ph dependent poly[2-(methacryloyloxyethyl)trimetylammonium chloride-co-methacrylic acid]hydrogels...

pH dependent poly[2-(methacryloyloxyethyl)trimetylammoniumchloride-co-methacrylic acid]hydrogels for enhanced targeteddelivery of 5-fluorouracil in colon cancer cells

R. K. Mishra • K. Ramasamy • N. A. Ahmad •

Z. Eshak • A. B. A. Majeed

Received: 27 June 2013 / Accepted: 23 December 2013

� Springer Science+Business Media New York 2014

Abstract Stimuli responsive hydrogels have shown enor-

mous potential as a carrier for targeted drug delivery. In this

study we have developed novel pH responsive hydrogels for the

delivery of 5-fluorouracil (5-FU) in order to alleviate its anti-

tumor activity while reducing its toxicity. We used 2-(meth-

acryloyloxyethyl) trimetylammonium chloride a positively

charged monomer and methacrylic acid for fabricating the pH

responsive hydrogels. The released 5-FU from all except

hydrogel (GEL-5) remained biologically active against human

colon cancer cell lines [HT29 (IC50 = 110–190 lg ml-1) and

HCT116 (IC50 = 210–390 lg ml-1)] but not human skin

fibroblast cells [BJ (CRL2522); IC50 C 1000 lg ml-1]. This

implies that the copolymer hydrogels (1–4) were able to release

5-FU effectively to colon cancer cells but not normal human

skin fibroblast cells. This is probably due to the shorter doubling

time that results in reduced pH in colon cancer cells when

compared to fibroblast cells. These pH sensitive hydrogels

showed well defined cell apoptosis in HCT116 cells through

series of events such as chromatin condensation, membrane

blebbing, and formation of apoptotic bodies. No cell killing was

observed in the case of blank hydrogels. The results showed the

potential of these stimuli responsive polymer hydrogels as a

carrier for colon cancer delivery.

1 Introduction

Colorectal cancer (CRC) is one of the most common

malignancies in developed countries. CRC remains one of

the common form of cancer with new cases and 500,000

deaths worldwide each year [1, 2]. The American Cancer

Society recently estimated 103,170 new cases in 2012 of

colorectal cancer in USA [3]. The high mortality associated

with CRC warrants more effective prevention along with

surgery, radiation and chemotherapy. Surgery continues to

have major role in CRC survival in early stage disease, by

removing detectable tumor; however residual microme-

tastases may cause relapse [4]. Chemotherapy is another

common option for treating colonic tumors but it is not

very effective because drugs do not reach the target sites at

effective concentration leading to increased dose size that

often results in enormous toxicity. It is a known fact that

most of the available chemotherapeutic drugs are taken up

non-specifically by all type of cells resulting in serious

consequences [5]. There has been enormous interest among

material scientists therefore to develop biomedical mate-

rials for controlled therapeutic delivery. An ideal formu-

lation should transport the drug specifically to colonic

region before it is released.

The common methods used for colon targeted delivery

of antibiotics are pro-drug approach, time dependent sys-

tems, pressure dependent systems, pH dependent systems,

and microbial triggered systems [6, 7]. pH sensitive poly-

mer hydrogels have gained significant attention as carriers

for colon targeted delivery of various therapeutic agents.

Smart or stimuli responsive hydrogels are materials which

R. K. Mishra (&) � A. B. A. Majeed

Brain Science Research Laboratory, Faculty of Pharmacy,

Universiti Teknologi MARA, 42300 Puncak Alam, Selangor,

Malaysia

e-mail: [email protected]

K. Ramasamy � N. A. Ahmad

Collaborative Drug Discovery Research Group, Faculty of

Pharmacy, Universiti Teknologi MARA, 42300 Puncak Alam,

Selangor, Malaysia

Z. Eshak

Microsopy Imaging Center, Faculty of Pharmacy, Universiti

Teknologi MARA, 42300 Puncak Alam, Selangor, Malaysia

123

J Mater Sci: Mater Med

DOI 10.1007/s10856-013-5132-x

can quickly respond to various triggers such as pH, tem-

perature, ionic strength, light, electric and magnetic field

[8, 9]. These responsive polymer hydrogels can be utilized

as smart drug delivery system (smart-DDS) in the field of

controlled drug release, in order to prolong and better

control over drug administration.

2 (methacryloyloxy) ethyl] trimethylammonium chloride

(MAETAC) is a positively charged monomer and a bifunc-

tional molecule containing both pH-independent cationic

head (quaternary ammonium) and a reactive methacryloyl

group [10]. Recently MAETAC received significant attention

as an excellent material for biomedical applications [11]. Poly

methacrylic acid (PMAAc) is an ionizable hydrophilic poly-

mer and has received considerable recognition as a pH sen-

sitive hydrogel [12]. PMAAc based formulations have shown

great potential for controlled drug delivery of various anti-

cancer agents [13] and were able to overcome multidrug

resistance in human breast cancer cells [14].

The aim of this work is to evaluate the potential of

poly[2-(methacryloyloxyethyl)trimetylammonium chlo-

ride-co-methacrylic acid] based novel pH responsive

hydrogels in colon cancer. The hydrogels are prepared by

simple redox copolymerization reaction and subsequently

loaded with 5-FU. The physicochemical properties of the

novel hydrogels were extensively investigated. In addition

in vitro 5-FU release, cellular uptake and cytotoxicity of

the hydrogels were investigated using human colon cancer

cell lines (HT29 and HCT116 cells).

2 Experimental

2.1 Materials

[2-(methacryloyloxyethyl) trimetylammonium chloride

(MAETAC) and methacrylic acid (MAAc) were purchased

from Sigma Aldrich (St. Louis, USA). Ammonium per-

sulfate (APS), N,N,N,N-tetramethyleneethylenediamine

(TEMED), N,N, methylenebisacrylamide (MBA) were

procured from Sigma Aldrich (St. Louis, USA). 5-FU a

model drug was purchased from Sigma (St. Louis, USA).

Deionized water was used for all copolymerization reac-

tions and in the preparation of buffer solutions.

2.2 Hydrogel synthesis

Five poly(MAETAC-co-MAAc) copolymer hydrogels

were synthesized with monomer feed ratios ranging from

50/50 to 90/10 (mol). The reactions as shown in Scheme 1

were carried out in aqueous medium using APS and TE-

MED as redox initiator at 41�C [15]. In a typical reaction,

five different monomer mixtures were prepared in chilled

condition using ice bath. Measured amount of MAETAC

was added in drop wise manner to requisite amount of

MAAc with continuous stirring on a magnetic stirrer. After

complete mixing the monomer mixtures were diluted with

predetermined amount of distilled water. The dilute

monomer mixture was taken in 100 mL flask equipped

with magnetic stirrer, a thermometer and a nitrogen inlet

system. The flask was kept submerged in thermostated

water bath at 41 �C on a magnetic stirrer. Nitrogen gas was

continuously purged inside the reaction mixture followed

by the addition of predetermined amount of MBA

(1 mol%), APS (0.5 mol%) and TEMED (3 mol%). To

accomplish the second phase of the reaction the individual

reaction mixtures were transferred to rectangular molds.

The molds 60/70/0.06 cm in dimension were prepared

using Teflon spacers along the three edges of a pair of glass

plates. The molds were then placed on thermostated water

bath at 41 ± 1 �C and dipped up to the height of reaction

mixtures. The nitrogen gas was purged into the reaction

mixtures through one of the orifices of the mold. After

10 min the nitrogen purging was stopped and orifices were

closed using paraffin grease. After 24 h of reaction the

hydrogels were removed from the molds, cut into pieces,

repeatedly washed up to three days to remove the unreacted

CH2 C

CH3

COOH

APS-MBA/TEMED

CH2 C

CH3

C O

O

(CH2)2

N

CH3

CH3CH3

+CH2 CH3

CH3

C O

O

(CH2)2

N

CH3

CH3CH3

CH2 C

CH3

COOH

)( m ( )n

Cl

Cl

MAAC

MAETAC

Poly (MAAC-co-MAETAC)

41oC/24 hours

Scheme 1 Synthesis of

Poly(MAAc-co-

MAETAC)copolymer hydrogel


123

monomers and dried under vacuum. The feed compositions

of five hydrogels and corresponding synthetic parameters

are as listed in Table 1.

The homopolymer of MAETAC and MAAc were syn-

thesized by the same method used for copolymer hydro-

gels. After completion of the polymerization step, the

PMAETAC homopolymer was obtained by precipitating in

excess of isopropyl alcohol and PMAAc was obtained as

solid translucent monolith.

2.3 FTIR-ATR spectroscopy

The infrared spectra of homopolymer and copolymer hydro-

gels were recorded within the range of 400-4,000 cm-1 as

KBr pellet on a Varian 640-IR FT-IR spectrophotometer.

Finely ground powder of the freeze dried samples was used to

prepare the KBr pellets.

2.4 Viscosity measurement

Intrinsic viscosity of the copolymers in 0.5 N NaCl solu-

tions at 25 �C was measured using a Ubbelhode viscome-

ter. The molar concentration of NaCl in the polymer

solution was maintained constant during the dilution pro-

cess. Intrinsic viscosity was determined using Huggins

equation maintaining the criteria of dilute solution.

g sp=c ¼ ½g� þ bc ð1Þ

where g is the intrinsic viscosity, gsp the specific viscosity,

b is the Huggins parameter and C is the polymer concen-

tration [16].

2.5 Wide angle X-ray diffraction (WAXD)

Dry copolymer hydrogels and homopolymers were ground

to fine powder. X-ray powder diffraction data were

collected on a XPERT PRO X-ray diffractometer (PAN

Analytical). The scanning 2h angle was from 5 to 65o.

2.6 Differential scanning calorimetry (DSC)

DSC of the copolymer hydrogels were recorded with

NETZSCH differential scanning calorimeter (DSC 200 F3)

at a 10 �C min-1 heating rate and N2 flow speed of

50 ml min-1.

2.7 Thermogravimetric Analysis (TGA)

Thermal stability of the copolymer networks were analyzed

with NETZSCH TG 209 F3 at a heating rate of

20 �C min-1 with nitrogen flushed at 100 ml min-1.

2.8 Environmental scanning electron microscopy

(ESEM)

A JEOL JSM-670 IF Environmental scanning electron

microscope (ESEM) was used to investigate the surface

morphology of the hydrogels. All the hydrogels (GEL1-5)

were allowed to swell in simulated intestinal fluid (SIF) and

stored in a deep freezer at -80 �C for 2 days. The samples

were then freeze dried at -50 �C using LABCONCO

(USA) freeze drying system for 3 days. Samples were kept

under vacuum before platinum sputtering treatment.

2.9 Swelling experiment

To investigate the pH responsive swelling behaviour the

dry gel discs *5 mm diameter were weighed and incu-

bated in buffered solutions of various pH value ranging

from 1 to 10 at 37 ± 1 �C. The ionic strength of each

buffer solution was adjusted to 0.2 M by the addition of

potassium chloride. Degree of swelling was determined by

gravimetric method. At equilibrium the swollen discs were

removed from the solution and reweighed after careful

removal of excess surface water. The swelling ratio (Q)

was calculated from the following equation

Q ¼ ðWf �WiÞ=Wi � 100 ð2Þ

where Wf and Wi were the final weight of the swollen disc

and initial weight of swollen disc respectively. Each

experiment was performed in triplicates.

2.10 Preparation of drug loaded hydrogels

5-Fluorouracil was incorporated into copolymer networks

by a swelling equilibrium method. The hydrogel discs

(GEL1-5) were allowed to swell in drug solution in SIF and

simulated gastric fluids (SGF) for 3 days at room temper-

ature. During this process, drug in the solvent was adsorbed

Table 1 Composition and intrinsic viscosity [g] of copolymer

hydrogelsa

S.

no

MAAc

(mol%)

MAETAC

(mol%)

Waterb

(mol%)

[g] (dL g-1)c

1 50 50 200 1.67

2 60 40 150 0.64

3 70 30 100 0.53

4 80 20 50 0.35

5 90 10 25 0.22

a The concentrations of MBA, SPS, and TEMED in the feed were 1,

0.5, and 3 mol% respectivelyb Molar percentage to total monomer contentc Intrinsic viscosity of copolymer hydrogels in sodium chloride

solution at 25 �C


123

onto the hydrogels. After incubation the discs were rapidly

washed with distilled water to remove the 5-FU that

adhered on the surface. The drug loaded hydrogels were

dried at room temperature for 48 h [17].

2.11 Determination of the entrapped 5-FU

To determine the actual drug entrapped in the hydrogel,

samples were placed in a 30 ml buffer solution and stirred

for 48 h. The solution was filtered and assayed by UV–Vis-

spectrophotometer at 266 nm [18]. The % of drug loading

and encapsulation efficiency (EE) was calculated using Eq.

(2) and (3) respectively.

Drug loading ð%Þ ¼Weight of drug in gel/weight of gel

� 100 ð3Þ

Encapsulation efficiency ð%Þ ¼ Actual loading=

Theoretical loading � 100ð4Þ

2.12 5-FU release from hydrogels

The 5-FU loaded dried hydrogel discs (GEL1-5) were

placed in a conical flask containing 50 ml physiological

fluid (SIF and SGF). The flask was kept at constant tem-

perature-shaking incubator at 37 �C at 100 rpm. At pre-

determined time points, 2 ml of solution was taken out and

replaced with the same amount of buffer solution in order

to maintain the same volume of solution. The percentage

cumulative release of 5-FU was analyzed at 266 nm by

using UV visible spectrophotometer. Each sample experi-

ment was repeated three times and final results were cal-

culated as an average [19].

2.13 Anticancer effect of released 5-FU

The human colon cancer cell lines HCT116 and HT29 were

obtained from American Type Culture Collection (Manas-

sas, VA) and was cultured in Roswell Park Memorial

Institute (RPMI) 1640 Medium (Sigma, Germany) supple-

mented with 10 % heat-activated fetal bovine serum (PAA

Laboratories, Austria) and 1 % penicillin/streptomycin

(PAA Laboratories, Austria). Cultures were maintained in a

humidified incubator at 37 �C in an atmosphere of 5 %

CO2. Cytotoxic activity of GEL-1-5 loaded with or without

5-FU at various concentrations (0.1–1,000 lg ml-1) fol-

lowing 72 h of incubation was assessed using the 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

(MTT) (Sigma, Germany) assay, as described by Mosmann

1983 [20] but with minor modifications. Assay plates were

read using a spectrophotometer at 520 nm. Data generated

were used to calculate the percentage of viable cells.

2.14 Confocal microscopy

HCT116 cells were plated at a density of 5 9 105cells cm-2

in 6-well culture dishes. Hydrogels loaded with or without

5-FU were then used to treat the HCT116 cells for 72 h of

incubation. The cells were then detached using trypsin and

centrifuged down at 2,000 rpm for 5 min. The supernatant

was discarded and 1 ml of 70 % cold methanol was resus-

pended into the cell pellet for 10 min to fix the cells. Meth-

anol was discarded and the cell pellets were washed twice

with cold phosphate buffer saline (PBS). Finally, the pellets

were resuspended with 10 ll of anti-fading (Fluoroguard)

along with 10 ll of 10 lg ml-1 acridine orange/propidium

iodide stains. Freshly stained suspension was dropped into a

slide and covered by a coverslip. The viewing was conducted

using 488 and 532 nm laser lines. Finally cells were

observed under Confocal laser scanning microscopy (Leica

TCS SPE Confocal Microscope) using a FITC filter.

2.15 Statistical analysis

All experiments were conducted three times independently

with triplicates and results were expressed as

mean ± standard deviation. Statistical analysis was

undertaken using Prism 6.0 (Graph Pad Software, USA).

Mean and standard deviations (SD) were calculated for all

quantitative data. Prior to analysis, the D’Agostino and

Pearson omnibus normality test was performed to confirm

as to whether the data were well-modelled by a normal

distribution. The one-way analysis of variance (ANOVA)

was then utilized to assess the significance of cytotoxicity.

A probability of P \ 0.05 was considered to be statistically

significant.

3 Results

3.1 Fourier transform infrared spectroscopy analysis

and intrinsic viscosity

Synthesized copolymer hydrogels was characterized by

FTIR-ATR spectroscopy to elucidate the chemical struc-

ture of the gels. Figure 1 shows the FTIR spectra of

polymer hydrogels (GEL1-5) and homopolymers (PMAAc

and PMAETAC). The IR spectrum of PMAAc homopol-

ymer (Fig. 1a) showed characteristic intense peaks at

1,700, 2,929 cm-1 due to mC=O, mC–H stretching vibration

group in the homopolymer [21]. Another significant signal

was observed at 3,352 cm-1 due to C–O–H stretching of

hydroxyl groups. Absorption bands at 3,374, 1,714, and

1,024 cm-1 that can be attributed to –OH stretching,

methacryloyl group and –OH bending peaks in the homo-

polymer (PMAETAC) were observed. An appearance of a


123

new band responsible for asymmetric vibration signals of

carboxylate anions was obtained at 1,552 cm-1. After

copolymerization of the MAAc with MAETAC, a more

intense peak characteristic of the methacroyl carbonyl

group from GEL-1 emerged at 1,717 cm-1. A decrease in

intensity of the carbonyl peak (1,710 cm-1) in GEL-5 that

Fig. 1 a FTIR spectrum of

PMAAc and PMAETAC,

b FTIR spectrum of copolymer

hydrogels (GEL-1 and GEL-5)


123

may be due to the higher ratio of MAAc in the hydrogel

was also observed (Fig. 1b). Intrinsic viscosity of the

hydrogels was measured and it shows reduction with

increase in MAETAC concentration in the copolymers

(Table 1).

3.2 X-ray diffraction analysis

Wide angle X-ray diffraction patterns of respective ho-

mopolymers and copolymer hydrogels are illustrated in

Figs. 2a, b. The X-ray diffractogram of PMAAc showed

two broad peaks at 2h that equalled to 15.91�and 31.11�due to the amorphous nature of the homopolymer [22].

Contrarily X-ray diffractogram of PMAETAC affirmed

semicrystalline nature with a sharp peak at 2h that equalled

to 19.28o. The XRD pattern of the copolymer hydrogels

confirmed their amorphous nature. Broad peaks in the

range of 2h that equalled to 16.86�–30.92o were observed

in the X-ray diffractograms.

3.3 Thermal properties

DSC traces of corresponding copolymer hydrogels

(GEL1-5) are presented in Fig. 3. It is evident from the

DSC thermogram of GEL-1 copolymer that there is no

glass transition temperature peak. A sharp endothermic

inflexion appeared at 178.5 �C which can be attributed to

the melting transition in the copolymer hydrogel. Another

broad melting endotherm was found at 230 �C that may be

related to enhance intra-molecular interaction between the

carbonyl groups in the copolymer [23]. The DSC thermo-

gram of GEL-5 gel again did not show any glass transition

peak. The GEL-5 thermogram showed a reduction in the

melting transition peak and the endothermic peak was

centred at 167.9 �C. The increase in MAAc concentration

in the gel reduced the melting temperature peak.

The TGA thermogram of PMAAc homopolymer and the

respective copolymer hydrogels (GEL1-5) are presented in

Fig. 4. The TGA thermogram of PMAAc showed a two-

step thermal degradation pattern. The first thermal degra-

dation event was evident at 258 �C where the sample lost

27.69 % of its original weight. The second weight loss step

was observed at 424 �C mainly due to thermal decompo-

sition of PMAAc chain. Diez-pena et al. [24] also sug-

gested a two-step degradation mechanism in PMAAc and

proposed that on heating, PMAAc would be converted to

poly methacrylic anhydride (PMAN), a six membered

glutaric anhydride type ring. The thermogram of GEL-5

showed similar thermal degradation behaviour with a

multiple degradation pattern (Fig. 4). The copolymer

hydrogel started to thermally degrade at 213 �C and

eventually lost 24.92 % of its sample mass. This step may

be associated with loss of bound water in the copolymer

hydrogel. It is worthwhile to mention that PMAETAC is

hygroscopic in nature therefore; it readily absorbs water

from the surrounding [21]. The second thermal degradation

event took place at 401 �C where the sample rapidly lost

52.67 % of its original weight. This may be associated with

rapid decomposition of quaternary groups from PMAE-

TAC and the removal of pendent copolymer chains at

higher temperature. The residual char yield was 2.20 % at

798 �C as compared to 0.59 % in the case of PMAAc that

showed enhanced thermal stability of the copolymer

hydrogel.

3.4 pH sensitivity

When a covalently cross-linked network is immersed in an

aqueous solution, the fluid penetrates the macromolecular

chains at a certain rate and decreases until it reaches the

equilibrium [25]. The diffusion mainly involves inward

migration of solvent molecules into pre-existing spaces

between the polymeric chains. The effect of pH andFig. 2 a X-ray diffractogram of PMAAc and PMAETAC, b X-ray

diffractogram of copolymer hydrogels (GEL1-5)


123

monomer composition on equilibrium swelling (Qs) of

copolymer hydrogels is depicted in Fig. 5. The increase in

pH of the solution enhanced the swelling ratio of all

hydrogels. A sharp transition at pH 7.4 and maximum

equilibrium swelling ratio was noticed at this pH in all the

studied hydrogels.

3.5 In vitro 5-FU release

5-FU is an antimetabolite of the pyrimidine analogue type,

with broad spectrum of activity against solid tumors

including breast and colorectal cancer. Due to its unique

structure it interferes with nucleoside metabolism and can

be incorporated into DNA and RNA, resulting in cytotox-

icity and cell death [26]. It has been suggested previously

that 5-FU has short plasma circulation half-life due to rapid

mechanism in the liver [27]. Therefore, effective admin-

istration of 5-FU by continuous intravenous infusion is

very important in providing and maintaining the desired

dose levels. It was recently reported that hydrogel could be

used as a delivery vehicle for 5-FU and had been found to

effectively minimize toxic effects [28]. The percentage

cumulative release profile and the calculated diffusional

exponent (n) value from the copolymer hydrogels are

illustrated in Fig. 6 and Table 2 respectively. The drug

loading was calculated and optimum drug loading was

Fig. 3 DSC thermogram of

GEL-1 and GEL-5 hydrogels

Fig. 4 TGA thermogram of

PMAAc homopolymer and

GEL-5 hydrogels


123

observed in the GEL-5 (73.2 %). It was observed that

an increase in MAAc composition in the hydrogels leads

to an increase in the percentage loading of 5-FU.

Copolymer hydrogels showed a biphasic release pattern.

The initial stage of the release study showed that all

hydrogels exhibited burst effect up to one hour and almost

24, 28, 34, 40, and 44 % of the drug was released from the

hydrogels.

Fig. 5 pH sensitivity of

copolymer hydrogels

Fig. 6 Drug release profile of

5-FU from hydrogels in PBS

(pH 7.4) at 37 �C. Data shown

are mean value of ± SD

(n = 3)

Table 2 Analysis of cumulative release from copolymer hydrogels

Sample identity n k 9 103 R2

GEL-1 0.54 2.25 0.98

GEL-2 0.58 2.95 0.98

GEL-3 0.67 3.15 0.97

GEL-4 0.82 4.24 0.99

GEL-5 0.96 4.64 0.99


123

3.6 Morphology observation

The ESEM images of copolymer hydrogels forming three

dimensional matrix in the process of freeze drying are

illustrated in Fig. 7. The photomicrographs of the hydrogel

without freeze drying showed clear non-porous surface

morphology. However when hydrogels were swelled in SIF

buffer and subsequently freeze dried, samples showed well

defined three dimensional pores on the surface. It can be

observed that GEL-1 showed pores all over the surface of

the gel and on higher magnification (2,0009) the gel

showed open channel pores interconnected with each other.

We observed that the hydrogel composition had a signifi-

cant impact on porosity and interconnectivity of the gel.

When we increased the MAAc concentration in the gel

from 50 to 90 mol ratio, the gel became highly macropo-

rous allowing faster entrapment of biological fluids inside

its three dimensional matrix.

3.7 Cytotoxicity and anticancer evaluation

The IC50 of hydrogels loaded with 5FU against two human

colon cancer cell lines (HCT116 and HT29) and a non-

tumorigenic fibroblast cell line BJ (CRL2522) is shown in

Table 3. The results demonstrated that the drug released

from all except the GEL-5 remained biologically active

against HT29 (IC50 = 110–190 lg ml-1) and HCT116

(IC50 = 210–390 lg ml-1). It is also interesting to note

that the hydrogel did not show any cytotoxicity

(IC50 C 1,000 lg ml-1) against BJ (CRL2522). This most

probably is influenced by the population doubling time

between colon cancer and normal cells which results in

different metabolic profiles of the cell types. It has been

reported that the population doubling time of BJ

(CRL2522) is 48 h [29] while that of cancer cells (HCT116

and HT29) to be between 16 and 24 h [30] resulting in a

reduced pH (due to higher lactic acid production) in the

Fig. 7 Surface morphology of copolymer hydrogels (freeze dried and without freeze dried) at different magnifications (9500, 91,000, 92,000

and 95,000)


123

cancer cell environment when compared to the normal

cells.

Expression of apoptosis as shown through the live dead

cells assay of the HCT116 treated with 5FU-loaded

hydrogels or blank hydrogels using the AO/PI staining

method is shown in Fig. 8. Colon cancer cells that were

treated with 5FU-loaded hydrogels for 72 h showed cell

rupture, chromatin condensation, membrane blebbing and

the formation of apoptotic bodies, these are features of

cells undergoing apoptosis (Fig. 9).

4 Discussion

The copolymer hydrogels synthesized by free radical aqueous

copolymerization method were obtained as transparent

cylindrical shape gels. In order to determine the macromo-

lecular dimensions of the copolymers the intrinsic viscosity in

sodium chloride solutions was determined. Since the synthe-

sized copolymers were polyelectrolyte in nature evaluation of

the intrinsic viscosity was performed in ionic solutions. It is

believed that the presence of inorganic salts in solution sup-

pressed the polyelectrolyte nature, resulting in additional

swelling of the macromolecular chains upon dilution that did

not permit extrapolation of reduced viscosity values to zero

Fig. 8 Confocal images of HCT116 cells showing the morphology of a non-treated cells, b cells treated with hydrogel loaded 5-FU and c cells

treated with blank hydrogel, d Cells treated with hydrogel sample, respectively

Table 3 The IC50 value of HCT116, HT29 and BJCRL2522 cell

against hydrogel sample with 5-FU

Hydrogels loaded

with 5-FU

IC50 (lg ml-1)

HCT116 HT29 BJCRL2522

1 200 150 C1,000

2 210 110 C1,000

3 330 120 C1,000

4 390 190 C1,000

5 700 C1,000 C1,000


123

concentration eventually allowing measurement of the

intrinsic viscosity [31]. We observed that reduction in

MAETAC concentration in the copolymer reduced the

intrinsic viscosity value. This may be attributed to the

unfolding of macromolecular chains due to high charge den-

sity of PMAETAC polymer [31]. FTIR studies were con-

ducted to confirm the copolymerization reaction between

MAETAC and MAAc. It was observed that two important

peaks responsible for methyl component of quaternary

ammonium groups in PMAETAC and tetralchilic nitrogen of

METAC residues were found at 1,476 and 951 cm-1 in the

polymer hydrogels [32]. The FTIR results confirmed the in-

terpolymeric complexation and copolymerization reaction

between MAETAC and MAAc monomers.

WAXD studies were performed to study the crystalline

and amorphous nature of the hydrogels. Incorporation of

more PMAAc content in the hydrogel feed significantly

disturbed the ordered arrangement of the crystals, which

resulted in amorphous nature of the hydrogels [24]. We

hypothesize that since PMAAc crystals are relatively small

in nature; they cannot be incorporated in the regular

structure of the crystalline region forcing them out of the

crystal packing and aggregation in the amorphous matrix

surrounding the crystals [33].

DSC is a useful tool for measuring the temperature and

energy variation involved in phase transitions of copolymer

hydrogels. The DSC result showed absence of glass transition

peak in thermogram of GEL-1 and GEL-5. It was reported by

Diez-pena et al. that PMAAc is a polymer that is very labile to

temperature from structural point of view, because its Tg lies

closer to its first degradation step. Hence the authors used an

indirect plasticization method to determine the Tg of PMAAc

homopolymer and P (NIPAM-co-MAA) copolymers [33].

Since we synthesized these copolymer hydrogels without any

plasticizer it was difficult to estimate its Tg. Tm peak was

evident in both the hydrogels but it decreased with increased

MAAc ratio. This may be attributed to the smaller fraction of

crystalline phase and larger fraction of the amorphous phase in

the copolymer network, which are mainly related to the

molecular entanglement and interpolymeric complexation

between MAETAC and MAAc. TGA analysis revealed a

slight improvement in thermal stability of GEL-5 as compared

to PMAAc.

Study of transport of molecules, as the swelling behav-

iour in different media compositions, is vital in under-

standing delivery systems in biomedicine and other

applications [34, 35]. It was earlier reported that swelling of

the hydrogels can be influenced by pH of the medium,

especially when ionisable groups are present in their

structure [36, 37]. We studied the swelling response of these

polymer hydrogels in different pH medium. The maximum

swelling of the hydrogels could be correlated with the dis-

sociation constant (pKa value 5.5), which causes an

improvement in the water uptake of hydrogels. The pure

PMAAc with pKa value around pH 5.5 directly influences

the sharp increase in swellability of the hydrogels. It is an

established fact that under basic pH conditions the –COOH

group of the copolymer hydrogels experiences electrostatic

repulsion which causes expansion of the polymer chains

[33]. The swelling ratio reached to a maximum at pH 7.4,

however after which a decrease in Qs was observed [38].

We observed that pH dependent nature of the hydrogels is

due to the presence of dominant PMAAc segments in the

hydrogels. Contrarily a reduction in the pH of the aqueous

solution modified the degree of ionization. Therefore, it

reduces the net ion concentration difference (osmotic

swelling pressure) which resulted in dehydration of the gel

(decrease in the volume due to polymer elasticity) to an

extent where further compression is limited by the excluded

volume of the polymer chains [33].

As the aim for controlled release application the pre-

pared polymer hydrogels were loaded with 5-FU an anti-

cancer drug. The hydrogels attained equilibrium in 24 h for

all systems and maximum 5-FU release from these

hydrogels was found to be 53.2, 58.9, 76.7, 88.4, and

93.2 % respectively. Burst effect was evident in all the

hydrogels that was attributable to crystallization of 5-FU

on the surface of the xerogel. After the burst phase, drug

was released from the hydrogel in a more controlled and

sustained manner. We found that hydrogel composition has

a significant impact on the release of 5-FU. As expected the

increase in the MAAc concentration in the gel enhanced

5-FU release from the gel. This observation can be corre-

lated with the swelling profile of copolymer hydrogels in

the present study. We hypothesize that when the 5-FU

loaded hydrogels are placed in pH 7.4 buffer, the solvent

Fig. 9 Confocal images of HCT116 cells treated with hydrogel

loaded 5-FU showed apoptosis characteristics such as chromatin

condensation (CC), membrane blebbing (MB) and the formation of

apoptotic bodies (AB)


123

diffuses into the outermost surface such that the macro-

molecular chains in the drug loaded device undergo rapid

chain relaxation due to electrostatic repulsion in charged

ionic groups of the hydrogel. This produces extensive gel

swelling, followed by release of 5-FU through water filled

macropores of the exterior swollen core [39]. It was

reported that drug release from hydrogel matrices is mainly

driven by two forces: diffusion effect and degradation or

erosion of the hydrogel. In the case of pH sensitive

hydrogels, the permeability and release rate of the drug are

largely affected by the type of releasing agent and amount

of water in the hydrogel [40]. We compared our results

with previous literature on similar drug delivery systems.

Zhao et al. [41] prepared biodegradable, thermo and pH

sensitive poly(N-isopropylacrylamide-co-methacrylic acid)

and incorporated BSA as a model protein drug for con-

trolled delivery. The authors found that BSA release was

dominated by diffusion and the increase in the MAAc

content which significantly enhanced the protein release

from the hydrogel at pH 7.4. Liang et al. [42] prepared

thermoreversible hydrogels based on methoxy poly(ethyl-

ene glycol)-grafted chitosan nanoparticles for drug delivery

system and found that the initial burst release was noticed

from the nanoparticle hydrogel and after 28 h all 5-FU had

been released.

The drug release kinetics (F) from different matrices

were determined by Ritger and Peppas [43]

F ¼ Mt=M1 ¼ Kt n ð5Þ

where k is a constant representing the apparent release rate

(%) that takes into account structural and geometrical

characteristics of the release device and n is the diffusional

exponent. The value of n is very important for investigating

the drug release mechanism from different matrices. In the

case of non-swelling matrices, the drug release is generally

expressed by Fickian diffusion, for which n = 0.5. In the

case of swelling matrices, the release mechanism is gen-

erally governed by a combination of swelling and erosion.

They follow non-Fickian release mechanism, for which n

generally ranges from 0.5 to 1. For most erodible matrices

the drug release follows Zero order kinetics for which

n = 1. Occasionally a value of n [ 1 is observed in few

reports, which have been regarded as super case-II kinetics

[44]. The calculated value of n (0.54–0.96) showed that the

release of 5-FU through the hydrogel followed a non-Fic-

kian release mechanism. This confirms that swelling/ero-

sion plays a vital role in diffusion of 5-FU through the

copolymer hydrogels.

ESEM studies were conducted to investigate the surface

morphology of the freeze dried polymer hydrogels. It is a

well-established fact that porosity has multiple functions in

maintaining the biological diffusion of fluids as well as to

increase cellular proliferation in the re-epithelization

phase. The pore size of the hydrogels was observed to

increase from 50 to 80 mol ratios. However a reduction in

the pore size was noticed in GEL-5 which could be

attributed to the incorporation of more hydrophilic com-

ponent (MAAc). The swelling ratio and water content

increased, which led to the reduction of polymer fractions

in the hydrogel. Similar findings were reported by Si et al.

[45] from their study on the porous nature of poly(NIPAM-

co-AAc) microspheres. The authors suggested that an

optimum acrylic acid weight percentage led to decrease in

the pore size of poly(NIPAM-co-AAc) microspheres. It is

hypothesized that the network structure formed during

polymerization may assume different size micropores

structure, depending on the swelling degree change in

different surroundings. The unique 3D porous network is

responsible for higher transport of water inside the

hydrogel and hence the polymer networks are stretched and

present larger porous structure [46, 47].

The potential of these pH sensitive hydrogels as anti-

cancer drug (5-FU) carrier for targeted release in vitro

using colon cancer cells (HCT116 and HT29) was also

investigated. It was observed that the hydrogel composition

had significant impact on anticancer activity against cancer

cells. It is worthwhile to remember here that PMAAc has a

pKa value 5.6–7 that is close to the tumor extracellular

pH (5.7–6.9) [48], hence these hydrogels may be interest-

ing candidates for selective delivery of anticancer agents

(5-FU) to colon cancer cells. It was reported recently that

pH responsive polymers can be used as agents to avoid

intracellular barrier. Moreover it is believed that tumors,

diseased and inflammatory tissues have lower pH than

normal tissues. These pH variations can be easily exploited

to deliver the chemotherapeutic drugs to specific intracel-

lular sites [49]. Enhanced permeation and retention (EPR)

effect is also the key factor which improves the delivery of

5-FU by taking advantage of leaky blood vessels of tumor

cells [50]. This implies that GEL (1-4) copolymer hydro-

gels are pH dependent and able to release effectively in

cancer cells but not normal cells.

Confocal laser scanning microscopy was employed to

investigate the apoptosis mechanism in HCT116 cells. In

contrast, the morphology of HCT116 cells treated with blank

hydrogels was similar to that of control cells (media only)

which were regular in shape with cellular contents still intact.

This further affirmed that blank hydrogels did not show any

cytotoxicity against BJ (CRL2522) and were therefore safe

and non-toxic. However after loading with 5-FU the hydro-

gel showed indication of apoptosis in treated HCT116 cells

such as cytoplasmic shrinkage and membrane blebbing [51].

Microscopic observation revealed cell rupture, chromatin

condensation and presence of apoptotic bodies which cor-

roborated the programmed cell death mechanism in the

HCT116 cells.


123

5 Conclusion

New pH sensitive hydrogels were synthesized by a simple

free radical copolymerization method in an aqueous milieu

at near ambient temperature. FTIR-ATR spectroscopy

showed chemical interaction between the two monomers

with the positively charged MAETAC being successfully

copolymerized with MAAc. Wide angle X-ray diffraction

analysis confirmed the amorphous nature of the polymer

hydrogels. It is hypothesized that increase in MAAc ratio

disrupted crystalline domains and long range order of the

crystals which lead to the amorphous nature of the

hydrogels. DSC showed reduction in Tm of the hydrogels

with increase in MAAc which may be ascribed to larger

fractions of amorphous than crystalline phase enhancing

interpolymeric complexation between the polymers. The

positively charged polymer hydrogels exhibited excellent

sensitivity towards the surrounding pH. SEM investigation

showed three-dimensional porous architecture embedded

inside the hydrogel monolith and this was highly influ-

enced by MAAc ratio in the hydrogels. In vitro release of

5-FU was evaluated under SIF conditions and hydrogel

composition significantly influenced the release pattern.

Biphasic 5-FU release was noticed in all the hydrogels

characterized by initial rapid release phase (burst effect)

and sustained release phase. The burst release was evident

in all the hydrogel formulations and GEL-5 showed max-

imum percentage cumulative 5-FU release (93.2 %) after

24 h. The hydrogels were able to selectively release 5-FU

to human colon cancer cell lines (HT29 and HCT116) as

compared to healthy skin fibroblast cells (BJCRL2522). It

is believed that enhanced permeability and retention effect

(EPR) plays a key role in improving the cell killing by

taking advantage of the leaky tumor vasculature. Once

accumulated inside the tumor mass the 5-FU is released

through diffusion controlled manner. However no cell

killing was noticed in blank hydrogels. Confocal laser

scanning microscopy analysis of HCT116 cells treated with

5-FU loaded hydrogels showed well defined cell apoptosis

mechanism which is a key factor in cancer treatment. The

pH sensitive hydrogels were non-toxic against BJCRL2522

cells. Our result demonstrated that these pH sensitive

hydrogel carriers could be used for colorectal cancer

therapeutics delivery.

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