34

CHAPTER 2

INVESTIGATION OF ANTIBIOTIC AND

ANTICANCER DRUG RELEASE FROM NANO

HYDROXYAPATITE AND AGAROSE COMPOSITES

2.1 INTRODUCTION

Nowadays, most of the people are affected by bone diseases such as

cancer, fracture and osteomyelitis (infection). Osteomyelitis is an

inflammation of the bone or bone marrow, usually caused by various

microorganisms such as bacteria and fungi. These microorganisms infect the

injuries and spread to the bones and adjacent areas through the blood stream

(Pineda et al 2006). There are several types of treatment for osteomyelitis and

cancer. They are chemotherapy, free tissue transfers, bone grafts, antibiotic

loaded biocompatible materials (beads, paste and solid) etc. This kind of

infections is a major problem in medical field due to poor accessibility of the

infected site by systemically administered antibiotics (Hendriks et al 2004).

For this reason, we require efficient and controlled manner of local drug

delivery system. From the last decade onwards, a lot of research is going on

to develop the novel drug storage and release systems. Nano biomaterials are

drug carriers that have greater efficiency, safety, biocompatibility and

effective relief from pain. They have a better therapeutic response due to

controlled and prolonged release (Di Silvio and Bonfield 1999). In recent

days, a large number of systems have been employed as local drug delivery

systems, such as biodegradable polymers (Di Silvio and Bonfield 1999),

35

xerogels (Yang et al 2002), hydrogels (Lin and Metters 2006) mesoporous

silica (Manzano and Vallet-Regi 2010), calcium phosphate cement

(hydroxyapatite) (Vallet-Regi et al 2006) and polymer composites

(Schnieders et al 2006).

Semisynthetic, orally absorbed broad spectrum antibiotic drug,

amoxicillin (AMX) has been extensively used against bacterial infections.

Slow and continuous release of drug during bone implantation is essential to

prevent infectious diseases. The possession of such property by HAp, other

calcium phosphates, porous HAp blocks and HAp coating on metals was

reported in literature (Joosten et al 2005, Kim et al 2005). 5-Fluorouracil

(5-Fcil) is an antineoplastic drug which is used in the treatment of cancer. It

is an acidic, water soluble and hydrophilic drug, used in the chemotherapy for

the treatment of solid tumours (Santos et al 2009).

Hydroxyapatite [(Ca10 (PO4)6(OH)2), HAp] is one of the major

inorganic compound of bone and teeth. Nanosized HAp has considerable

potential as remodeling implants, prosthetic bone replacement and for protein

and drug delivery systems (Nair et al 2008). Surface modified nanosized

HAp with different morphology (sphere, rod, needle and plate) can exist in

mesoporous form. They are used as efficient drug carriers for the local drug

delivery of a variety of pharmaceutical molecules because of their

biocompatibility, osteoconductivity, nontoxicity and non-inflammatory

properties (Vallet-Regi et al 2006). But, HAp is very brittle and could not be

used for load bearing bone replacements. The powder and block forms of

HAp ceramics are used as defect and crack fillers in bones. Powder ceramics

are best for filling small and irregular defects. However, there will be

migration of HAp particles from the implants. It is difficult to handle and

keep the implant compactly in the defect site (Holmes and Hagler 1987).

36

Thus, it is necessary to mix a suitable binder with the HAp granular material

to overcome these problems.

Agarose is a natural, thermally responsive polysaccharide that is

widely used in biological sciences (e.g., microbial cultivation and gel

electrophoresis) (De Lange et al 2011). It acts as gelling agent leading to

strong gels and allows fast room temperature polymerization. It is a linear

polymer that behaves like a hydrogel (Cabanas et al 2006). The hydrogel is a

biomaterial having physical properties similiar to that of living tissues. This

resemblance was due to its high water content, soft rubbery consistency and

low interfacial tension with lower decomposition temperature (Roman et al

2008). It is widely used as scaffold for hard tissue implants and drug delivery

systems.

HAp composites have attracted much attention because of its better

biocompatibility, mechanical properties and bone bonding ability. The

presence of HAp in the composite material improves the growth of osteoblast

cells, resulting to better osteoconductivity (Liu et al 1998). Recently,

composites of ceramics with natural degradable polymers could be used as

bone fillers (Verheyen et al 1993). Composites based on degradable

biopolymers such as collagen (Hsu et al 1999), fibrin glue, gelatin, chitosan,

alginate and hydroxy-propyl-methyl cellulose with inorganic powders were

used as bones (Sivakumar and Rao 2003). Tabata et al (2003) reported that

HAp/agarose composites showed better healing nature than pure HAp.

HAp/agarose scaffolds have been investigated in combination with stem cells

for generating a variety of replacements of cartilage, heart and nerves. Studies

have demonstrated the suitability of agarose scaffolds for promoting stem

cells to differentiate into chondrocytes (Xu et al 2005). In this chapter, the

synthesis of nanosized HAp/agarose composite powder by sol-gel method is

discussed. In this method, the pH of the sol-gel was maintained constant using

37

pH stat that was followed by microwave treatment. The effect of calcination

process and their biological and drug release behavior were investigated.

2.2 EXPERIMENTAL METHODS

2.2.1 Material Preparation

The powder form of nanosized HAp/agarose composite was

prepared by ethanol based sol-gel technique followed by microwave

treatment. Calcium nitrate tetrahydrate (Ca(NO3)2.4H2O, Merck), diamonium

hydrogen phosphate ((NH4)2HPO4, Merck), agarose (SRL), ethanol (Merck)

and ammonia solution (Merck) were used for the synthesis without further

purification. 0.3 M diammonium hydrogen phosphate was dissolved in

250 mL of ethanol and heated to about 85 ± 5 °C and subjected to vigorous

stirring. Then, 1 wt % of agarose was added to phosphate solution. 0.5 M

calcium nitrate tetrahydrate was dissolved in 250 mL of ethanol which was

added to phosphate/agarose solution at constant flow rate (2 mL/min) under

continuous stirring. After mixing, the resulting sol was subjected to vigorous

stirring for 3 h. The pH of the solution was maintained at 10.5 using ammonia

by pH stat (Radiometer analytical) (Figure 2.1) at constant temperature (85 ±

5 °C) during the entire reaction process. After one day aging, the resulting

sol-gel was washed with deionized water. The sol-gel was centrifuged at 3000

rpm using micro centrifuge (Eltek, Model: Labspin Tc 4815S) equipment and

dried at 70 °C using digital vacuum oven (Insref, Model-IRI-021). Before

aging a portion of the prepared sol-gel was subjected to microwave treatment

at 900 W for 30 min using domestic microwave oven (Whirlpool,

model:magicook, 900W). The final product was washed with deionized water

for the removal of organic solvent and excess/unreacted nitrate substance and

dried at 70 °C using hot air oven. The obtained powders were subjected to

calcination at 700 °C for 2 h. After calcination, the colour change was

observed on the powders from brown to white, due to the removal of agarose.

38

Experimental flow chart for the preparation of HAp/agarose composite is

shown in Figure 2.2. Hereafter, the samples as-synthesized, as-synthesized

calcined at 700 °C, microwave treated and microwave treated calcined at 700

°C are referred as SAS, SAS700, SMWT and SMWT700, respectively.

Figure 2.1 pH stat used for the preparation of nano HAp/agarose

composite

Figure 2.2 Experimental flow chart for the preparation of nano

HAp/agarose composite

Sol-gelpH=10.5 maintained by pH stat

0.5 M Ca(NO3)2.4H2O+ Ethanol

0.3 M (NH4)2HPO4 + EthanolStirring and heating at 85 °C

As-synthesized Microwave treated

Calcined 700 °C (2 h)

1 wt % Agarose

39

2.2.2 Characterization

Powder X-ray diffraction was measured using Siemens D500

diffractometer. The SAS, SAS700, SMWT and SMWT700 samples were

analyzed using CuK radiation source ( = 1.5406 ) with 40 kV and 20 mA.

The pattern was recorded in the range of 5 to 70 with increment steps of

0.02 /s. Fourier transform infrared (FTIR) spectra were recorded in the range

of 400 to 4000 cm-1 with Perkin-Elmer spectrum RXI FTIR system using KBr

pellet technique. Surface morphology and the elements present in the

samples, were studied by scanning electron microscope coupled with energy-

dispersive X-ray spectroscope (SEM-EDX) ESEM Quanta 400 FEG, FEI;

gold–palladium [80:20] - sputtered samples; EDX detector: S-UTW-Si (Li)

(Mondejar et al 2007). The specific surface area was determined by the

Brunauer-Emmett-Teller (BET) method using micromeritics (model-ASAP

2020 V3.00 H) surface area analyzer. The pore size and volume were

obtained from the BJH absorption/desorption isotherm.

2.2.3 In vitro Dissolution Study

Phosphate buffer saline (PBS) was prepared by dissolving the

chemicals in deionized water by the following order NaCl, KCl, KH2PO4,

NH2HPO4 and its pH was adjusted to 7.4 using HCl. The SAS, SAS700,

SMWT and SMWT700 samples were made into pellet of 8 mm diameter and

1 mm thickness. These pellets were immersed into 20 mL of PBS in plastic

containers incubated at 37 °C for different time intervals (one to four weeks).

Finally, the weight difference between before and after soaking into PBS were

measured using weighing (Sartorius) balance with an accuracy of ± 0.01 mg

at the end of the experiment (fourth week (W)). The pH of the dissolution

medium was measured after removing the samples (1W, 2W, 3W and 4W)

from the PBS solution.

40

2.2.4 In vitro Bioactivity Study

In vitro bioactivity of SAS, SAS700, SMWT and SMWT700

samples were investigated to form bone like apatite on the surface using

simulated body fluid (SBF). It was prepared using reagent grade chemicals

dissolved in the order of NaCl, NaHCO3, KCl, Na2HPO4.2H2O, MgCl2.6H2O,

CaCl2.2H2O, Na2SO4, TRIS buffer and 1M HCl in deionized water. Finally,

HCl was used to adjust pH 7.4 at 37 °C (Kokubo and Takadama 2006). All

the samples were made into a pellet (8 mm diameter and 1 mm thickness)

with constant weight (100 mg) and it was soaked into 20 mL of SBF in plastic

container at 37 °C for a period of one to four weeks. The solution was

renewed once in two days and it’s pH was measured using pH meter. After

incubation, the samples were gently washed with deionized water and dried at

37 °C for analysis. The samples were weighed using weighing (Sartorius)

balance with an accuracy of 0.01 mg before and after soaking in SBF. Apatite

deposition on the surface of the pellet was investigated by SEM.

2.2.5 Haemolysis Test

Fresh human blood was collected in a sterile centrifuge tube which

contains heparin to avoid the clot formation. 100 mg of SAS, SAS700,

SMWT and SMWT700 of pellets were equilibrated by 1 mL of sterile saline

and incubated at 37 ºC for 12 h. After incubation the saline was removed.

Then, 250 µL of human blood was added on the pellets and incubated for 20

min. Finally, 5 mL of saline was added on each sample to stop the haemolysis

and it was incubated for 1 h. The positive and negative controls were

produced by adding 250 µL of human blood in distilled water and saline

incubated at 37 ºC for 1 h. All the samples were centrifuged at 1000 rpm for

5 min. The absorbance (optical density) of the supernatant solution was

recorded at 545 nm using UV-Vis spectrophotometer (Shimadzu, UV-1601). The

41

percentage of haemolysis was calculated as per the formula and their values

were plotted in the graph.

[OD (test) OD (negative control)]Haemolysis (%) = × 100 [OD (positive control) OD (negative control)]

The accepted norm of haemolysis percentage is (i) Highly

haemocompatible (<5 % haemolysis), (ii) Haemocompatible (within 10 %

haemolysis) and (iii) Non-haemocompatible (>20 % haemolysis) (Pal and Pal

2006).

2.2.6 Drug Loading/Release

In vitro drug release experiment was carried out on antibiotic

(Amoxicillin, AMX) and anticancer (5-Fluorouracil, 5-Fcil) loaded HAp

pellets. AMX maximum absorption wavelength ( max) was 230 nm obtained

with the help of UV-Vis spectrophotometer (Shimadzu UV-1601). The

standard graph was obtained by plotting concentration versus absorbance.

A best linear fit with regression of 0.9998 was taken for the drug release

calculation. For, 5-Fcil maximum absorption wavelength ( max) was 265 nm

and the standard graph regression value was 0.9994.

The SAS, SAS700, SMWT and SMWT700 samples were mixed

with AMX and 5-Fcil in the ratio 1:0.5 using mortar and it was made into

8 mm pellet using hydraulic press with constant pressure (2 tons). The drug

loaded pellets were immersed in 200 ml of PBS with pH 7.4 in a conical flask

with shaking speed of 100 rpm at 37 °C using incubator-cum-orbital shaker

(Niolab). From the dissolution medium, 1 mL of AMX and 5-Fcil solutions

were removed at various time intervals and it was replaced by equal volume

of PBS. The quantity of drug release was determined using

UV-Vis spectrophotometer ( max = 230 nm for AMX and 265 nm for 5-Fcil).

42

All samples were performed in triplicate and its mean value of the data was

used to plot the graph.

2.2.7 Antimicrobial Activity Test

The bactericidal effect of SAS, SAS700, SMWT and SMWT700

with AMX drug loaded pellets were investigated using Gram negative

bacteria Escherichia coli (E.coli, MTCC 2939) and Gram-positive bacteria

Staphylococcus aureus (S. aureus, MTCC 3381), Staphylococcus epidermidis

(S. epidermidis, MTCC 3382). A qualitative diffusion disk test was carried

out using 105 colony forming units (CFU/mL) of the E.coli, S. aureus and

S. epidermidis. The culture was added to the plate and it was spread

uniformly on Muller-Hinton agar plates. After spreading, same size (8 mm

diameter) of without and with AMX loaded SAS, SAS700, SMWT and

SMWT700 were placed on the culture plate. The plates were incubated at

37 °C for 24 h. The inhibition zone was noticed and it was measured every

3 h and the image was documented.

2.2.8 Cell Viability Test

Human osteoblast-like cell line was derived from human

osteosarcoma (MG-63 cells obtained from NCCS-Pune). MG-63 cells were

cultured in 25 cm2 cell culture flask at 37 °C in Dulbecco’s Modified Eagles

Medium (DMEM) (Invitrogen, USA) supplemented with 10 % fetal bovine

serum (DMEM) (Invitrogen, USA) and 1 % penicillin and streptomycin in a

5 % CO2 incubator. When the cells had grown to confluence, they were

detached by trypsin/EDTA (0.05 % w/v trypsin/0.02 % EDTA) (Sigma) and

used for cell culture study on HAp samples. The SAS, SAS700, SMWT and

SMWT700 samples (pellet form with 8 mm diameter and 1 mm thickness)

were kept into a 48-well plate (Axygen, California). All the samples were

sterilized before starting the biocompatibility study. The grown MG-63 cells

43

were seeded (density of 5000 cells/cm2) on all samples with five trials for

each and compared with induced toxic agent (TritonX). It was incubated for

one day to allow cell adherence at 37 °C in 5 % CO2 atmosphere. Then,

50 µL of MTT reagent (5 µg/mL) was added to each well and incubated for

4 h at the same condition. Finally, 500 µL of dimethylsulfoxide (DMSO)

(Sigma-Aldrich) was added for dissolving the formasan crystals and

absorbance was measured at 570 nm in an ELISA (Thermo scientific) reader.

The cell culture medium was used as a negative control and a TritonX toxic

agent as a positive control.

2.3 RESULTS AND DISCUSSION

2.3.1 XRD Analysis

The XRD patterns of SAS, SAS700, SMWT and SMWT700 were

as shown in Figure 2.3(a-d). All the patterns were in good agreement with

JCPDS data (09-0432) corresponding to pure phase of HAp. The SAS

showed broad peaks of low intensity indicating the presence of nanosized

particles (Figure 2.3(a)). When, it was subjected to calcination the intensity

increased, well resolved and reduction in peak broadening was found on

SAS700 sample (Figure 2.3(b)). Increase in crystallinity and crystallite size

was due to the calcination process and decomposition of agarose. The

intensity of the planes (211), (112), (100), (101), (200) and (111) of the

SMWT increased (Figure 2.3(c)) compared to SAS sample. In addition,

SMWT700 also showed enhanced intensity (Figure 2.3(d)) and it was similar

to the SMWT.

The crystallite size (L) was calculated from XRD spectrum using

Scherrer formula for all samples. L = K 1/2 cos , Where, K is a constant as

0.9, is the X-ray wavelength (1.5406 Å), is the full width at half maximum

and is the diffraction angle at the particular plane (Rusu et al 2005).

44

Crystalline planes of the samples were indexed and its full width at half

maximum was analyzed by XRDA software (Desgreniers and Lagare, 1994).

The crystallite size was calculated for all crystalline planes in the XRD

patterns of the samples and its average values are tabulated (Table 2.1).

Compared to the XRD patterns of SAS700 and SMWT700 samples, the

microwave treated sample (SMWT) appeared to have higher crystallinity and

less crystallite size than calcinated samples. During calcination, temperature

might not be uniform through out the matrix, but it is uniform during

microwave treatment. It might be the reason for higher crystallinity during the

microwave treatment. The crystallite size of SAS and SMWT was less than

SAS700 and SMWT700 samples. Further, the crystallinity was examined by

the relation, Xc = 1-(V112/300 I300) where I300 is the intensity of (300) plane and

V112/300 is the intensity of the peaks appearing between (112) and (300) (Rusu

et al 2005, Landi et al 2000).

Table 2.1 Lattice parameters, average crystallite size and crystallinity

of SAS, SAS700, SMWT and SMWT700

Samples

codeLattice parameters

Average

crystallite size

(± 1 nm)

Crystallinity (± 2%)

Xc =

1-(V112/300/I300)

KA =

z ×(Xc)1/3

SAS a and b = 9.44±0.04 (Å)c = 6.87±0.01 (Å)V = 530.69±1 (Å3)

38 90 88

SAS700 a and b = 9.30± 0.02 (Å)c = 6.86±0.01 (Å)V = 513.55±2 (Å3)

45 98 97

SMWT a and b = 9.42 ± 0.02 (Å)c = 6.87 ± 0.01 (Å)V = 528.61±1 (Å3)

33 95 98

SMWT700 a and b = 9.42 ± 0.01 (Å)c = 6.87 ± 0.01 (Å)V = 528.75±1 (Å3)

43 98 98

45

10 20 30 40 50 60 70

41

35

11304

50

22

14

33

1

31

332

2

00

44

10

32

12

13

31

222

220

31

13

3113

10

301

202

3001

12

21

12

10

10

2

00

2

11

1

20

0

10

1

10

0

Two theta (degree)

(a)

(b)

41

35

11

304

50

22

14

331

31

33

220

04

41

032

12

13

3122

22

203

11

3

31

131

0

30

120

23

001

12

21

121

01

02

00

2

111

200

101

100

(c)

413

51

13

04

50

221

433

1

31

33

220

04

41

032

121

33

122

22

20

31

13

3113

10

3012

02

30

01

12

211

210

10

200

2

11

12

00

10

1

10

0

Inte

nsity (

a.u

)(d)

41

3

511

30

45

02

21

43

31

31

33

2200

44

10

321

21

331

2222

20

31

13

31

131

0

30

120

230

011

2

211

21

01

02

00

2

11

12

00

10

1

100

Figure 2.3 XRD patterns of (a) SAS (b) SAS700 (c) SMWT and

(d) SMWT700

Crystallinity has increased in the order of SAS, SMWT, SAS700

and SMWT700 samples (Table 2.1). A verification can be done with

empirical relation between Xc and z ie. z × (Xc)1/3 = KA, Where Xc is the

degree of crystallinity, z the full width at half maximum of (002) plane in

(degree-2 ), KA is a constant at 0.24 (Rusu et al 2005, Landi et al 2000). In

both method of calculation, almost same crystallinity has been found for all

samples.

46

2.3.2 FTIR Analysis

The FTIR spectra of SAS, SAS700, SMWT and SMWT700

samples were as shown in Figure 2.4 (a-d). In SAS sample, the high intense

broad envelope between 2600 and 3750 cm-1 was ascribed to the OH stretch

of hydroxyl groups along with the NH stretch of NH4+. A sharp peak at 1386 cm-1

was assigned to C-O-C stretch of the agarose and N-O stretch of NO3- (Anee

et al 2003, Sanchez-Vaquero et al 2010). The bending mode of water was

observed at 1638 cm-1. The sharp band at 1034 cm-1 was assigned to the

triply degenerate asymmetric P-O stretching mode ( 3). The low intensity

peak at 962 cm-1 was due to non-degenerate P-O symmetric stretching mode

1). The peak at 825 cm-1 was assigned to NO3- bending mode. The peak at

637 cm-1 was attributed to hydroxyl stretch vibration. Well resolved peaks at

606 and 562 cm-1 were attributed to the triply degenerate of O-P-O bending

mode ( 4). The doubly degenerate of O-P-O bending mode was observed at

475 cm-1. SAS spectrum provides the evidence for the formation of

HAp/agarose composite (Figure 2.4(a)).

For SAS700 sample, the intensity of peak decreased at region

between 2600 and 3750 cm-1. The reduction of peak intensity at 1638, 1386

and 3467 cm-1 was good evidence for the loss of water, decomposition of

agarose and NH4+, respectively. Compared to SAS spectrum, the sharp peak

observed at 3570 cm-1 was attributed to hydroxyl stretch in HAp crystal. The

phosphate peaks at 1034 and 637 cm-1 were resolved which confirmed the

improved crystallinity (Guo and Xiao 2006). In addition, the low intensity

peaks at 1460 and 1415 cm-1 were attributed to the carbonate group vibration.

It might be due to agarose decomposition and chemisorption of atmospheric

CO2 on the HAp during the thermal treatment (Kuriakose et al 2004).

47

4000 3500 3000 2500 2000 1500 1000 50087

58

25

82

5

14

60

14

15

56

260

66

37

56

26

066

37

56

26

06

637

96

29

62

96

2

10

34

10

34

10

34

10

98

109

810

98

13

83

138

6

16

38

16

38

16

38

20

40

20

40

20

40

236

723

67

23

67

35

70

35

70

357

0

378

83

78

837

88

34

67

318

5

378

8

23

67

20

40

163

8

13

86

10

98

103

4

96

2

637

60

6

(d)

(c)

(b)

(%)

Tra

nsm

itta

nce

Wavenumber (cm-1)

(a)

56

2

Figure 2.4 FTIR spectrum of (a) SAS (b) SAS700 (c) SMWT and

(d) SMWT700

A low intense peak at 1383 cm-1 was assigned to N-O stretch of

NO3- and agarose which confirmed the presence of agarose gel after

microwave treatment (Figure 2.4(c)). Compared to SAS sample, the peak at

1383 cm-1 was of low intense indicating the decomposition of agarose during

microwave treatment. In addition, the reduction in intensity of phosphate

peaks at 606, 562 cm-1 and water molecule vibration at 637 cm-1 were due to

loss of phosphate group from the crystal lattice. The absence of agarose peak

at 1383 cm-1 indicates the decomposition of agarose gel on microwave

treatment (Figure 2.4(d)).

48

2.3.3 SEM and EDX Analysis

SEM micrographs and EDX of SAS, SAS700, SMWT and

SMWT700 were shown in Figure 2.5(a-d). The nano rods of length 10 to 100 nm

and width 5 to 20 nm were observed on SAS sample (Figure 2.5(a)). SAS700

showed agglomerated particles with non-uniform interconnected pores (100 to

150 nm), due to the decomposition of agarose and nitrate (Figure 2.5(b)). For

SMWT, the size of HAp/agarose rods (length 5-70 nm and width 5-30 nm)

were found to reduce compared with SAS sample. When SMWT was

subjected to calcination at 700 °C (SMWT700), irregular pores (100-250 nm)

with continuous network was observed. The aspect ratio (length/diameter)

was 2 to 5 and 1 to 2.3 for SAS and SMWT samples respectively. All the

samples were subjected to elemental analysis using SEM-EDX. Atomic Ca/P

ratio of SAS, SAS700, SMWT and SMWT700 was found to be 1.66, 1.68,

1.54 and 1.73 respectively (Table 2.2). Microwave treatment may produce

the calcium deficient HAp which was shown in EDX data of SMWT sample.

This may be due to loss of calcium during microwave treatment.

Table 2.2 The elemental data analyzed by EDX

SamplesWeight (± 1%) Atomic (± 1%) Ca/P

ratioCa P O C Ca P O C

SAS

SAS700

SMWT

SMWT700

32.79

36.98

31.65

43.37

15.21

16.99

15.93

19.38

43.37

37.15

43.62

28.45

8.62

8.88

8.79

8.79

17.26

20.36

16.58

25.65

10.37

12.11

10.80

14.83

57.22

51.23

57.25

42.16

15.15

16.31

15.37

17.36

1.66

1.68

1.54

1.73

49

(a)

(b)

Figure 2.5 SEM micrographs with EDX spectrum of (a) SAS (b) SAS700

(c) SMWT and (d) SMWT700

50

(c)

(d)

Figure 2.5 (Continued)

51

2.3.4 BET Analysis

The results of nitrogen adsorption/desorption isotherms of SAS,

SAS700, SMWT and SMWT700 samples were as shown in Figure 2.6(a-d).

All samples showed type IV isotherms and the typical H1-hysteresis loops,

demonstrating the properties of typical mesoporous materials (Haber 1991).

The BET surface area, pore volume and pore size of all samples were

presented in Table 2.3. SAS contain agarose and its surface area was 52 %

higher than SAS700. Microwave treated (SMWT) sample showed surface

area nearly to the SAS. SMWT700 showed 47 % decrease of the surface area

compared to SMWT sample. The variation in surface area for SAS and

SAS700 samples was also reflected in their pore volume. Similarly, SMWT

showed pore volume more than SMWT700. The pore of 17-34 nm diameter

was observed in all the samples. SAS sample showed lower pore size than

other samples. This is due to the presence of higher amount of agarose which

was mixed with HAp and it was confirmed by high intensity peak of agarose

(1386 cm-1) in FTIR spectrum (Figure 2.4(a)). The nanosized pore of

HAp/agarose composite could be useful for drug delivery as well as to enhance

the bone bonding ability.

Table 2.3 Pore size, volume and surface area for SAS, SAS700, SMWT

and SMWT700

Sample

code

Pore size (Dp)

(± 1 nm)

BET Surface area (SBET)

(± 1 m2/g)

Pore volume (Vp)

(± 0.002 cm3/g)

SAS

SAS700

SMWT

SMWT700

17.17

25.28

24.01

34.28

47.795

25.045

41.692

19.709

0.323

0.277

0.450

0.205

52

0.0 0.2 0.4 0.6 0.8 1.0

0

50

100

150

200

0

50

100

150

2000

100

200

3000

50

100

150

(a)

Qua

ntity

Adsorb

ed (

cm

3/g

)

Relative Pressure (P/Po)

(b)

(c)

(d)

Figure 2.6 N2 adsorption/desorption isotherms of (a) SAS (b) SAS700

(c) SMWT and (d) SMWT700

2.3.5 In vitro Bioactivity Study

In vitro bioactivity of SAS, SAS700, SMWT and SMWT700

samples were tested for a period of one to four weeks. The pH of the SBF

decreased initially from 7.4 to ~7.2±0.05 in one week for SAS and SMWT

samples, indicating the partial dissolution of the samples. Subsequently, pH

increased to ~ 8.1±0.05 in four weeks, suggesting the formation of apatite

layer on the pellets when immersed in SBF. SAS700 and SMWT700 samples

showed no decrement in pH of SBF solution. SBF soaked SAS and SMWT

showed 8 and 10 mg of weight loss in the first week. The weight loss occurs

due to the dissolution of agarose in SBF solution from SAS and SMWT

samples. No weight loss was observed on SAS700 and SMWT700 samples.

All samples showed gradual increment of weight up to four weeks (after one

week of immersion in SBF) (Figure 2.7). It means that apatite layer was

formed over the surface of the pellets. The weight increment of SAS, SAS700,

53

SAS SAS700 SMWT SMWT700

0

20

40

60

80

100

120

1W

4W4W4W

3W3W3W 2W2W

2W

1W

1W0W0W0W4W

3W2W

1W

We

igh

t (m

g)

0W

SMWT and SMWT700 was 2, 16, 14 and 15±1 mg, respectively. The SAS and

SMWT samples showed initial resorbablity followed by bioactivity.

Figure 2.7 Weight difference of SAS, SAS700, SMWT and SMWT700

before and after soaking in SBF (W represent week)

Micrographs of all samples before soaking in SBF solution showed

heterogeneous surface (Figure 2.8(a), (c), (e) and (f)). After soaking the

samples for two weeks in SBF, it exposed the spherical apatite along with

micro pores formation on the surface of SAS (Figure 2.8(b)). Spherical apatite

particles (1-5 µm) were observed on the entire pellet surface for all samples

(Figure 2.8(b), (d), (f) and (h)). Apatite formation increased with an increase

in soaking time. Finally, the dense layer of apatite was observed on the SAS,

SAS700, SMWT and SMWT700 samples. SMWT enhanced the apatite

formation compared to SAS sample and it was evident from the weight

difference graph (Figure 2.7).

54

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Figure 2.8 SEM micrographs of (a) SAS (b) after SBF soaked SAS

(c) SAS700 (d) after SBF soaked SAS700 (e) SMWT (f) after

SBF soaked SMWT (g) SMWT700 and (h) after SBF soaked

SMWT700

55

Figure 2.9 Mechanism of apatite formation on the surface of all samples

Figure 2.9 illustrates the mechanism of apatite formation on the

surface of HAp/agarose composite. The process of formation of apatite

depends on the negativity of the surface, i.e. PO43- and OH- constitutes the

negative group, while Ca2+ constitutes the positive group of HAp

(Figure 2.9(a)). The more the PO43- and OH- are exposed on the surface, the

greater the negativity of the surface. These negative ions attract the positive

Ca2+ ions from the SBF, thereby forming a layer of positive charge i.e.

calcium-rich amorphous calcium phosphate (ACP) (Figure 2.9(b)). Now the

layer becomes positive with respect to the surrounding SBF, which is enough

to attract negative PO43- and OH- from the SBF and thereby forming calcium-

poor ACP (Figure 2.9(c)). This gradually crystallizes to bonelike apatite. The

grown bonelike HAp appears to stabilize their surfaces in SBF (Figure 2.9(d))

(Kim et al 2005).

56

SAS SAS700 SMWT SMWT700

0

1

2

3

4

5

6

7

8

9

10

4W

4W

3W

3W

3W

4W

3W

2W

2W

2W

2W

1W

1W

1W

We

igh

t (m

g)

1W

0 1 Week 2 Week 3 Week 4 Week

0.0

0.1

0.2

0.3

7.2

7.3

7.4

pH

SAS

SAS700

SMWT

SMWT700

2.3.6 In vitro Dissolution Study

The in vitro dissolution of SAS, SAS700, SMWT and SMWT700

samples were investigated in PBS. The experiment was carried out in

triplicates for each sample. The pH of the PBS was gradually decreased from

7.4 to ~7.1±0.05 for all samples which was measured periodically for four

weeks (Insert of Figure 2.10). This indicated that partial dissolution takes

place from the pellets surface. The weight loss indicated overall dissolution of

the samples. The SAS showed higher dissolution compared to other samples

due to the presence of agarose and nitrate. The weight loss of SMWT was

40 % less than SAS sample. This may be due to the variation of agarose

quantity in SMWT sample.

Figure 2.10 Weight loss graph of SAS, SAS700, SMWT and SMWT700

(Insert pH variation) during the dissolution test

57

(d)

(b)

(c)

(a)

The SEM micrographs of SAS and SMWT before and after soaking

in PBS were shown in Figure 2.11(a-d). Before dissolution test, the surface of

SAS and SMWT showed small sized pores. SAS and SMWT samples showed

rough surface along with increase in pores size (Figure 2.11(b) and (d)). There

was no significant change on the SAS700 and SMWT700 pellet surface.

Figure 2.11 In vitro dissolution studies using PBS (a and b) before and

after dissolution of SAS, (c and d) before and after

dissolution of SMWT

2.3.7 Haemolysis Test

The percentage of haemolysis was less than one for all samples

(Figure 2.12). The results suggested that all samples were haemocompatible,

therefore it could be used for implantation, wound-dressing or local drug

delivery system (Pal and Pal 2006).

58

Figure 2.12 Percentage of haemolysis for SAS, SAS700, SMWT and

SMWT700

2.3.8 In vitro Drug Release

The cumulative percentage of AMX (antibiotic) release profiles

from SAS, SAS700, SMWT and SMWT700 samples as a function of release

time in PBS were as shown in Figure 2.13(a). SAS showed initial burst

release of about 60 % within 10 h, due to the dissolution of agarose in PBS.

Subsequently, controlled drug release was observed. The slow release was

attributed to the strong interaction between mesoporous surface of HAp and

the drug. SMWT released 20 % of drug at 1 h then it showed controlled

release. Each hour, it released 2 to 3 % of the drug, the controlled and

extended drug release was noted upto 125 h (100 %). When compared with

SAS, SMWT had low surface area and large pore size. However, SMWT

sample showed controlled and extended drug release. This may be due to high

crystallinity and reduction of particle size. SAS700 and SMWT700 samples

showed 77 and 92 % rapid release within 30 h. The controlled release was

extended upto 152 h for SMWT700 sample (Figure 2.13(a)). This is due to

high crystallinity of HAp particles which was produced by microwave

treatment along with calcination (Melville et al 2008).

SAS SMWT SAS700 SMWT700

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

(%)

of h

aem

oly

sis

59

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

0

20

40

60

80

100

(%)

of cum

ula

tive A

MX

re

lease

T ime (h)

SAS

SAS700

SMW T

SMW T700

0 20 40 60 80 100 120 140

0

20

40

60

80

100

(%)

of cu

mula

tive

AM

X r

ele

ase

T ime (h)

SMW T

(a)

0 10 20 30 40 50 60 70 80 90 100

0

20

40

60

80

100

Time (h)

(%)

of cum

ula

tive 5

-Fcil

rele

ase

SAS

SAS700

SMW T

SMWT700

0 20 40 60 80 100

0

20

40

60

80

100

Time (h)

(%)

of cu

mu

lative

5-F

cil re

lea

se

SMWT

(b)

Figure 2.13 In vitro drug release of (a) AMX and (b) 5-Fcil drug

(Insert SMWT drug release profile)

The cumulative percentage of drug release profiles for the 5-Fcil

(anticancer) drug loaded SAS, SAS700, SMWT and SMWT700 samples were

shown in Figure 2.13(b). Rapid release, followed by controlled release was

60

observed. SAS, SAS700, SMWT and SMWT700 samples showed burst

release of 79, 84, 87 and 95 % respectively at 10 h. 100 % of release was

observed on SMWT700 within 36 h. SAS700 released 95 % at 25 h then it

showed stable value upto 50 h. SAS and SMWT showed controlled release of

96 and 98 % at 80 h respectively. Gradual increase in the percentage of drug

release upto 95 h on SMWT sample was observed. The SAS and SMWT lead to

a significant decrease in 5-Fcil release. It indicates a stronger bonding of

5-Fcil to SAS and SMWT sample due to the presence of agarose. In addition,

high surface area and low pore size also contributes to the decrease in drug

release. Prolonged 5-Fcil drug release for cancer chemotherapy could

probably circumvent drug resistance which is an important impediment in

cancer therapy.

2.3.9 Antimicrobial Activity

AMX loaded samples exhibited good inhibitory effect against

S. aureus, S. epidermidis and E. coli (Table 2.4). There was no inhibition zone

around without drug incorporated samples. From these results, it is concluded

that amoxicillin drug possess resistance against S. aureus, S. epidermidis and

E.coli species. The efficacy against E. coli showed highest zone of inhibition for

all samples when compared with S. aureus and S. epidermidis (Figure 2.14).

Table 2.4 The zone of inhibition of drug incorporated samples against

E. coli, S. epidermidis and S. aureus

Samples

Diameter of inhibition zone (± 1 mm)

E.coli S. aureus S. epidermidis

9h 12h 15h 24h 9h 12h 15h 24h 9h 12h 15h 24h

SAS 45 47 52 55 38 40 41 42 45 47 52 55

SAS700 48 49 51 54 40 40 41 41 50 51 51 53

SMWT 47 50 53 55 40 42 43 44 41 45 45 49

SMWT700 49 49 51 52 45 46 46 46 45 49 50 53

61

(a)

(b)

(a) (a)

(b) (b)

(c) (c) (c)

(d) (d) (d)

E. coli

E. coli

E. coli

E. coli

S. aureus

S. aureus

S. aureus

S. aureus

S. epidermidis

S. epidermidis

S. epidermidis

S. epidermidis

Figure 2.14 Inhibition zone of without and with AMX loaded (a) SAS

(b) SAS700 (c) SMWT and (d) SMWT700 samples against

E. coli, S. aureus and S.epidermidis

62

SAS SAS700 SMWT SMWT700

0.0

0.2

0.4

0.6

0.8

1.0

Op

tica

l d

ensity

2.3.10 Cell Viability

The osteoblast behavior on the HAp/agarose composite was

investigated using MTT test. The percentage of viable cells was greater than

70 % for all the samples (Figure 2.15). The results suggested that there was

no toxicity on SAS, SAS700, SMWT and SMWT700 samples.

Figure 2.15 Cell viability study of SAS, SAS700, SMWT and SMWT700

2.4 CONCLUSION

Nano rods of HAp/agarose composite (10-100 nm) was synthesized

by sol-gel technique at low temperature with constant pH followed by

microwave treatment. HAp/agarose composite was confirmed by XRD and

FTIR. Microwave treated SMWT showed increase in crystallinity and

decrease in particle size (30 %) compared to the SAS. The calcined samples

showed interconnected porous structure which could help osteointegration

when used as an implant. The presence of pores allows the circulation of

physiological fluid to help the new bone formation which further improves

the mechanical fixation of the implant at the implantation site. Further, the

porous bioceramics could be used for local drug delivery system. The specific

63

surface areas of the as-synthesized and microwave treated samples were

higher (~ 50 %) than that of calcined samples. In vitro bioactivity tests

revealed initial absorption and subsequently exhibited bioactivity. Controlled

antibiotic and anticancer drug release was observed with microwave treated

samples. SMWT showed prolonged antibiotic release upto 125 h, which is

two times higher than SAS sample. Anticancer drug showed faster release

than the antibiotic. The antibacterial activity results showed that the entire

drug incorporated samples are strongly active against the most common

bacterial strains. It may be used as an implant material and for reconstructive

surgery applications. All samples showed haemocompatability and had no

cytotoxicity.