faculty of resource science and technology and characterization of cellulose-based...biomedik...
TRANSCRIPT
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)