Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil

In the present study, the nanocomposite of alginate - modi fied bentonite has been synthesised for the controlled release applica- tions of anti-cancerous drug 5-FU.. The precursors used [r]

Original Article

Synthesis and characterization of a clay-alginate nanocomposite for

the controlled release of 5-Flurouracil

Department of Chemistry, Fatima Mata National College, Kollam, 691001, India

a r t i c l e i n f o

Article history:

Received 7 January 2019

Received in revised form

29 July 2019

Accepted 6 August 2019

Available online xxx

Keywords:

Bentonite

Controlled release

Nanocomposite

Kinetics

Alginate

a b s t r a c t

The scope of the present study is the synthesis and characterization of a nanocomposite based on natural bentonite clay and sodium alginate as a drug delivery system The nanocomposite was prepared by the grafted copolymerization of alginate, acrylamide and modified bentonite The characterization of the nanocomposite was carried out using FTIR, XRD, SEM, TG/DTA, Zeta potential, DLS and TEM analysis A hydrophilic anticancer drug 5-Flurouracil was chosen as the model drug to investigate the loading and release of the nanocomposite Swelling profile study revealed that maximum swelling was occurred at

pH 6.8 Thefitting of Peppas's kinetic model was analysed at pH 6.8 and the release kinetics was found to

be morefitted to Korsemeyer-Peppas kinetic model having R2¼ 0.9840 Human Colorectal Adenocar-cinoma cells-HT 29 was used for analysing cell viability The percentage of cell viability decreases from 46.65% to 20.12% when the concentration increases from 2.5mg/ml to 10mg/ml As an alternative to in-vivo models the chick embryo chorioallantoic membrane (CAM) study was conducted The study showed the better biocompatibility and non-toxicity of the nanocomposite

© 2019 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an

open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

1 Introduction

In cancer treatment, in order to eradicate tumour, the

thera-peutics must be delivered in high doses to ensure sufficient and

sustained therapy However, the sustained therapy in high dose is

causing damages to healthy tissues such as liver, kidney and bone

marrow along with the targeted cells Therefore it is desirable to

develop stimuli-responsive controlled drug delivery systems

(CDDS) CDDS work on the principle that drug is delivered only

on the exposure to external stimuli thereby reducing the

pre-mature release of drugs The study on controlled drug release has

been getting wide acceptance from the researchers due to its

main advantages such as high drug efficiency, continuous release,

and reduced side effects compared with conventional drugs in

dosage[1,2]

Recently preparation, characterization and applications of

controlled drug delivery materials prepared from biopolymer/

inorganic compounds have much sought after owing to their

peculiar properties such as biodegradability, controlled release

characteristics and high encapsulation efficiency [3,4] Most

hydrogels are prepared by the copolymerization of different vinyl monomers containing hydrophilic side groups with natural poly-saccharides as well as their derivatives Apart from various ad-vantages such as excellent biocompatibility, biodegradability and nontoxicity, they suffer from disadvantages of low strength This disadvantage can be overcome by using natural clays asfiller Clay minerals that predominantly have properties governed by smectites are called bentonites Montmorillonite is a major con-stituent of most bentonites (typically 80e90 wt%), the remainder being a mixture of mineral impurities including quartz, cristobalite, feldspar and various other clay minerals depending on the geological origin This group of clay minerals has a dioctahedral or tricotcahedral 2:1 layer structure, with isomorphous substitution that leads to a negative layer charge of less than 1.2 per formula unit Interlayer spacing varies between 10 and 15 Å and are generally dependent on the nature of the exchangeable cation and relative humidity Montmorillonites are dioctahedral smectites with layer charges predominantly in octahedral and tetrahedral sites, respectively The general formula of the montmorillonite group can be represented as (Mxþ)ex[(Si8)tet

(M(III)4-xM(II)x)octO20(OH)4]x, where Mþis the exchangeable cation pre-sent in the interlayer (e.g Naþ) and M(III) and M(II) are non-exchangeable octahedrally trivalent and divalent cations (e.g Al3þ and Mg2þ) respectively, and the layer charge is 0.5< x<1.2[5,6] The surface property of bentonite can be enhanced by introducing

* Corresponding author.

E-mail address: mdmullassery@gmail.com (M.D Mullassery).

Peer review under responsibility of Vietnam National University, Hanoi.

Contents lists available atScienceDirect Journal of Science: Advanced Materials and Devices

j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j s a m d

https://doi.org/10.1016/j.jsamd.2019.08.001

2468-2179/© 2019 Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license ( http:// creativecommons.org/licenses/by/4.0/ ).

Please cite this article as: R Surya et al., Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil, Journal of Science: Advanced Materials and Devices, https://doi.org/10.1016/j.jsamd.2019.08.001

silylated amino functional groups[7] Tuneable pore size is a

pre-requisite for the successful loading and the release of the drug,

the release kinetics may be slower than expected if the pore size of

the carrier is very small[8]

Clays exhibit many interesting features with active sites such

as hydroxyl groups, Lewis and Brønsted acidity, and

exchange-able interlayer cations In addition to the high aspect ratio of

clay minerals and the small dimensions of the individual layer

render them particularly attractive in several areas of material

science[9]

Clay minerals were proposed as fundamental constituents of

several modified therapeutic carriers and had different purposes

and acted through various mechanisms Although clay minerals

and polymers were often used in the pristine form as a single drug

carrier, they did not meet all the requirements The preparation of

polymer-layered silicate composite offered the possibility of

improving the properties of individual components Different

biodegradable and biocompatible polymers are suitable drug

car-riers which can release therapeutics at a constant, predetermined

rate[10] Alginate is a widely used mucoadhesive and

biodegrad-able natural polymer for the controlled release of drugs But the low

efficiency in trapping water-soluble drugs in alginate is one of the

problems for developing a drug delivery system[11] To improve

the drug entrapment efficiency and thereby modulating the drug

release it is desirable to incorporate water-insoluble materials like

bentonite Thus alginate-clay composite formed would decrease

the drug release by increasing the drug absorption capacity in the

composite matrix

Present study explores the utilisation of a nanocomposite

prepared by the copolymer reaction of alginate and natural

so-dium bentonite for the controlled release application of

5-Flurouracil

2 Materials and methods

2.1 Materials

Sodium alginate (SA), 5-Flurouracil (5-FU) and

ethyl-eneglycoldimethacrylate (EGDMA), fetal bovine serum (FBS),

3-(4,5-dimethythiazol-2-yl)2,5-diphenyl tetrazolium bromide

(MTT), Dulbecco's Modified Eagle's medium (DMEM), phosphate

buffered saline (PBS) were purchased from Sigma Aldrich

Bentonite clay was procured from Ashapura Clay Mines (India),

which was further converted to sodium bentonite (NaeB) by

immersing it in 1M NaOH Acrylamide (AM) was received from

Merck Life Solence Pvt Ltd Ceric ammonium nitrate (CAN),

DMSO and methanol were purchased from Merck Specialities Pvt

Ltd 3-Aminopropyltriethoxysilane (APTES) was procured from

Spectrochem Pvt Ltd NCCS, Pune, India supplied HT 29 cell line

Distilled water with specific conductivity of less than 1mScm1

was used throughout the study

2.2 Synthesis of the drug delivery system involves the following

steps

2.2.1 Preparation of 3-Aminopropyltriethoxysilane bentonite

(APSB)

About 5.0 g of NaeB was dried at 60C for 24 h It was dispersed

in 100 ml ethanol Separately a solution with 5.0 g of

3-Aminopropyltriethoxysilane (pur) was dissolved in 100 ml

sol-vent This solution was added to the dispersion containing sodium

bentonite The dispersion was stirred at 50 C under magnetic

stirring for 72 h After the complete stirring, the product was

centrifuged and was washed with distilled water, dried at 80C and

grained tofine powder

2.2.2 Preparation of alginate-modified bentonite nanocomposite (ABNC)

About 1.0 g of sodium alginate was dissolved in 100 ml water taken in a three-neck RBflask, which was equipped with a reflux condenser, magnetic stirrer, and a nitrogen line Nitrogen was purged into the solution for 30 min, after that the solution was heated to 60C To this, an appropriate amount of ceric ammo-nium nitrate was added After 10 min, a mixture of a suitable amount of acrylamide and modified bentonite (APSB) and the cross-linking agent EGDMA were added The above mixture was magnetically stirred for 2 h at 450 rpm and at 60C The pH was adjusted to 7.0 by using 1 M NaOH solution and was precipitated

by using a methanol-water mixture (5:1) The sample was dried at

60 C to a constant weight The product was milled and had a uniform particle size

2.3 Characterization The FTIR spectral analysis was performed to characterize the presence of functional groups present in NaeB, APSB, ABNC nanocomposite and the spectrum was recorded in the scanning range between 400 and 4000 cm1 using a Bruker-spectrophotometer (Germany) X-ray diffraction study was used

tofind out the crystallinity of a substance XRD measurement was carried out on a Rigaku Geigerflex X-ray diffractometer with Ni filtered Cu Karadiation at 40 kV, 20 mA and a diffraction angle of

2qscanning from 1to 100 The thermal analyses were made on

a Metler Toledo Star system under nitrogen atmosphere with a heating rate of 20C min1 A Philips model XL 30 CP scanning electron microscope (SEM) was used to take micrographs In this instrument, cryofreezing method was used for taking SEM pho-tographs at 15 kV and 20 kV with a working distance of 6 mm, in which frozen samples were coated with a thin layer of gold to make the surface conductive towards electron beam The samples were ultrasonicated (PCi Electronics, Mumbai, 230 V, 50 Hz) for a

definite period before the Dynamic Light Scattering (DLS) analysis and were performed by BI-200SM multiangle dynamic/static laser scattering instrument (Brookhaven, USA) The Zeta potential (z) of the samples was measured using Horiba SZ-100 equipped with a

532 nm Diode Pumped Solid State (DPSS) laser, operated at a temperature of 25C TEM images of the sample were recorded using JEOL-1200 TEM instrument

The cell viability of the prepared sample in HT 29 cell line was measured using MTT assay at different concentrations of 2.5, 5.0, 10.0, 20.0 and 40.0mg/ml A temperature-controlled water bath shaker (Lab line, India) with a temperature variation of±1.0C was used for the controlled shaking experiments

All pH measurements of the solution were carried out using a

pH meter (Systronics modelm362, India) The absorbance mea-surements of the 5-FU solution were performed on a UV-Visible spectrophotometer (Systronics, India) at 266 nm Accurate weights of samples were taken using electronic balance (Shimadzu, Japan)

2.4 Swelling study The swelling study was mainly carried out at two different physiological conditions of simulated gastric pH (1.2) and intestinal fluid pH (7.4) About 0.1 g of the nanocomposite was taken in a previously weighed tea bag and immersed in buffer solutions of pH 1.2 and 7.4 for 24 h at physiological temperature of 37C to attain the equilibrium The samples were then dried in an air oven at 50C until there was no change in the dried mass of the sample The percentage of equilibrium water uptake can be calculated as equation(1)

Swellingð%Þ ¼WwetWdry

2.5 Drug encapsulation studies

About 50 ml of 5-FU solution at a concentration of 5.0 mg/ml

was added to 0.1 g of ABNC in a stoppered bottle; the pH was

adjusted to 5.0 using phosphate buffer and stirred for 4 h at

1000 rpm The composite was further washed with distilled water

to remove the loosely bound drug molecules on the surface and

dried The drug encapsulation efficiency (DEE) was calculated using

the equation given below

DEE¼ðTotalamount of 5  FlurouracilÞ  ðFree 5  FlurouracilÞ

Total amount 5 Flurouracil

 100%

(2) The total amount of 5-Flurouracil indicates the initial

concen-tration of the drug and free 5-Flurouracil indicates the

concentra-tion of the drug in the supernatant after encapsulaconcentra-tion onto ABNC

The concentration (mg/mL) of 5-FU was measured by using the

UV-Visible spectrophotometer at 266 nm by comparing the value with

a standard calibration curve

2.6 In vitro drug release study

Release study of 5-FU from the ABNC was studied under two

different physiological pH conditions of 1.2 and 7.4 It is known that

pH values differ in different tissues and cellular compartments

within the human body such as that in the gastrointestinal tract

Most tumour tissues, as well as inflamed or wound tissues, exhibit a

pH that differs from the pH value of 7.4 found in normal tissues The

change in pH along the gastrointestinal tract from acidic to basic in

the intestine has been utilized to explore the pH-responsive

com-posite for oral drug delivery Therefore the swelling profile of the

composite in aqueous solution was carried out at two different pH

conditions of 1.2 and 7.4 by varying time intervals About 0.1 g of

5-FU loaded ABNC was placed in 100 ml of buffer solution at

physi-ological temperature At specific intervals, about 2 ml of the

su-pernatant solution was taken and the concentration of released

drug was measured using UV-Visible spectrophotometer at

266 nm

% of drug release¼ amount of drug released

Total amount of drug loaded 100 (3) The in vitro release kinetics was analysed using the

Korsmeyer-Peppas kinetic relation[12]equation(4)

Mt

where Mtis the amount of drug released at time t and M is the

amount of the drug released completely K is the rate constant and

n is the diffusion exponential

2.7 Cell line and cell culture conditions

HT 29e Human Colorectal Adenocarcinoma cells were taken as

the cell line The HT cells were cultured in DMEM, supplemented

with 10% FBS and kept at 37C in a humidified 5% CO2incubator

(NBS Eppendorf, Germany) The cells were trypsinized with

buff-ered saline solution contains 0.25% trypsin and 0.03% EDTA The

cells were plated to the culture plate for 24 h

2.8 Cytotoxicity analysis/MTT assay MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity which is based on the reduction of the yellow coloured water-soluble tetrazolium dye MTT to formazan crystals Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple colour, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570 nm Briefly, seeded using a

200ml cell suspension in a 96-well plate at a cell density of 20,000 cells per well without the test agent Allow the cells to grow for about

12 h Add appropriate concentrations of the test agent (2.5mg/ml,

5mg/ml, 10mg/ml, 20mg/ml and 40mg/ml) Incubate the plate for

24 h at 37C in a 5% CO2atmosphere After the incubation period, the plates are taken out from the incubator, the spent media is removed and MTT reagent is added to afinal concentration of 0.5 mg/ml of total volume Wrap the plate with aluminium foil to avoid exposure

to light Return the plates to the incubator and incubate for 3 h Remove the MTT reagent and then add 100ml of solubilisation so-lution (DMSO) Gentle stirring in a gyratory shaker will enhance the dissolution Occasionally, pipetting up and down may be required to completely dissolve the MTT formazan crystals especially in dense cultures Read the optical density on a spectrophotometer at 570 nm and 630 nm used as the reference wavelength The cell viability % can

be calculated by (Equation (5)) From the % of cell viability, it is possible to calculate the % of cytotoxicity (Equation(6))

Cell viability %¼Optical density of controlOptical density of test  100 (5)

Cytotoxicity %¼ 100 e cell viability % (6)

2.9 Chorioallantoic membrane (CAM) assay protocol The anti-angiogenic activity of the drug 5-FU loaded in the nanocomposite was analysed by CAM assay Fertilized chick embryos were collected from Kerala State Poultry Farm, Kudappanakunnu, Trivandrum (India) and were incubated in a humidified incubator at

37C After four days of incubation, a small window on the eggshell

of about 2 cm width was introduced Afixed concentration of the sample was prepared in PBS and introduced through the hole by a sterilized needle on the eighth day The window was tightly sealed by wax and the incubation was continued After eleven days, the eggs were taken out and the window was opened to see the formation of blood vessels and photographs were taken using a digital camera All experiments were conducted under the sterilized condition in order

to avoid the contamination[13] 2.10 Statistical analysis All the results were expressed as mean ± standard deviation (SD) Statistical analysis was performed with origin 8.0 (Origin eLab Corporation- USA)

3 Result and discussion 3.1 Synthesis and characterisation of the drug delivery system 3.1.1 Synthesis of the drug delivery system (ABNC)

In the present study, the nanocomposite of alginate - modified bentonite has been synthesised for the controlled release applica-tions of anti-cancerous drug 5-FU The precursors used for the Please cite this article as: R Surya et al., Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil, Journal of Science: Advanced Materials and Devices, https://doi.org/10.1016/j.jsamd.2019.08.001

synthesis of the drug delivery system are sodium alginate,

acryl-amide and silylated bentonite

During the synthesis of the DDS, one of the precursors, NaeB

was silylated using 3-Aminopropyltriethoxysilane in order to

in-crease the interlayer spacing NaeB was silylated by exchanging

intercalated water molecules with 3-aminopropyltriethoxysilane

The water molecules present in the ethanol medium catalysed the

interlayer surface silylation on NaeB[6]

During the first step of the synthesis, ceric ions induced the

sodium alginate (SA) through a redox initiation method Ceric

ammonium nitrate can act as redox initiator both in aqueous and

acidic conditions (~0.1 N HNO3)[14] Due to the redox reaction,

alginate is converted into corresponding alginate radicals These

macromolecular radicals of alginate initiate the acrylamide

monomer molecules followed by grafted copolymerization on the

alginate back bone In the presence of the cross linking agent

EGDMA, the crosslinked grafted copolymer was formed

In the next step, the layer structured inorganic filler of the

modified bentonite was introduced into the polymeric matrices

followed by the strong intermolecular hydrogen bonds between

theeOH group present in the clay and the electronegative groups

present in the cross-linked polymer By the alkaline hydrolysis

using NaOH, the amide groups present in the cross-linked polymer

was converted into carboxylate anions The scheme of preparation

of the nanocomposite was presented as supplementary

information S1

3.1.2 Fourier transform infrared spectroscopy (FTIR)

Fourier transfer infrared spectroscopy was one of the simplest

methods for the characterization and identification of functional

groups present in a compound Additional information regarding

the structure and the chemical bonds between chemical species

was obtained by carrying out by the infrared spectroscopy in

4000-400 cm1wavenumber range By comparing the IR spectra of the

starting materials with the final modified composite, it was

possible to ascertain whether the required modification was

incorporated

The FTIR spectra of NaeB, APSB, ABNC and 5-FU-loaded ABNC

are shown inFig 1 In NaeB, the band at 3630 cm1corresponds to

the stretching vibrations of the hydroxyl group in MgeOHeAl and

AleOHeAl The bands at 3420 cm1and 1628 cm1are due to the

stretching vibrations and bending vibrations of the HeOeH bonds

of water molecules intercalated in the clay minerals[15] There is

an intense band at 980 cm1present in both NaeB and APSB due to

the SieO stretching in SieOeSi in clay materials In APSB there are

two bands observed at 2930 cm1 and 2860 cm1, due to the

stretching asymmetric and symmetric vibrations ofeCH2groups

And also the two bands observed at 1555 cm1and 1490 cm1are

corresponding to the bending vibrations of eNH2 and eCH2

respectively[16]

Sodium alginate showed asymmetric and symmetric stretching

vibrations at 1627 and 1415 cm1, due to the carboxyl anion and the

peak at 1034 cm1 was due to oxygen stretching in cyclic ether

bridge The presence of a broadband at around 3450 cm1

corre-sponds toeOH stretching vibrations[11]

From the spectrum of ABNC, it can be observed that the

chem-ical structure of ABNC is similar to alginate, which is the major

fraction in its composition The eOH stretching vibration gets

decreased in intensity than that of pure alginate and also it gets

shifted to 3462.2 cm1, indicating the participation ofeOH groups

in the composite formation Band corresponds to 1059 cm1

in-dicates the formation ofeCH-O-CH2during the grafting The strong

band at 1047 cm1is due to SieOeSi stretching which is found to

be more sharp with a decrease in intensity may be due to the better

participation ofeOH groups on the clay Presence of bands at 1459

and 1552 cm1 are due to the symmetric stretching and asym-metric stretching modes ofeCOO-group The FTIR spectrum of the drug-loaded composite material resembles the superimposition of the spectra corresponding to the drug and to the ABNC This simi-larity between the spectra indicates the interaction between the drug and the composite is rather weak

3.1.3 X-ray diffraction (XRD)

Fig 2shows the XRD pattern of NaeB, APSB, ABNC and 5-FU-L-ABNC The XRD pattern of NaeB showed a characteristic (001)

reflection peak at 2q¼ 6.89corresponding to the d-spacing value

of 13.02 A

̊

(Fig 2) Silylation of NaeB had resulted in the shift of d001

of Na-bentonite to lower 2q value from 6.89 to 4.17, which had resulted in an increase in the basal spacing d001from 13.02 Ato 21.15 A[16] The peak at 2q ¼ 20.63 Awas attributed to (002)

reflection[17] Interaction of alginate with silylated bentonite had not resulted in any change in the XRD pattern of silylated bentonite, but the spectra was broadened due to the dispersion of clay into the polymeric matrix The spectra of the drug-loaded system was again

Wavenumber (cm-1)

400 900 1400 1900 2400 2900 3400 3900

Na-alginate APSB ABNC 5-Flurouracil 5-FU-L-ABNC

Fig 1 FT-IR of Na-alginate, APSB, alginate-clay nanocomposite, 5-Flurouracil and 5-Fu-L-nanocomposite.

5-FU-Loaded-nanocomposite Alg-clay nanocomposite

APSB

Na-B

2 Theta (degree)

Fig 2 XRD of NaeB, APSB, Alg-clay nanocomposite, and 5-Fu-Loaded- nanocomposite.

broadened, indicated the loading of drug molecules into the

poly-meric system[18]

3.1.4 Thermo analytical techniques (TG/DTA)

Fig 3shows the thermal analysis of ABNC and 5-FU-L-ABNC

Alginate powder DTA curve showed that three weight loss events at

252 C, 374 C and 450 C which are associated with alginate

decomposition The weight loss above 600 C may due to the

destruction of the carbon skeleton

The drug-loaded composite showed four weight-loss events at

50C, 260C, 342C and 464C The initial weight loss at 50C was

due to the loss of physisorbed drug weakly bonded on the surface

and absorbed water The second weight loss at 269C was due to

the drug decomposition The third weight loss at 342C may be due

to the strongly bonded drug molecules The weight loss at 464C

may due to the alginate decomposition The decrease in % weight

loss at a higher temperature for the composite was 1.613%, whereas

for alginate the decrease in percentage was 2.93% This fact

attri-butes to the extra thermal stability of the composite compared to

the alginate

3.1.5 Zeta potential analysis

The stability of the colloidal particles depends on the magnitude

of zeta potential[19] Dispersion of particles with zeta potential

higher thanþ30 mV or lower than 30 mV is considered as stable

Zeta potential of modified clay, APSB is slightly positive in an

aqueous medium The slightly positive charge means the overall

instability of the nanoparticles Due to the lack of stabilization, the

particles may aggregate to form a precipitate and later they

dispersed in water[20].Supplementary information S2shows the

zeta potential of ABNC and 5-FU-L-ABNC ABNC shows the zeta

potential value of39.8 mV indicates the better stability of

nano-composite The high negative zeta potential of the composite is due

to the repulsion betweeneCOO-groups on the surface After the

loading of 5-FU, the zeta potential is changed to45.4 mV, implied

the successful loading of the drug molecule with appropriate

sta-bility It was reported that 5-FU was negatively charged[21] The

encapsulation of negatively charged drug molecule may cause

electrostatic repulsion with the anionic part of the composite and

which may lead to change in the zeta potential to45.4 mV

3.1.6 Particle size determination of the nanocomposite

Particle size determination can be done by dynamic light

scat-tering (DLS), transmission electron microscopy (TEM) and field

emission scanning electron microscopy (FE-SEM) analysis It was

observed that the particle size by DLS analysis was larger than that

obtained from TEM because DLS gave the hydrodynamic size of the

particles The nanostructure of the ABNC composite was confirmed

by TEM analysis (Fig 4) The particle size of the DDS was calculated with the help of IMAGE J software Using the software scale the image with the magnification mentioned (200 nm) and then picking the particles using the selection tool to get the area of a single particle Then after selecting a minimum of 10 points the software will calculate the average, maximum and minimum area

of the entire particle From the area obtained the average radius and diameter of the particles can be calculated For the minimum area

of 4910 nm2, the diameter obtained was 80 nm and for the maximum area of 14,577 nm2, it showed a diameter of 136 nm For the average value of 8988 nm2, the particle diameter was found to

be 106 nm Thus from the TEM data using the IMAGE J software the approximate diameter lies in the range of 100 nm for the DDS particles

From the TEM analysis of the drug delivery system, the average diameter of DDS was around 100 nm The DLS of nanocomposite and 5-FU-nano composites were 115.1 nm and 228.5 nm indicating encapsulation of the drug (S3) From SEM analysis, the increase in particle size from the precursor to thefinal product gives a positive evidence of successful synthesis of nanocomposite and loading of 5-FU (Fig 5) In SEM, each and every sample can be distinguished based on their surface morphology

3.2 Drug encapsulation study

Fig 6shows the encapsulation profile at variable pH conditions The maximum drug encapsulation efficiency was found to be at pH 5.0 (89.8%) This may due to the fact that at pH 5.0 the nano-composite can be appropriately swollen So the drug molecules can enter the inner gallery where it can form H-bond or electrostatic attraction with the modified clay Better encapsulation was observed within the pH of range 4.5e5.5 At very low pH conditions the swelling of the composite was extremely low therefore the entry of drug molecules into the DDS was limited Also at pH from 7

to 10, the encapsulation efficiency was found to be too low Since at alkaline pH condition 5-FU attains mono or di-anionic tautomeric form[22,23] This may lead to anioneanion repulsive interaction and preventing the entry of drug molecules into the drug delivery system

3.3 Swelling kinetics and swelling capacity of the nanocomposite

It was reported that the swelling kinetics of a nanocomposite gel depends on the variable factors such as swelling capacity, the particle size of composite and the composition of the composite

[24].Fig 7depicts the swelling capacity of the composite under

-1.991 -1.491 -0.991 -0.491 0.009 0.509 1.009

60 70 80 90 100

Weight % (%) Derivative Weight % (%/m

Temperature (°C)

5-Fu -L-Nanocomposite

-3.243 -2.743 -2.243 -1.743 -1.243 -0.743 -0.243 0.257

50 60 70 80 90 100

Weight % (%) Derivative Weight % (%/m

Temperature (°C)

ABNC

Fig 3 TG/DTA of alginate-clay nanocomposite and 5-Fu-L- nanocomposite.

Please cite this article as: R Surya et al., Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil, Journal of Science: Advanced Materials and Devices, https://doi.org/10.1016/j.jsamd.2019.08.001

variable pH conditions by taking stock solutions of NaOH and HCl.

During the study, it was found that the swelling ratio of the

com-posite was found to increase with the increase in pH After pH 3.0

the swelling profile was changed This may due to the ionization of

eCOOH group leading to anioneanion repulsion [25] The maximum swelling was found to be at pH 6.8, after that there was a gradual decrease in the swelling ratio due to screening effect of excess sodium ions, which may reduce the anioneanion repulsion Fig 4 TEM of alginate-clay nanocomposite.

Fig 5 FE-SEM images of NaeB, APSB, alginate-clay nanocomposite and 5-Fu-L-nanocomposite.

[26] It was reported that the swelling behaviour of the polybasic

composite was influenced by the composition as well as the pKa of

the carboxylic group present[26,27] In order to attain the better

swelling of the composite, the pH of the medium should be greater

than the pKa of the carboxylate groups in the composite Moreover,

alginate polymer composite has the tendency to attain the

maximum swelling nearly at neutral pH conditions than at acidic/

alkaline pH The pKa of alginic acid lies between 3.4 and 4.4 and at

above pH 5.0 most of the carboxylic groups get protonated Above

the pH of 7.4, the swelling of the composite was found to be

retarded due to the screening effect Therefore, the maximum

swelling was occurred within the pH range of 6.5e7.0 The size of

the composite also influences the swelling index Nanocomposite

can absorb more and more water molecules due to the large surface

area, so the nanogels can swell easily

In order to study the dependence of the composition of the

nanocomposite gel on the swelling kinetics, various proportions of

precursors were taken From the studies, it was observed that the

swelling property of the composite was increased with increase in

the concentration of SA It was observed that, by varying the

con-centration of SA from 0.1 to 0.9 g, the swelling capacity of the

com-posite was getting increased by ~10 times Further, an increase in the

concentration of SA would decrease the swelling capacity This was

because SA dispersion in water resulted in the increase in viscosity

hence it was very difficult for the monomer units of acrylamide to

reach the active sites But on increasing the amount of the clay the swelling of the composite was found to decrease due to the more cross-linkage with the polymer The optimum amount of SA: NB was approximately 1: 1 And also the concentration of the crosslinking agent had a profound effect on the swelling % It was seen that the cross-linking of the nanocomposite increased with increase in the concentration of EGDMA, but there was a possibility for lowering the absorption of water by the polymer due to the high extent of cross-linking This tendency was not at all favouring the better swelling property of nanocomposite gel The optimisation of the concentra-tion of crosslinking agent was determined by taking variable con-centrations of EGDMA and studied its influence in the swelling and encapsulation properties of the composite and the results were presented astable in S4 From the table, it could be shown that the optimised concentration of EGDMA for better swelling behaviour was found to be 2.5 mol/l The equilibrium swelling of the nano-composite was attained within 30 min The maximum swelling un-der optimum conditions (AAme 4.83 mol/l, SA- 0.0462 mol/l and EGDMA- 2.5 mol/l) was found to be 280 g/g at pH¼ 6.8

3.4 In vitro release study

Fig 8shows the in vitro release study carried out at two different physiological pH conditions of simulated gastric pH of 1.2 and simulated intestinal pH of 7.4 The pH-responsive nature of alginate leads the scientist to develop new drug delivery systems using alginate as the starting material The in vitro release studies of 5-FU from alginate-chitosan-MMT has been reported by Azhar et al

[28] In that study, 5-FU was loaded in MMT and further it was coated with alginate followed by chitosan; with the maximum release of drug was occurring at pH 7.4, and the time taken for 50% (I50) release was 8 h Iliescu et al.[11]reported a DDS of alginate-MMT composite beads for the release of the drug irinotecan According to them, the MMT-drug hybrid was synthesized and was again coated with alginate Maximum release of drug was occurring at pH 7.4

In the present study, the maximum release of drug was occur-ring at pH 7.4 About 81.5% of drug release was occuroccur-ring within

12 h, with the I50of 6 h In our study, we synthesized a grafted hybrid of alginate and modified bentonite and further it was loaded with 5-FU From the swelling and in vitro release study of the nanocomposite, it was clear that the swelling profile of the com-posite had a direct influence on the in-vitro release The maximum swelling of the composite occurred at pH 6.8 (~7.0) and the in-vitro release occurred at a pH of 7.4, this may due to the peculiarity of alginate component in the nanocomposite The equilibrium swelling was attained within 60 min and about 35% of drug release

0

20

40

60

80

100

Temperature = 37 oC

pH

Fig 6 Encapsulation of 5-Flurouracil at different pH (triplicates for each sample

were analyzed and each datum point represents the mean value ±standard

devia-tion; n ¼ 3).

150 175 200 225 250 275

6 6.2 6.4 6.6 6.8 7

pH

0 50 100 150 200 250

1 2 3 4 5 6 7 8 9 10

pH

Fig 7 Swelling study of the alginate e clay nanocomposite at variable pH conditions (triplicates for each sample were analyzed and each datum point represents the mean value

±standard deviation; n ¼ 3).

Please cite this article as: R Surya et al., Synthesis and characterization of a clay-alginate nanocomposite for the controlled release of 5-Flurouracil, Journal of Science: Advanced Materials and Devices, https://doi.org/10.1016/j.jsamd.2019.08.001

was occurring within 1 h This is a good evidence for the influence

of swelling property on in-vitro release kinetics

The in-vitro drug release study was analysed using

Korsmeyer-Peppas kinetic equation (Fig 8) In Peppas equation, the value of

k gives the idea about the interaction of drug on to the drug

de-livery system Smaller the value of K, weaker is the interaction

between the drug and drug delivery system[12] The value of n

determines the release mechanism If the value of n  0.43, the

drug release is mainly due to the diffusion of drug known as Fickian

diffusion If the value of n¼ 0.85, the drug release is mainly due to

the swelling of the polymer and if the value between is 0.43 and

0.85, it accounts for the non-Fickian diffusion In the non-Fickian

diffusion, the release kinetics is controlled by both the diffusion

and swelling process The fitting of Peppas's kinetic model was

analysed at pH 7.4 and the release kinetics was found to be more

fitted to Korsemeyer-Peppas kinetic model having R2¼ 0.9840 The

value of n¼ 0.6223 means, the release of the drug follows

non-Fickian mechanism The release of the drug depends both on the

diffusion of drug and the swelling of the composite The value of

K¼ 0.0250, indicates the weaker interaction between the drug and

the drug delivery system The release of the drug may due to both

the swelling of alginate covering and also due to the diffusion of

drug from the modified clay

3.5 Cell viability assay (MTT assay) The cell viability was analysed by MTT method using HT 29e Human Colorectal Adenocarcinoma cells, in which it was incu-bated to 48 h under variable concentration of 2.5 mg/ml to 40.0mg/ml The % cell viability of above 80 was considered as cytocompatible and non-toxic[29] The % of cell viability of the drug delivery system was not below 80% even at higher con-centration of 40.0mg/ml The % of cell viability was found to be decreased with increase in the concentration Fig 9 represents the profile of cell viability assay and the % of cell activity (cell toxicity/cell viability) of the drug delivery system In the case of the drug-loaded composite, the % of cell viability was decreased from 46.65% to 20.12% when the concentration was changed from 2.5mg/ml to 10mg/ml This was due to the fact that the % of cell toxicity was increased by the factor of 26.53%, when the concentration was increased 4 times The IC 50 value means the drug concentration required to achieve 50% inhibition time, which is found to be 10.21mg/ml for the 5-FU-loaded composite and 18.64% for Flurouracil Due to the larger IC 50 value of 5-FU-loaded composite than the free 5-Flurouracil, the cellular drug release may be better for 5-FU-loaded composite than pure drug

0

10

20

30

40

50

60

70

80

90

pH =7.4

pH = 1.8

Time (min)

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2

0

y = 0.6223x - 1.6018

R2 = 0.9840

n = 0.6223

K = 0.0250

Log (time) in minute

Fig 8 In-vitro release profile of 5-Flurouracil from alginate-clay nanocomposite and the kinetic profile fitted with Korsmeyer-Peppas kinetic model ((triplicates for each sample were analyzed and each datum point represents the mean value ±standard deviation; n ¼ 3).

Fig 9 The % of cell viability of DDS (alginate-clay nanocomposite), Drug (5-Flurouracil) and Drug-L- DDS (5-Fu-L-nanocomposite And the % of activity of alginate-clay nano-composite in HT 29 e Human Colorectal Adenocarcinoma cells cell line.

3.6 In vivo study model- CAM assay

Angiogenesis is the development of new blood capillaries from

pre-existing capillaries and post-capillary venules[30] Among the

different kinds of angiogenic diseases, tumour growth and

metathesis is the most drastic and dangerous one A tumor

stim-ulates the blood capillaries to enhance its own growth and

spreading A permanent remedial to prevent the tumour growth is

the retardation of blood vessels Drugs like 5-FU generally called

anti-angiogenic drugs can prevent the tumors from growing their

blood vessels So the tumour can be starved by blocking oxygen and

nutrients

The chick embryo chorioallantoic membrane (CAM) is an

extraembryonic membrane with dense capillary blood vessels

CAM is a better alternative of animals in vivo models to study both

angiogenesis and antiangiogenesis Thefirst in vivo angiogenesis

study using CAM was reported in 1913 There are so many

advan-tages of CAM than the mammalian models such as low cost, easy to

carry out, reproducibility and easy availability of materials, etc[31]

During the analysis, three sets of eggs were taken for the study

of 1) Controlled 2) ABNC and 3) 5-FU-loaded ABNC The image of

the controlled study shows the angiogenic nature of CAM as shown

inFig 10 The growth of blood capillaries are so dense and can be

visualized clearly Similarly, there was no drastic differentiation

between ABNC and the control It shows the better biocompatibility

and non-toxicity of the nanocomposite By examining the images of

5-FU-loaded nanocomposite, the blood vessels are found to be

much more retarded indicating the potent drug release by the

nanocomposite without any premature leakage

4 Conclusion

Alginate-modified bentonite nanocomposite (ABNC) was

syn-thesized by combining the copolymer of alginate-acrylamide using

EGDMA as crosslinking agent followed by silylated bentonite

(APSB) for the controlled release of 5-FU The composite was

characterized by FTIR, XRD, SEM, and thermal analysis Swelling

studies of the composite proposed that the maximum swelling of

the composite occured at a pH of 6.8 The profile of the

encapsu-lation study suggests that the encapsuencapsu-lation efficiency gets

decreased with the increase in pH The release kinetics is controlled

by both the diffusion and swelling process Thefitting of Peppas's

kinetic model was analysed at pH 6.8 and the release kinetics was

found to be morefitted to Korsemeyer-Peppas kinetic model having

R2¼ 0.9840 The cell viability was analysed by MTT method using

HT 29e Human Colorectal Adenocarcinoma cells In the case of the drug-loaded composite, the % of cell viability was decreased from 46.65% to 20.12% when the concentration was changed from 2.5mg/

ml to 10 mg/ml This indicated that the % of cell toxicity was increased by the factor of 26.53%, when the concentration was increased 4 times The chick embryo chorioallantoic membrane (CAM) study is a better alternative to animals in in-vivo models The study showed the better biocompatibility and non-toxicity of the nanocomposite By examining the images of the 5-FU-loaded nanocomposite, the blood vessels are found to be much more retarded indicating the potent drug release by the nanocomposite without any premature leakage

Acknowledgments The authors are expressing sincere gratitude to The Head, Department of Chemistry, Fatima Mata National College, Kollam for providing laboratory facilities The corresponding author thanks UGC, New Delhi for financial assistance in the form of Minor Research Project (2324-MRP/15e16/KLKE015/UGC-SWRO) The authors sincerely acknowledging the services rendered by Indian Institute of Science, Bangalore for their assistance in the charac-terization of the samples

Appendix A Supplementary data Supplementary data to this article can be found online at

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