge oxygen

for about 30min. The free radical copolymerization was

carried out at room temperature for 4 days. After

copolymerization, the solid copolymer slab was cut into

circular disks using punches. The samples were immersed in

deionized water for 6 days to remove the unreacted

monomers. Finally, the samples were dried in vacuum at

258C to a constant weight and stored for further use. The feed

composition for the preparation of hydrogels is listed in

Table 1 and the samples are designated as PMGx.

Fourier transform infrared (FIIR)measurementsFIIR spectroscopy was used to confirm the structure of

semi-IPN. The hydrogel samples and GGwere analyzed on a

Bruker EQUNINOX FIIR spectrophotometer in the region of

400–4000 cm�1. Before themeasurements, the dried hydrogel

samples were crushed down (KBr, pellet).

Morphology of semi-IPN xerogelsThe equilibrium-swollen gels were frozen at �808C for 12 hr

in refrigerator, then freeze-dried and fractured. The fractured

specimens were covered with gold vapor, and then the

morphology of the fractured surface of the xerogels were

observed by field emission scanning electron microscopy

Table 1. The feed composition of semi-IPN hydrogels

Sample GG (g) MAA (g) MBA (g)

PMG0 0 1 0.02PMG1 0.4 1 0.02PMG2 0.4 1 0.04PMG3 0.4 1 0.06PMG4 0.4 1 0.08PMG5 0.4 1 0.1PMG6 0.2 1 0.02PMG7 0.1 1 0.02

Copyright # 2007 John Wiley & Sons, Ltd.

(FE-SEM; FEI Sirion 200) with an acceleration voltage of

10 kV.

Swelling studiesThe dry hydrogel was immersed in the swelling medium at

378C. At regular time intervals, the gels were removed from

the medium, the weight of the swollen hydrogels was

determined after the removal of the surface water through

blotting with filter paper. The equilibrium-swelling ratio was

calculated by the following equation:

SR ¼ Ws �Wd

Wdð1Þ

when the swollen hydrogels reached a constant weight, the

SR was considered to be the equilibrium. Wd and Ws was

the weight of xerogels and equilibrium-swollen gels,

respectively.

In vitro degradation of hydrogelsThe enzymatic degradation of the hydrogels was performed

in a flask filled with 150ml of phosphate buffer (pH 7.4,

0.1mol/L, 378C) which contained a certain quantity of freeze

dried rat cecum content (from male Sprague–Dawley rats,

about 250 g). The degradation experiments were conducted

by incubating the hydrogel mass in buffer placed in a

thermostatic rotary shaker (HQ45Z, Chinese Academy of

Sciences instrument Corp. Ltd., Wuhan, China) and by the

determination of the weight loss after recovery of the

samples at predetermined time intervals. The buffer solution

was changed every 2 days to maintain enzymatic activity.

After a predetermined time, the samples were removed from

the solution, washed thoroughly with distilled water, and

then dried below 608C. The degradation was assessed by

measuring the weight loss ratio (R%), which was defined as

the following equation:

R ð%Þ ¼ W1 �W2

W1ð2Þ

where, W1 and W2 were the weights of the gel before and

after degradation, respectively.

In vitro loading and release of 5-ASAof the hydrogelsIn order to determine the actual amount of drug entrapped

into the hydrogel, various sample synthesized were washed

in 1L of distilled water taken in five installments of 200ml

each and the absorbance of each solution was measured at

275 nm. In this way, the actual drug entrapped in the

K2S2O8 (g) NaHSO3 (g) H2O (g)

0.02 0.01 100.02 0.01 100.02 0.01 100.02 0.01 100.02 0.01 100.02 0.01 100.02 0.01 100.02 0.01 10

Polym. Adv. Technol. 2008; 19: 371–376

DOI: 10.1002/pat

Figure 2. FT-IR spectrum of hydrogel (a) 5-ASA-unloaded,

(b) 5-ASA-loaded hydrogel.

Colon-specific drug delivery 373

hydrogel was calculated using entrapment efficiency (E%),

which was defined as the following equation:

E ð%Þ ¼ 100ðM1 �M2ÞM1

ð3Þ

where, M1 was the amount of 5-ASA added to the reaction

mixture before the polymerization process. M2 was the

amount of drug lost during washing of drug-loaded

hydrogels from the initial loading.

In vitro release of 5-ASA of the hydrogels was carried out

by a method from the literature.11 Briefly, dehydrated 5-ASA

loaded hydrogels were immersed at 378C in vials containing

20ml of phosphate buffer solution with or without addition

of rat cecum content. For those added rat cecum content, the

above solution was bubbled with nitrogen for 5min to obtain

anaerobic conditions, and the vials were closed, tightly

sealed, and incubated in a thermostatic rotary shaker

(HQ45Z, Chinese Academy of Sciences Instrument Corp.

Ltd., Wuhan, China) at shaking speed of 50 rpm. At periodic

intervals, 1ml of solutionwas pipetted out and replacedwith

equal volume of the same dissolution medium, centrifuged

and the released drug was analyzed at 275 nm with a UV

spectrophotometer. Then the weight of the release drug was

calculated with the standard equations of curves.

RESULTS AND DISCUSSION

FTIR characterization of hydrogelsFigure 1 shows FT-IR spectra for no-semi-IPN Hydrogel

PMG0, Semi-IPN Hydrogel PMG1 and GG. From FTIR

spectra of GG, it can be seen that the band at 1073 cm�1 was

the characteristic absorption band of C–O groups of primary

alcohol. From FT-IR spectra of hydrogel PMG1 and PMG0, it

is found that the absorption of two gels are similar. However,

there is a new absorption band at 1073 cm�1 in gel PMG1

comparingwith that in gel PMG0. This indicates that GGwas

incorporated into the gel system and the semi-

interpenetrating polymer network structure was formed.

As shown in Fig. 2, a comparison of FTIR spectra of both

5-ASA-unloaded and 5-ASA-loaded hydrogel samples

indicates that there is a decrease in the intensity in region

3000–4000 cm�1 due to absorption of 5-ASA. This could be

Figure 1. FT-IR spectrum of hydrogel PMG0 (a), Semi-IPN

Hydrogels PMG1 (b), and guar gum (c).

Copyright # 2007 John Wiley & Sons, Ltd.

attributed to the absorption of 5-ASA in the polymer through

hydrogen bonding interaction.

Swelling properties of semi-IPN hydrogelsThe swelling kinetics of different semi-IPN hydrogels were

investigated at both pH 2.2 and pH 7.4 buffer solutions, to

simulate the stomach and colon conditions, and the results

are shown in Figs 3 and 4, respectively. The equili-

brium-swelling ratio at pH 7.4 are almost an order higher

compared with those of the same semi-IPN hydrogels at pH

2.2, implying their swelling property is highly dependent on

pH values. This may be attributed to the fact that hydrogels

are in compact collapsed status (non-ionized status) in pH 2.2

(the pH in the stomach) due to the hydrogen bonding

interactions between the carboxylic groups of MAA present

along the polymer chains. However, the hydrogels swell in

great degree at pH 7.4 (the pH in the colon) because of the

electrostatic repulsion among the charged –COO� groups

due to the ionization of the carboxylic groups.

The influence of the cross-linking degree on the swelling

kinetics of hydrogels PMG1, PMG2, PMG3, PMG4, and

PMG5 is shown in Fig. 3. Obviously, an increase in the degree

of cross-linking leads to the decrease of swelling degree in

both pH 2.2 and pH 7.4 buffer solutions, although the

hydrogels have the same comonomers. The influence of the

GG content on the swelling kinetics of hydrogels PMG1,

PMG6, and PMG7 is shown in Fig. 4. It can be seen that the

values of SR decrease with an increase the quantity of GG,

which leads to a more chain entanglements.

In vitro degradation of semi-IPN hydrogelsIn vitro degradation of semi-IPN hydrogel consisted of

poly(methacrylic acid) and GG were studied by incubation

with rat cecum content. This results from that degradation

produced by cecum bacterial caused part mass loss when the

larger quantity of GG was introduced into gel system. In

order to confirm this idea, degradation was assessed by

measuring the weight loss ratio (R%). The degree of

degradation of hydrogels in vitro is shown in Table 2. In

the hydrogel synthesis experiment, we found the copoly-

merization reacted completely when the free radical

copolymerization was carried out at room temperature for

4 days. Thus, the feed composition of semi-IPN hydrogels

could represent the original composition of semi-IPN gel

after copolymerization approximately. For hydrogels with

the same content of GG and different concentration of

Polym. Adv. Technol. 2008; 19: 371–376

DOI: 10.1002/pat

Figure 3. Swelling kinetics for semi-IPN hydrogels PMG1 (&), PMG2 (*), PMG3 (~), PMG4 (!),

and PMG5 (^) in pH 2.2 and pH 7.4 buffer solutions.

Table 2. Degree of degradation of hydrogels in vitro

Sample

Degradation (%)

1 day 2 days 5 days

PMG1 8.1 15.4 26.3PMG2 7.2 11.2 20.1PMG3 6.3 10.1 14PMG4 5.4 9.2 11.2PMG5 3.1 5.5 8.2PMG6 2.1 4.3 7.5PMG7 0.2 3.2 5.3

374 S. Li and X. Liu

cross-linking agent (MBA), the higher concentration of MBA,

the lower the degradability (see hydrogel PMG1, PMG2,

PMG3, PMG4, and PMG5). In comparison with hydrogel

PMG5 and 6, hydrogel PMG7 that has the lower content of

GG shows lower the degree of degradation. In other words,

the degree of degradation also depended on the content of

GG. As the content of GG in the gel decreases, the value of R

also decreases. This mainly may be due to the fact with

decrease in the GG concentration within the gel matrix, the

chances of binding of enzyme molecules with substrate

decreases, thus resulting in a decrease in the degree of

degradation of gel matrix. Similar results were also found in

our previous work.11

The degradation of the hydrogels was further confirmed

by SEM micrographs of the gels before and after enzymatic

degradation (Fig. 5). Comparing with that before degra-

dation in Fig. 5 (left), the pore size after degradation in Fig. 5

(right) is larger and there are many fragments of the residues

on the pore walls. These results indicate that the

b-D-mannopyranose bonds of GG in the gel networks have

been cleaved through enzymatic degradation by rat cecum

content.

Drug loading and release from semi-IPNhydrogelsAll entrapment efficiency (%) of hydrogel PMG1 were found

above 85% (Table 3). This indicates that drug loading into the

Figure 4. Swelling kinetics for semi-IPN hydrogels

and pH 7.4 buffer solutions.

Copyright # 2007 John Wiley & Sons, Ltd.

hydrogels is sufficiently high in this way. However, the little

loss occurred could be attributed to that the drug present on

the surface is loosely bound and it was lost on washing.

As stated above, the Semi-IPN hydrogel exhibited

excellent pH sensitivity. It showed that minimum swelling

in pH 2.2 andmaximum swelling in pH 7.4. This implies that

the hydrogel system has potential to be used as oral

colon-specific drug delivery system. Therefore, a very low

extent of swelling in pH 2.2 and a high extent of swelling in

pH 7.4 indicate that the drug inside the semi-IPN hydrogel

might be protected from the gastric enzyme before its entry

into the colon. In the colon, drug would be largely released

because of their high extent of swelling, which allows colonic

enzyme to permeate into the gels and high degradation

occurs. In order to confirm this, the release dynamics of

PMG1 (&), PMG6 (*), PMG7 (~), in pH 2.2

Polym. Adv. Technol. 2008; 19: 371–376

DOI: 10.1002/pat

Figure 5. SEM images of semi-IPN hydrogel PMG1before degradation (left), and

after 5 days degradation by rat cecum content (right). All images were 100� original

magnification.

Colon-specific drug delivery 375

5-ASA loaded semi-IPN gel PMG115.69 samplewas studied in

pH 2.2 and pH 7.4 (without rat cecum contents). Results, as

depicted in Fig. 6, there is less than 3.5mg 5-ASA/g gel

(cumulative release: 22.3%) of the drug release from Semi

IPN hydrogel PMG115.69 in pH 2.2 within 36 hr. However,

there is 8.2mg 5-ASA/g gel (cumulative release: 52.3%) of

the drug release in pH 7.4 within 36 hr. This may be

attributed to the fact that the carboxyl groups being in the

non-ionized status in pH 2.2 (the pH in the stomach), and the

drug release is lower because of the hydrogen bonding.

However, the hydrogel swells in great degree at pH 7.4 (the

pH in the colon) due to ionization of carboxyl groups in the

gel. Therefore, there is higher release of drug at in the

medium of pH 7.4.

The mechanism of 5-ASA released from the gel matrices

was simulated using a simple equation.12 F¼Mt/M1¼ ktn,

where Mt is the percentage of drug released at time t, k is a

Table 3. Entrapment efficiency (%) of hydrogel PMG1

Sample code

Amount of drug

Entrapmentefficiency (%)

Loadedinitially (mg)

Retained inhydrogel (mg)

PMG1a8.79b 15.0 12.75 85

PMG112.0 20.0 17.40 87PMG115.69 25.0 22.75 91

aHydrogel sample code.bAmount of drug in milligram present per gram dry gel.

Figure 6. 5-ASA released profiles for PMG115.69 in pH 2.2

and in pH 7.4 buffer solutions with or without cecal contents.

Copyright # 2007 John Wiley & Sons, Ltd.

release rate constant, and n is an exponent of release, which is

calculated as the slope of linear regression lines fitted to the

ln F versus ln t. The values of the exponent of release n

calculated according to above method were found to be 0.52

and 0.68 in the medium of pH 2.2 and pH 7.4, respectively.

The values clear indicate that the hydrogel follows Fickan

diffusion controlled release mechanism in pH 2.2, while in

pH 7.4 follows non-Fickan diffusion and chain relaxation

controlled mechanism.12

Figure 6 also shows release behavior of the 5-ASA-loaded

gel PMG115.69 in the presence of rat cecum bacterial in the

pH 7.4 buffer solutions. Approximately 13.5mg 5-ASA/g gel

(cumulative release: 86.0%) were released in the presence of

rat cecum bacterial within 36 h. High release of 5-ASA is

attributed to the degradation of b-D-mannopyranose bonds13

of GG in the semi-IPN gel network by rat cecum bacterial.

CONCLUSIONS

The hydrogels consisted of poly(methacrylic acid) and GG

were prepared in aqueousmedium at room temperature. The

studies on the swelling behavior of hydrogels reveal their

sensitive response to pH change. The hydrogels can be

degraded by rat cecum bacterial and the degree of

degradation depended on the cross-linking density and

content of GG. The model drug 5-ASA can be loaded up to

85% by embedding into the hydrogel network during the

polymerization process. The results of in vitro 5-ASA release

indicate that the release is controlled by swelling and

degradation of the hydrogels. It shows that a minimum

release of 5-ASA is due to the complex hydrogen-bonded

structure of the hydrogels in the medium of pH 2.2. The

enzymatic degradation of GG in the hydrogels by cecal

bacteria can accelerate the release of 5-ASA in pH 7.4

medium.

AcknowledgmentsThis research was supported by the Natural Science Founda-

tion of Huangshi Institute of Technology (Project No.

07yjz01R) and the Innovative Team of Huangshi Institute

of Technology. Authors also gratefully acknowledge the

reviewers of this article.

Polym. Adv. Technol. 2008; 19: 371–376

DOI: 10.1002/pat

376 S. Li and X. Liu

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Polym. Adv. Technol. 2008; 19: 371–376

DOI: 10.1002/pat