Faculty of Resource Science and Technology and Characterization of Cellulose-Based...Biomedik ABSTRAK Sellulosa ialah polimer material yang bersifat boleh diperbaharui, mudah didapati

SYNTHESIS AND CHARACTERIZATION OF CELLULOSE-BASED

NANOPARTICLES AND AEROGEL FOR BIOMEDICAL

APPLICATION

Fiona Beragai anak Jimmy

Master of Science

(Physical Chemistry)

2014

Faculty of Resource Science and Technology

SYNTHESIS AND CHARACTERIZATION OF CELLULOSE-BASED

NANOPARTICLES AND AEROGEL FOR BIOMEDICAL

APPLICATION

FIONA BERAGAI ANAK JIMMY

A thesis submitted

in fulfillment of the requirements for the

Master of Science

Faculty of Resource Science and Technology

UNIVERSITI MALAYSIA SARAWAK

2014

DECLARATION

No portion of the work referred to in this dissertation has been submitted in support of an

application for another master of qualification of this or any other university/institution of

higher learning.

…………………………………………………

Fiona Beragai anak Jimmy (11021717)

Department of Chemistry (Physical Chemistry)

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

i

ACKNOWLEDGEMENTS

First and foremost, I wish to express my deepest gratitude to my supervisor Dr. Chin

Suk Fun for her patience and endless encouragement throughout the course of my study. I

would also like to extend my gratitude to my co-supervisor, Assoc. Prof. Dr. Pang Suh Cem

for his valuable suggestions and support along the way.

I wish to thank all staffs and lab technicians from Faculty of Resource Science and

Technology, UNIMAS, for their assistance and co-operations. It is also my pleasure to

acknowledge Ministry of Higher Education for their financial supports under Fundamental

Research Grant Scheme (FRGS), grant no.: 01(17)746/2010 and MyBrain15 (MyMaster)

scholarship.

I wish to thank my friends Akmar, Ain, Aressa, Nabil, Ying Ying, Lee Ken, Li Shan

and all others for their kindness and support. All the joyful moment together will forever be

treasured and missed. Last but not least, I am forever grateful for I have been blessed with

such supportive grandmother, the late Mdm. Sudan anak Begam, my parents, brothers and

relatives who always inspire me to go further, provide me with all my needs and encouraged

me throughout my study. Thank you and God bless.

ii

Synthesis and Characterization of Cellulose-based Nanoparticles and Aerogel for

Biomedical Application

ABSTRACT

Cellulose is the most abundant renewable material available worldwide and its non-toxic

feature has propelled its popularity in pharmaceutical industries due to its biodegradability and

biocompatibility. In this study, cellulose nanoparticles with mean particle sizes ranging from

70 to 360 nm were synthesized from commercial facial cotton was developed for drug delivery

application. The effects of synthesis parameters such as concentration of cellulose solution,

ratio of solvent/non-solvent, water-in-oil (w/o) microemulsion and surfactant on the particle

size of cellulose nanoparticles formed were studied. Methylene blue (MB) as a hydrophilic

model drug was loaded into cellulose nanoparticles with different mean particle sizes. In drug

loading study, it was apparent that cellulose nanoparticles with the smallest mean particle size

(70 nm) has the highest loading efficiency (89 %) of MB. The drug release study also unveiled

that cellulose nanoparticles with the smallest mean particle size (70 nm) has the fastest release

rate where 100 % of MB was released within 52 hours. Apart from being synthesized as

nanoparticles, cellulose also showed a great potential as aerogels for drug delivery application.

This was due to their surface properties such as its porosity and high surface area, which

influence the drug loading/adsorption behavior. From this study, cellulose aerogels were

successfully synthesized from cellulose fibers isolated from sugarcane bagasse (SCB) and it

was apparent that cellulose aerogel with cellulose solution with the lowest concentration (1

w/v %) produced the highest BET surface area of 525 m2/g. In drug loading study, cellulose

aerogel with the highest BET surface area recorded the highest loading capacity and the fastest

release profile of MB, with 100 % release within 23 hours.

iii

Sintesis Dan Pencirian Nanopartikel dan Aerogel Berasaskan Selulosa Untuk Kegunaan

Biomedik

ABSTRAK

Sellulosa ialah polimer material yang bersifat boleh diperbaharui, mudah didapati dan tidak

bertoksik, disamping ianya juga bersifat biodegradasi dan bioserasi. Ciri-ciri tersebut

menjadikan selulosa sering kali digunakan dalam industry farmaseutikal. Kajian penyelidikan

ini adalah mengenai penggunaan selulosa nanopartikel dalam aplikasi pengawal pelepasan

ubat. Selulosa nanopartikel yang bersaiz 70 hingga 360 nm telah dihasilkan daripada kapas

muka komersial dan digunakan sebagai agen pengawal pelepasan ubat. Kesan seperti

kepekatan larutan selulosa, perkadaran antara larutan/bukan larutan, mikroemulsi air kepada

minyak dan kehadiran surfaktan terhadap penghasilan saiz selulolsa nanopartikel telah dikaji.

Biru metilen (MB) adalah model ubat yang bersifat hidrofilik telah dimuatkan ke dalam

selulosa nanopartikel yang berbeza saiz untuk mengkaji kesan kadar pemuatan ubat MB dan

kadar pengelepasan ubat MB dalam penyelidikan pengawal pelepasan ubat. Hasil kajian

mendapati MB yang dimuatkan ke dalam nanopartikel tang bersaiz kecil (70 nm) mempunyai

kadar kapasiti pemuatan ubat yang tertinggi (89 %) dan juga menunjukkan kadar pengelepasan

ubat yang cepat iaitu 100 % MB dibebaskan dalam masa 52 jam. Selain nanopartikel, selulosa

juga dihasilkan dalam bentuk aerogel sebagai agen pengawal pelepasan ubat. Hal ini kerana

sifat aerogel yang berpori dan mempunyai luas permukaan yang besar untuk membantu kadar

pemuatan dan penyerapan ubat. Dalam penyelidikan ini, selulosa aerogel telah Berjaya

dihasilkan daripada hampas tebu menunjukkan bahawa selulosa yang mempunyai larutan

selulosa yang rendah (1 w/v %) menghasilkan selulosa yang mempunyai luas permukaan yang

tertinggi iaitu 525 m2/g. Selulosa yang mempunyai luas permukaan yang tinggi ini

merekodkan kadar pemuatan yang tinggi dan kadar pengelepasan ubat MB yang terpantas,

dengan kadar pengelepasan 100 % ubat MB dalam tempoh 23 jam.

iv

TABLE OF CONTENTS

Page

Acknowledgements i

Abstract ii

Abstrak iii

Table of Contents iv

List of Tables viii

List of Figures ix

List Abbreviations x

List of Symbols xi

CHAPTER 1 INTRODUCTION

1.1 Background 1

1.2 Objectives 5

1.3 Scopes of Study 6

CHAPTER 2 LITERATURE REVIEW

2.1 Cellulose 7

2.2 Cellulose Isolation 9

2.3 Applications of Cellulose 12

2.4 Cellulose Nanoparticles 13

2.5 Synthesis Methods for Cellulose Nanoparticles 14

2.5.1 Nanoprecipitation 16

2.5.2 Microemulsion 17

2.6 Cellulose Aerogel 19

v

2.7 Controlled Release Agents 21

CHAPTER 3 SYNTHESIS AND CHARACTERIZATION OF CELLULOSE

NANOPARTICLES FOR CONTROLLED RELEASE OF

HYDROPHILIC DRUG

3.1 Introduction 24

3.2 Materials and Method 26

3.2.1 Materials 26

3.2.2 Chemical Pretreatment 26

3.2.3 Dissolution of Cellulose 27

3.2.4 Synthesis of Cellulose Nanoparticles 27

3.2.4.1 Water-in-Oil (w/o) Microemulsion 28

3.2.5 Characterization of Cellulose Nanoparticles 28

3.2.6 Drug Loading Efficiency Evaluation 28

3.2.7 Drug Release Analysis 29

3.3 Results and Discussion 30

3.3.1 Effect of Cellulose Concentration 30

3.3.2 Effect of Solvent/Non-solvent Ratio on Mean Particle Size 32

3.3.3 Water-in-Oil (w/o) Microemulsion Method 34

3.3.3.1 Effect of Ratio of Oil:Co-surfactant 34

3.3.3.2 Effect of Surfactant Concentration 35

3.3.4 Drug Loading Analysis 37

3.3.5 Drug Release Study 38

3.4 Conclusion 40

vi

CHAPTER 4 SYNTHESIS AND CHARACTERIZATION OF CELLULOSE

AEROGEL AS CONTROLLED RELEASE CARRIERS

4.1 Introduction 41

4.2 Materials and Methods 42

4.2.1 Materials 42

4.2.2 Isolation of Cellulose Fibers 43

4.2.3 Preparation of Cellulose Aerogels 44

4.2.3.1 Drug Loading Capacity 44

4.2.3.2 Drug Release Studies 45

4.2.3.3 Swelling Studies 46

4.2.4 Characterization of Samples 46

4.3 Results and Discussion 47

4.3.1 Effect of Cellulose Concentration 49

4.3.2 Drug Loading Capacity 52

4.3.3 Drug Release Studies 53

4.4 Conclusion 56

CHAPTER 5 CONCLUSION AND RECOMMENDATIONS

5.1 Concluding Remarks 57

5.2 Recommendations for Future Works 58

REFERENCES 59

APPENDIX A 73

APPENDIX B 76

APPENDIX C 80

APPENDIX D 83

vii

APPENDIX E 85

APPENDIX F 89

viii

List of Tables Page

Table 2.1 Chemical composition of some cellulose sources 10

Table 4.1 BET surface area from the resultant cellulose aerogels 50

ix

List of Figures

Page

Figure 2.1

Molecular structure of cellulose 7

Figure 2.2

Schematic of the tree hierarchical structure 9

Figure 2.3 Schematic presentation of the nanoprecipitation methods applying

(a) dialysis membrane and (b) the dropping technique under stirring

16

Figure 2.4 Schematic representation of the three microemulsion systems (a)

water-in-oil (w/o) microemulsion (b) oil-in-water microemulsion (c)

bicontinuous microemulsion

18

Figure 3.1 SEM micrographs of (a) cellulose fiber isolated from facial cotton,

(b) cellulose nanoparticles prepared from 0.01 w/v % of cellulose

solution and (c) TEM micrograph of cellulose nanoparticles

prepared from 0.01 w/v % of cellulose solution

30

Figure 3.2 Effect of cellulose concentrations on mean particles sizes of

cellulose nanoparticles

31

Figure 3.3 Effect of solvent/non-solvent ratio on mean particle size of cellulose

nanoparticles

32

Figure 3.4 Effect of various oil:co-surfactant ratio on the mean particle size of

cellulose nanoparticles

34

Figure 3.5 Mean particle size of cellulose nanoparticles synthesized in the

presence of various concentrations of Tween-80

35

Figure 3.6 Loading efficiency of MB onto various sizes of cellulose

nanoparticles

37

Figure 3.7 Release profile of MB from cellulose nanoparticles as a function of

time

38

Figure 4.1 FTIR spectra of the (a) pure cellulose, (b) sugarcane bagasse and (c)

cellulose fibers isolated from SCB

47

Figure 4.2 SEM micrographs of (a) cellulose fibers isolated from SCB and

cellulose aerogels produced from (b) 1, (c) 2, (d) 3, (e) 4 and (f) 5

w/v % of cellulose solution

49

Figure 4.3 Effect of BET surface area of cellulose aerogel (w/v %) on loading

capacity of MB onto cellulose aerogels

52

Figure 4.4 Release profile of MB loaded cellulose aerogels

53

Figure 4.5 Swelling ratio of MB loaded cellulose aerogels 54

x

List of Abbreviations

BET Brunauer-Emmet-Teller

CMC Carboxymethyl cellulose

CNPs Cellulose nanoparticles

CO2 Carbon dioxide

DP Degree of polymerization

FTIR Fourier Transform Infrared Radiation

HCl Hydrochloric acid

H2SO4 Sulphuric acid

KBr Potassium bromide

MB Methylene blue

NaOH Sodium hydroxide

NMMO N-methylmorpholine-N-oxide

NTU NaOH/thiourea/urea

o/w Oil-in-water

PBS Phosphate buffer solution

SCB Sugarcane bagasse

SEM Scanning electron microscope

TEM Transmission electron microscope

UV Ultraviolet

w/o Water-in-oil

xi

List of Symbols

Abs Absorbance

cm Centimeter (10-2

m)

°C Degree celcius

g Gram

mg Miligram

mg/mg Milligram over miligram

mg/mL Miligram per milliliter

mL Mililiter (10-3

L)

mM Milimolar (10-3

M)

M Molarity

nm Nanometer

% Percentage

% T Percentage of transmittance

w/v % Weight over volume

1

CHAPTER 1

INTRODUCTION

1.1 Background

Efficient drug delivery systems are essential in the field of medicine and healthcare. By

loading drugs within a drug delivery system, premature degradation of drug molecules can be

prevented and drug uptake can be improved (Zhang et al., 2013; Kamel, 2007). Furthermore,

it is possible to manipulate a drug delivery system so that precise amount of drug is delivered

to targeted disease sites at predetermined rate over a desirable period to subsequently improve

the therapeutic efficacy and reduce the non-specific side effects (Zhang et al., 2013).

Cellulose is a favorable precursor material for drug delivery carriers since it is low cost,

bioresorbability, and biocompatible in nature. Cellulose is a polydisperse linear polysaccharide

consisting of β-D-glucopyranose units linked by glucoside bond at their C1 and C4 hydroxyl

groups (Heinze & Liebert, 2001). Cellulose consists of two structure regions, which are the

crystalline region and the amorphous region, while the abundant hydroxyl groups present

along the cellulose molecule skeleton gives them an extended network of hydrogen bonds

(inter- and intramolecular bonds) (Qiu & Hu, 2013).

Nanotechnology has promoted the advancement of drug delivery system by creating

nanocarriers that are precise and stimulus-responsive. The capability to control the shape and

sizes of nanoparticles through the application of nanofabrication technologies, both top-down

and bottom-up, gives way into the development of an effective nanoparticulate drug delivery

system. Nanoparticles with particle sizes in the range of 10 to 1000 nm have been used to

improve the pharmacokinetic and pharmacodynamic properties of different types of drugs.

2

They have proven capabilities to deliver drugs at a controlled and sustained rate to the site of

action (Kamel, 2007; Mohanraj & Chen, 2006).

Designing nanoparticles for drug delivery system must take into consideration

properties such as the particle size, surface properties, and the release kinetics of encapsulated

drug in order to reach the specific sites at a therapeutically optimal rate and dose regimen

(Mohanraj & Chen, 2006). In this regard, nanoparticles that are biodegradable, biocompatible,

and nontoxic are very favorable for such drug delivery systems, and those derived from

polysaccharides (e.g., starch, cellulose, and chitosan) are of great interest. Their applications

as controlled release carriers have gained much attention since they provide better flexibility

in obtaining desirable drug release profile, are low cost, can be easily modified through simple

chemical reactions, and degrade readily in the human body (Sonia & Sharma, 2011). For

instance, Yallapu et al. (2012) formulated curcumin loaded cellulose nanoparticles for prostate

cancer which assay have shown improved anti-cancer efficacy compared to free curcumin. On

the other hand, Aswathy et al. (2012) developed a cell specific nanoparticle based on

carboxymethyl cellulose (CMC) where the folate group was attached to the nanoparticles for

specific recognition of cancerous cells and fluorouracil (5FU) was encapsulated as the model

drug. The multifunctional nanoparticles were targeted at human breast cancer cell, MCF7, and

their study showed that the folate-conjugated nanoparticles were more efficient compared to

non-conjugated nanoparticles.

In addition to nanoparticles, aerogel is another promising drug delivery carriers.

Aerogels are materials with porous structure and high surface area, which have transformed

them into an ideal carrier material for drug delivery (Sehaqui et al., 2011b). The drug loading

efficiency and release profile of aerogels are affected by the surface area and pore volume or

3

network structure of the aerogels. Most aerogels are fabricated from silica or pyrolized organic

polymers, which caused the structure to be very brittle and have limited practical applications.

However, these problems can be partially relieved using cellulose-based aerogels since the

long nanofibrils of native cellulose in aerogels are able to provide the needed strength and

flexibility due to the fibrillar morphology and strong molecular interactions of cellulose

through, for example, van der Waals and hydrogen bonds (Kettunen et al., 2011; Aulin et al.,

2010). Cellulose-based aerogels offer wide array of advantages as they are bioresorbability,

renewable, and does not require the usage of harmful solvents during processing; they are

usually prepared through supercritical drying with carbon dioxide (CO2) or lyophilization

(freeze-drying) to induce fast drying and maintain the porous structure. These are achieved by

avoiding pore collapse with the assistance of liquid surface tension (Tchang et al., 2012;

Gavillon, 2008).

Cellulose-based aerogels are gaining its spot in drug delivery studies due to its highly

porous nature, lightweight, high surface area, and abilities to provide enhanced drug

bioavailability and drug loading capacity. Aerogels with high specific surface area are

preferred in drug delivery studies since they are capable of achieving maximum drug

adsorption in the matrices and faster rate of release of drugs (García-González et al., 2011).

Sehaqui et al. (2011b) prepared cellulose aerogels with a specific surface area as high as 153

to 284 m2/g. García-González and co-workers (2012) produced starch aerogel with specific

surface areas of 34 to 120 m2/g that are capable of achieving high loading capacity (1.1 x 10

-3

g/m2) of ketoprofen as a model drug.

In this study, the cellulose nanoparticles were prepared from cellulose that was isolated

from facial cotton. The cellulose aerogels were prepared from cellulose isolated from

4

sugarcane bagasse (SCB). Both of the isolated cellulose fibers were dissolved in an

environmental friendly sodium hydroxide/thiourea/urea (NTU) aqueous-based solvent system

to form homogeneous cellulose solution. Cellulose nanoparticles with mean particles sizes

ranging from 70 to 680 nm were formed by controlled precipitation of cellulose solution into a

non-solvent (ethanol), whereas cellulose aerogels with various surface morphology were

formed by supercritical drying of the wet gel derived from different concentrations of

cellulose solution. The effect of particle sizes of cellulose nanoparticles and surface

morphology of cellulose aerogels on their drug loading capacity and drug release profile were

explored by using methylene blue (MB) as a model hydrophilic drug (Huang & Lowe, 2005).

5

1.2 Objectives

The objectives of this study are:

a. To prepare cellulose nanoparticles from commercially available facial cotton

b. To study the effect of particle size on the drug loading capacity and drug release

profile of cellulose nanoparticles

c. To synthesize cellulose aerogels from the fibers isolated from sugarcane bagasse (SCB)

d. To investigate the loading efficiency and drug release profile of cellulose aerogels

6

1.3 Scopes of Study

The scopes of this study include the synthesis and characterization of cellulose-based

nanoparticles from commercial facial cotton and cellulose aerogels from sugarcane bagasse

(SCB) fibers for drug loading efficiency and controlled drug release studies. Chapter 1

describes the background and justification of this study. Chapter 2 provides an introduction of

cellulose-based nanoparticles and aerogels along with an overview of their recent development.

Chapter 3 describes the formulation of different sizes of cellulose nanoparticles through

controlled precipitation and the effect of particle size on the drug loading efficiency and drug

release profile. Chapter 4 illustrates the synthesis and characterization of cellulose-based

aerogels from cellulose fibers isolated from sugarcane bagasse (SCB) as well as the drug

loading efficiency and drug release profile using a hydrophilic model drug. The final chapter

(Chapter 5) depicts the concluding remarks and recommendations for future research works.

7

CHAPTER 2

LITERATURE REVIEW

2.1 Cellulose

An inexhaustible amount of cellulose which can be available worldwide has made its

popularity soared for industrial applications as an alternative route to counter the problems

arising from high demands for nonrenewable and limited petroleum supplies (Brinchi et al.,

2013; Cao et al., 2009). The increasing demand for environmentally friendly and

biocompatible products has also helped to boost the popularity of cellulose and other

polysaccharides. Cellulose can be obtained from various sources such as wood fibers (Sehaqui

et al., 2011a), cotton (Siro & Plackett, 2010), wheat straw (Chen et al., 2011), coconut husk

fibers (Rosa et al., 2010), sugarcane bagasse (Mandal & Chakrabarty, 2011), sesame husk

(Purkait et al., 2011), hemp fiber (Ouajai & Shanks, 2009) and banana rachis (Zuluaga et al.,

2009).

Figure 2.1: Molecular structure of cellulose (taken from Heinze & Liebert, 2001)

8

Cellulose is a polydispersed linear homopolysaccharide consisting of β-D-

glucopyranose units linked by glucoside bonds at their C1 and C4 hydroxyl groups. As shown

in Figure 2.1, there are three types of functional units in the cellulose backbone: a non-

reducing end to the left, a reducing end to the right and an anhydroglucose unit (AGU) in the

center. The C-2, C-3, and C-6 atoms are the three reactive hydroxyl groups of the polymer,

which give way to the conversions of primary and secondary alcoholic -OH groups. These

structures have contributed to the strong hydrogen bonding patterns observed in cellulose

molecules, which in turn controls the physical properties of cellulose (Heinze & Liebert, 2001).

Cellulose does not exist as an isolated individual molecule (Brinchi et al., 2013). On

the contrary, it is a group of individual cellulose chain-forming fibers, which are in fact packs

of microfibrils, made from stacks of elementary fibrils (protofibrils) as indicated in Figure 2.2.

The molecular structure of cellulose brought forth two types of solid state representation; high

order (crystalline) and low order (amorphous). As to date, there are five known

interconvertible polymorphs of cellulose: I, II, IIII, IVI, and IVII (Brinchi et al., 2013). Two

most common crystalline forms of cellulose are Cellulose I, which is thermodynamically less

stable; and Cellulose II, which is a more stable structure. Cellulose I is known as a native

cellulose and it exists as two suballomorphs, Iα (triclinic structure) and Iβ (monoclinic

structure); while Cellulose II, which generally occurs in marine algae, forms when Cellulose I

is treated with aqueous sodium hydroxide. Cellulose III is the product of liquid ammonia

treatments from Cellulose I or II, and further thermal treatments will form Cellulose IV

(Brinchi et al., 2013; Moon et al., 2011; Habibi et al., 2010). However, different sources of

celluloses give different structures and degree of crystallinity (DP) (40 to 60 %) as dictated by

the biosynthesis conditions (Habibi et al., 2010).

9

Figure 2.2: Schematic of the tree hierarchical structure (taken from Moon et al., 2011)

2.2 Cellulose Isolation

The importance of developing environmentally friendly polymer composites or green

composites has drawn the attention of scientists to make use of cellulose, which is one of the

most abundant natural polymers (Haafiz et al., 2013). Cellulose plays an important role in

higher plants by reinforcing elements in the cell wall, co-existing with lignin and

hemicellulose, and is widely available in agro-industrial residues. However, the relative

content of lignin and cellulose differs according to the species of the biomass used (refer to

Table 2.1). Table 2.1 shows the chemical composition of cellulose, hemicellulose and lignin

from different sources.

10

Table 2.1: Chemical composition of some cellulose sources (taken from Hon, 1996)

Source

Composition, %

Cellulose Hemicellulose Lignin Extract

Wheat straw 30 50 15 5

Bagasse 40 30 20 10

Softwood 40-44 25-29 25-31 1-5

Hardwood 43-47 25-35 16-24 2-8

Flax (retted) 71.2 20.6 2.2 6.0

Jute 71.5 13.6 13.1 1.8

Henequen 77.6 4-8 13.1 3.6

Ramie 76.2 16.7 0.7 6.4

Cotton 95 2 0.9 0.4

High efficiency in isolation of cellulose is important as they are used as raw material

for making paper, fuel, and other industrial applications. However, the numerous inter- and

intramolecular hydrogen bonds present in cellulose causes cellulose to be more resistant

towards dissolving in most traditional solvents (Zhong et al., 2013). Thus, chemical

pretreatment is required to remove impurities from cellulose, such as lignin and hemicellulose,

by disrupting the inter- and intramolecular hydrogen bonding and loosening the crystalline

structure, which all leads to an enhancement of cellulose solubility in solvents (Brinchi et al.,

2013; Chen et al., 2011).

Haafiz et al. (2013) managed to remove microcrystalline cellulose (MCC) from an oil

palm empty fruit bunch (OPEFB) fiber-total chlorine free (TCF) pulp by employing the acid

hydrolysis method. Further characterization with the Fourier transform infrared (FT-IR)