formulating curcumin in a biodegradable polymeric

Formulating curcumin in a biodegradable polymeric composite

material: A step towards wound healing applications

Maryam Shahnia

http://orcid.org/0000-0001-5565-881X

Submitted in total fulfillment of the requirements for the degree of

Master of Philosophy

April 2017

Faculty of Veterinary and Agricultural Sciences

The University of Melbourne

i

Declaration

To the best of my knowledge and belief, this thesis contains no material previously published by

any other person except where due acknowledgement has been made. The thesis also contains no

material which has been accepted for the award of any other degree or diploma in any university.

Also, this thesis does not exceed 50,000 words and complies with the provisions set out for the

degree of Master of Philosophy by the University of Melbourne.

Maryam Shahnia

March 2017

ii

Acknowledgements

I would like to thank my supervisor Dr Ken Ng, who guided me through the project. Many thanks

to my advisory committee Professor Paul Taylor, and Associate Professor Brian Leury. My special

thanks go to Professor Paul Taylor, the chair of my committee for the time, and energy he spared

to me, and for his profound understanding, and great moral support at all times. I am also highly

indebted to Professor Jean-Pierre Scheerlinck for his time and helpful insights and suggestions

about my project.

I gratefully thank the University of Melbourne for providing me MIRS, and MIFRS scholarships,

and also funding, and providing all the laboratory facilities. I am thankful to our lab managers,

Carolyn Selway, Michelle Rhee, and especially Kathryn Mistica who provided continuous and

great support during my project. I also appreciate Baker IDI heart, and diabetes research institute,

for providing the cell lines, and would like to thank the kindness of Dr Iska Carmichael for helping

me in getting microscopic images.

I would like to thank my family who have always stood by me, and supported me, with their

endless love, while I was pursuing my studies. Last but not least, I would like to thank all those

who assisted me with a smile or made me smile during this journey.

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Dedication

To my beloved Mother and Father,

for their unconditional love, encouragement and support in my entire life

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Abstract

The natural process of wound healing typically consists of four distinct but overlapping phases

which include, hemostasis (platelet aggregation and blood clot formation), inflammation

(migration of blood cells), proliferation (angiogenesis or blood vessel formation), and remodelling

(reorganisation of collagen and scar tissue formation). However, in diabetic patients, this elaborate

well-programmed process becomes disrupted, and there is an urgent need for compounds and

formulations that can improve wound healing in these cases. A variety of natural components,

including curcumin, have been identified as wound-healing agents. Curcumin, is a yellow

hydrophobic natural polyphenolic pigment derived from the rhizomes of the herb Carcuma longa,

which has been identified as the active principal of turmeric. The inability to efficiently deliver

curcumin in a soluble form presents a chief challenge for its clinical use. Here we characterised,

and optimised different biodegradable and biocompatible formulations of curcumin encapsulated

particles, in order to enhance the efficiency of curcumin wound healing effect.

The size of the optimised curcumin particles ranged from 1286 to 1485 nm, with an encapsulation

efficiency of 75%. The zeta potential exhibited values in the range of (-7.2) to (-7.96) with the PDI

of 0.4. Physical characterisation using TEM imaging ensured the successful fabrication and

encapsulation of curcumin in the polymeric matrix, which had been fabricated in rod shape.

Release profile occurred in a biphasic manner including an initial burst, followed by a sustained

release trend for curcumin particles. In vitro cytotoxicity assays along with microscopic imaging

confirmed safety of the applied concentration of curcumin particles below 25 µg/ml. Moreover,

the results of cellular uptake study validated the internalisation of curcumin particles. Overall this

thesis, elucidated the developed biocompatible and biodegradable formulations for curcumin

v

encapsulation do have the potential to be employed as a drug delivery vehicle for curcumin. Further

validation of the potential of this preparation to enhance wound healing is still needed.

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Table of contents

Chapter 1: Introduction ................................................................................................................. 1

1.1. Turmeric and its active ingredients .................................................................................. 1

1.2. Curcumin .......................................................................................................................... 2

1.2.1. Pharmacological activity of curcumin .......................................................................... 4

1.2.2. Limitations of curcumin application as a bioactive agent ............................................ 5

1.2.3. Micro- and nanoparticles .............................................................................................. 5

1.2.4. Liposomes ..................................................................................................................... 6

1.2.5. Micelles ........................................................................................................................ 7

1.2.6. Niosomes ...................................................................................................................... 8

1.2.7. Cyclodextrins ................................................................................................................ 9

1.2.8. Dendrimers ................................................................................................................. 10

1.2.9. Nanogels ..................................................................................................................... 11

1.2.10. Polymeric-based Particles........................................................................................... 12

1.2.10.1. Chitosan .................................................................................................................. 13

1.2.10.2. Alginate ................................................................................................................... 14

1.3. Wound classification ...................................................................................................... 17

1.4. Natural wound healing mechanism ................................................................................ 18

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1.5. Diabetes .......................................................................................................................... 24

1.6. Diabetic foot ulcer .......................................................................................................... 26

1.7. Effects of curcumin on different phases of wound healing and mechanism of action... 30

1.7.1. Effect of curcumin on inflammation .......................................................................... 30

1.7.2. Effect of curcumin on the proliferative phase of wound healing ............................... 31

1.7.2.1. Effects of curcumin on the fibroblast proliferation................................................. 31

1.7.2.2. Effects of curcumin on the stage of granulation tissue formation .......................... 32

1.7.2.3. Effect of curcumin on the stage of collagen deposition .......................................... 32

1.7.2.4. Effect of curcumin on the stage of apoptosis .......................................................... 33

1.7.3. Effect of curcumin on the stage of wound contraction ............................................... 33

1.8. Research gap .................................................................................................................. 34

1.9. Research hypotheses ...................................................................................................... 35

1.10. Research aims ............................................................................................................. 35

Chapter 2: Materials and methods............................................................................................... 37

2.1. Materials ......................................................................................................................... 37

2.2. Equipment ...................................................................................................................... 37

2.3. Preparation of blank alginate/chitosan/PF 127 particles ................................................ 37

2.3.1. Preparation of stock solutions ..................................................................................... 37

2.3.2. Preparation of the blank particles ............................................................................... 38

2.3.3. Preparation of curcumin loaded particles ................................................................... 38

viii

2.4. Optimisation of the procedure parameters ..................................................................... 40

2.5. Physicochemical characterisation of the particles .......................................................... 40

2.5.1. Particle size, zeta potential and polydispersity index ................................................. 40

2.5.2. Morphology determination by transmission electron microscopy (TEM) ................. 40

2.5.3. Encapsulation efficiency of loaded curcumin ............................................................ 41

2.5.4. In-vitro curcumin release studies ................................................................................ 42

2.6. Cell culture ..................................................................................................................... 43

2.7. Routine cell culture ........................................................................................................ 43

2.8. Preparation of cell culture for treatments ....................................................................... 44

2.9. Drug preparations for cell treatments ............................................................................. 45

2.10. Cell viability assay...................................................................................................... 45

2.11. Microscopic cell toxicity ............................................................................................ 46

2.12. Cellular uptake by confocal microscopy imaging ...................................................... 47

2.13. Statistical analysis....................................................................................................... 47

Chapter 3: Results and discussion ............................................................................................... 49

3.1. Physicochemical experiments ........................................................................................ 49

3.1.1. Effects of methanol (%) rinse on particles ................................................................. 49

3.1.2. Effects of PF127 ......................................................................................................... 50

3.1.3. Effects of curcumin concentration on zeta potential .................................................. 54

3.1.4. Effects of curcumin concentration on particle size .................................................... 57

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3.1.5. Morphological characterisation of the particles ......................................................... 60

3.1.6. Release kinetics .......................................................................................................... 61

3.2. Cellular experiments ...................................................................................................... 69

3.2.1. In vitro validation of particle cytotoxicity .................................................................. 70

3.2.1.1. Effect of particles on FEP-188 keratinocyte cell viability ...................................... 70

3.2.1.2. Microscopic cell toxicity......................................................................................... 71

3.2.2. Cellular uptake by confocal microscopy imaging ...................................................... 73

Chapter 4: Conclusions and future works ................................................................................... 75

References ..................................................................................................................................... 78

x

List of Figures

Figure 1. a: Turmeric plant, b: Turmeric rhizome, c:Turmeric powder (Palve and Nayak 2012),

and d: Chemical structure of the three curcuminoids (Aggarwal et al. 2003) ................................ 3

Figure 2. Curcumin encapsulated in liposome and the mechanism of entrance. Adapted from

(Ghalandarlaki et al. 2014) under creative commons rules ............................................................ 7

Figure 3. The connection of curcumin to cyclodextrin. Adapted from (Ghalandarlaki et al. 2014)

under creative commons rules ...................................................................................................... 10

Figure 4. Structure of chitosan. Adapted from (Albuquerque et al. 2016) under creative common

rules ............................................................................................................................................... 14

Figure 5. Structure of alginate, adapted from (Rinaudo 2014) under creative commons rules ... 15

Figure 6. Schematic process of wound healing process............................................................... 24

Figure 7. Prevalence of diabetes in the world in 2010 and 2030. Adapted from (J. E. Shaw et al.

2010) ............................................................................................................................................. 26

Figure 8. Wound healing process in normal and diabetic wounds. Reproduced with permission

from (Moura et al. 2013) ............................................................................................................... 29

Figure 9. Schematic process of curcumin particle fabrication ..................................................... 39

Figure 10. Standard curve of curcumin in methanol .................................................................... 42

Figure 11. a: curcumin particle solution, b: curcumin in distilled water, c: curcumin particle in

PBS in dialysis tube ...................................................................................................................... 43

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Figure 12. Variation in curcumin encapsulation efficiency by variation of PF127 concentration,

and methanol rinse (MR) at a: 0.1% curcumin concentration, and b: 0.2% curcumin. All data

represent mean ± SD (n =3) and *p value < 0.05 ......................................................................... 50

Figure 13. Variation in curcumin encapsulation efficiency, and particle size by variation of

PF127 concentration and methanol rinse (MR) at a: 0.1% curcumin concentration, and b: 0.2%

curcumin. All data represent mean ± SD (n =3), and *p value < 0.05 ......................................... 52

Figure 14. Variation in PDI, and zeta potential by variation of PF127 concentration and

methanol rinse (MR) at a: 0.1% curcumin concentration, and b: 0.2% curcumin. All data

represent mean ± SD (n =3), and *p value < 0.05 ........................................................................ 54

Figure 15. Zeta potential plot of curcumin particles a: empty particles (blank) .......................... 56

Figure 16. Variation in particle size by variation of PF127, and methanol rinse concentration at

different concentrations of curcumin. All data represent mean ± SD (n =3), a and b represent p

value < 0.05 between different curcumin concentrations and * represent the p value < 0.05 within

groups ............................................................................................................................................ 57

Figure 17. Size distribution of intensity for curcumin particles a: empty particles (blank) b:

formulation with 20% methanol rinse, c: formulation with 50% methanol rinse ......................... 59

Figure 18. TEM images of a: curcumin+ NaAlg+CaCl2+chitosan+PF127, b: curcumin+

NaAlg+CaCl2+chitosan, c: NaAlg+CaCl2+ chitosan+PF127 (blank), d: NaAlg+CaCl2+ chitosan

(blank) ........................................................................................................................................... 61

Figure 19. Release curves of curcumin particles. All values represent mean ± SD (n =3), and *p

value < 0.05 ................................................................................................................................... 62

Figure 20. Release kinetics of optimised 20% methanol rinse formulation ................................ 65

Figure 21. Release kinetics of optimised 50% methanol rinse formulation ................................ 67

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Figure 22. Cell toxicity curve of curcumin in DMSO, curcumin particles, free particles, and

control. All values represent mean ± SD (n =3), and *p value < 0.05 .......................................... 70

Figure 23. Qualitative intracellular uptake of curcumin particles, and free curcumin in 0.4%

DMSO by keratinocytes over 3 days ............................................................................................ 72

Figure 24. Confocal images of keratinocytes over 8 hours treated by a - e: curcumin particles, f:

normal view of the keratinocytes, g: free curcumin in PBS, and h: empty particles ................... 74

xiii

List of Tables

Table 1. Estimated parameters for encapsulated curcumin obtained after fitting the release

experimental data to five kinetic models ...................................................................................... 68

Chapter-1: Introduction Page 1

Chapter 1: Introduction

This chapter presents a literature review on curcumin properties and its application as a

valuable bioactive in drug delivery and pharmaceutical sciences, and the recent

investigations, which established numerous approaches to improving the limitations of

curcumin application in the mentioned fields. This chapter further outlines the hypotheses,

the existing gaps in the application of curcumin as a wound healing agent, and aims of this

research.

1.1. Turmeric and its active ingredients

The isolation of antibiotic-resistant pathogens, coupled with increased awareness of people

about the side effects of synthetic drugs have resulted in the search for different formulations

of natural medicinal plants.

One of these highly impressive medicinal plants is turmeric, which belongs to the

Zingiberaceae family with the scientific name of Curcuma longa L. (Goel et al. 2008).

Turmeric has other names such as Jiang Huang, harida, and Indian saffron and obtained from

the rhizomes of the plant. The history of turmeric dates back to 3000 BC in Asia where people

had used an ointment based on it for relieving food poisoning effects (Anand et al. 2008). It

has been used for centuries in the Ayurvedic system of medicine as a blood purifier

(Aggarwal et al. 2003). Turmeric has been used in home remedies for the treatment of skin

diseases and softening of rough skins in the shape of bath soaps and creams in India.

Furthermore, the curing effects of turmeric on cuts, bruises and wounds have been known


over centuries. Turmeric was introduced to Europe by Arab traders in the 13th century and its

use as an exceptional herb attracted the spotlight of Westerners due to its broad spectrum of

medicinal advantages. Turmeric consists of 3-5% curcuminoids, which include curcumin

(deferuloyl methane), as the most important fraction, desmethoxycurcumin, and

bisdemethoxycurcumin. Among these components, curcumin serves a great role in biological

and therapeutic activities of turmeric. Figure 1 shows turmeric along with the chemical

structure of the three curcuminoids (Aggarwal et al. 2003, Goel et al. 2008).

1.2. Curcumin

Curcumin is a hydrophobic component which was isolated in 1815 for the first time, but after

around 158 years the chemical structure was determined in 1973 by Roughly and Whiting

(Roughley and Whiting 1973). The chemical structure of curcumin consists of two rings,

both of which have a methoxy ether group in the ortho position.

Curcumin is soluble in ethyl alcohol, propylene glycol and glacial acetic acid but insoluble

in water. The maximum absorbance of curcumin is at 425 nm. Curcumin has been

acknowledged as a nontoxic chemical for humans as a result of many studies (Shoba et al.

1998, Qureshi et al. 1992). Curcumin is responsible for the yellow colour of turmeric. The

melting point of curcumin is 176-177°C, and it makes a reddish-brown salt with alkali (Braga

et al. 2003).


Figure 1. a: Turmeric plant, b: Turmeric rhizome, c:Turmeric powder (Palve and Nayak 2012), and d: Chemical structure of the three curcuminoids (Aggarwal et al. 2003)

a

b

c d


1.2.1. Pharmacological activity of curcumin

According to many clinical and preclinical studies conducted on curcumin during the past

decades, curcumin has been broadly acknowledged as a “wonder drug of the future”, due to

its extraordinary potential in preventing and treating several incurable illnesses (Epstein et

al. 2010).

Over the last 50 years, curcumin has been investigated for its anticancer properties. These

studies included the effect of curcumin on breast cancer (Carroll et al. 2008), colon cancer

(Shpitz et al. 2007, Moos et al. 2004), stomach cancer (Ikezaki et al. 2000), liver cancer

(Chuang et al. 2000), and oral cancer (Azuine and Bhide 1992) for which curcumin showed

chemotherapeutic effects.

Other studies included the effects of curcumin on diseases such as Alzheimer (Pan et al.

2008), gastric ulcer (Prucksunand et al. 2001), rheumatoid arthritis (Dcodhar et al. 2013),

diabetes (Usharani et al. 2008), psoriasis (Heng et al. 2000), bowel syndrome (Bundy et al.

2004) and HIV (Balasubramanyam et al. 2004), which revealed the potential of curcumin as

a useful therapeutic agent.

Further studies have investigated the effects of curcumin on the cardiovascular system, as

well as pulmonary and metabolic diseases (Aggarwal and Harikumar 2009). Also, studies

indicated the antimicrobial (Basniwal et al. 2011), antiviral (Zandi et al. 2010), antioxidant

(Binion et al. 2008), anti-inflammatory (Liang et al. 2009) and wound healing effects of

curcumin extensively (Panchatcharam et al. 2006, Sidhu et al. 1998).


1.2.2. Limitations of curcumin application as a bioactive agent

An ideal drug delivery system must be safe and deliver the encapsulated compound

efficiently at the site of action without exhibiting any adverse effects. Another central aspect

of drug delivery which should be taken into consideration is the release properties of the

system, which should happen in a desirable manner in order to show effective impacts

(Soppimath et al. 2001).

A large number of studies have investigated the biological and pharmacokinetic

characteristics of curcumin in animals, and to a lesser extent in humans (Epstein et al. 2010,

Ghalandarlaki et al. 2014). The majority of which have indicated that the poor solubility of

curcumin in water has restricted its full potential because it restricts bioavailability, stability,

and gastrointestinal absorption. Therefore, a wide range of research have been conducted to

improve the pharmacokinetic properties of curcumin in the past decades by developing

various formulations and structures to convey it efficiently to the targets (Ghalandarlaki et

al. 2014).

Different structures have been used for curcumin delivery to date which include:

1.2.3. Micro- and nanoparticles

Nano- and microparticles are small particles in the range of nanometer and micrometre

respectively including the core and the membrane as the main parts. The most significant

particularity of micro- and nanoparticles is their remarkably small size which effectively

increases the surface area to volume ratio, enabling them to penetrate the target efficiently

(Brannon-Peppas 1995).

Nano- and microparticulate systems for drug delivery have made a tremendous advance

towards enhancing bioavailability of hydrophobic drugs. These systems have given scientists


an opportunity to deliver the drugs specifically to the targeted sites, and overcome poor

solubility, bioavailability, inefficient permeability, rapid elimination and extensive

metabolism. They also protect the drug from migrating away from the target sites or being

cleared out of the body through biological degradation, as well as decreasing the required

dose due to their ability to maintain the applied quantity through proper encapsulation

(Brannon-Peppas 1995).

Micro- and nanoparticles have been extensively used in different clinical and non-clinical

trials in sustained and controlled patterns. Typically, drugs are trapped inside the particles or

adsorbed to the particle surface. Drug release from the particulate system generally is

specified with an initial burst release attributed to the drug adsorbed on the particle surface,

followed by a sustained and controlled release featuring the release of the trapped drug due

to particle degradation (Brannon-Peppas 1995).

1.2.4. Liposomes

Liposomes are artificial and synthetic vesicles described for the first time in 1965, with a

globular character that are formed of an aqueous core and amphiphilic lipid bilayer and

classified based on their lamellarity and size. Small unilamellar vesicles (SUV) are of 20-100

nm size, while large unilamellar vesicles (LUV) having a size of greater than 100 nm and

large multilamellar vesicles (MLV) having a size of greater than 0.5 µm (Faraji and Wipf

2009, Cho et al. 2008). They are stated to behave as immunological adjuvants and drug

carriers (Aqil et al. 2013). Liposomes can be applied for encapsulation of drugs with broadly

varying solubility or lipophilicity entrapped either in the aqueous core of the phospholipid

bilayer or at the bilayer interface (Aqil et al. 2013). In addition, they are able to deliver drugs


into cells by fusion or endocytosis, and practically any drug regardless of its solubility can

be entrapped in liposomes (Aqil et al. 2013) (Figure 2).

Figure 2. Curcumin encapsulated in liposome and the mechanism of entrance. Adapted

from (Ghalandarlaki et al. 2014) under creative commons rules

1.2.5. Micelles

Micelles are nanosized vesicular membranes which become soluble in water by gathering the

hydrophilic heads outside in contact with the solvent and hydrophobic tails inside, which is

known as emulsification (Aqil et al. 2013). They arrange themselves in a spherical form in

aqueous solutions with a very narrow range of 10 to 100 nm in size which increases their

stability in biological fluids. The functional properties of micelles are based on amphiphilic

block copolymers, which provides a nanosized core or shell structure in the aqueous media

(Jones and Leroux 1999). The hydrophobic core area stays as a pool for hydrophobic drugs

and is stabilised by the hydrophilic shell. Polymeric micelles act as transporters of water-


insoluble drugs such as curcumin, which can increase the drug’s efficiency by targeting

definite cells or organs leading to accumulation of fewer drugs in healthy tissues and

consequently decreased toxicity. Therefore, occasionally higher doses can be administered

(Jones and Leroux 1999).

1.2.6. Niosomes

Niosomes are microscopic vesicular constructions that are formed by self-association of

nonionic surfactants of alkyl or dialkyl polyglycerol ether category with cholesterol, which

were first established in the 70s (Cho et al. 2008). Niosomes can provide a container for drug

molecules with wide solubility range owing to the presence of amphiphilic, hydrophilic and

lipophilic moieties in their constitution (Azmin et al. 1985). Their architecture is similar to

liposomes in vivo and can be applied as an effective alternative to liposomal drug carriers,

because of their good stability, low cost, and ease of storage for different industries including

pharmaceutical, cosmetic and food applications (Azmin et al. 1985). Niosomes can be

applied in a number of potential pharmaceutical and therapeutic applications, such as

anticancer and anti-infective drug targeting agents (Aqil et al. 2013). They have the capacity

to improve the therapeutic indices of drugs by restricting their action on the target cells. They

also improve oral bioavailability of drugs which are poorly absorbed such as curcumin to

design a potential drug delivery system and boost the skin penetration of drugs (Jain et al.

2005).


1.2.7. Cyclodextrins

Cyclodextrins (Cds) are a group of complexes created from sugar molecules bound together

in cyclic oligosaccharides (Menuel et al. 2007). Cds are produced from starch by using an

enzymatic switch and are generally used in agriculture and in environmental engineering in

food, drug delivery systems, and chemical industries (Menuel et al. 2007, Davis and Brewster

2004). Cds form water-soluble inclusion complexes with small molecules and portions with

large complexes (Davis and Brewster 2004, Menuel et al. 2007). Cds consist of an interior

hydrophobic surface, which can provide a place for the residence of poorly water-soluble

molecules and an external hydrophilic area, which makes its solubility possible in the

aqueous setting. Additionally, Cds can augment bioavailability of insoluble drugs such as

curcumin by rising drug solubility and dissolution. Cds are also able to increase the

permeability of hydrophobic agents by making them accessible at the surface of the

membrane’s biological barrier (Aqil et al. 2013). According to the literature, Cds help

improve, the hydrolytic stability of curcumin, the protection against decomposition, the

bioavailability, and the molecular dispersion compared to the free curcumin without altering

their pharmacokinetic characteristics (Tomren et al. 2007, Yadav et al. 2010, Darandale and

Vavia 2013) (Figure 3).


Figure 3. The connection of curcumin to cyclodextrin. Adapted from (Ghalandarlaki et al.

2014) under creative commons rules

1.2.8. Dendrimers

Dendrimers were introduced in the mid-1980s, and are referred to as synthetic proteins,

which are created with structural controls that match traditional biomolecules. They are a

group of greatly split globular polymers which have different chemical and surface-related

properties. They have controlled structures with a globular shape with much higher accuracy.

They also have a single molecular weight instead of a distribution of molecular weights


typically observed in traditional linear polymers (Namazi and Adeli 2005). The dendrimer

structures are consisted of a core, branched interiors, and numerous groups of surface

functionals and act as a platform allowing extra substrates to be added in a highly controlled

way (Longmire et al. 2008). This nano space acts as a separate environment, which decreases

the payload related toxicity. The exceptional structure, dense spherical form controllable

surface, monodispersity and size properties of dendrimers result in outstanding

characteristics for drug delivery applications. Furthermore, the biocompatibility of

dendrimers increases their effectiveness in molecular imaging, which can be enlarged by

functionalization with small molecules of lower generation branch cells with anionic or

neutral groups rather than similar branch cells of higher generations that have groups of the

cationic surface. There are studies suggesting a potential use for them in curcumin delivery

(Yallapu et al. 2011, Sarbolouki et al. 2012, Alizadeh et al. 2012, Babaei et al. 2012).

1.2.9. Nanogels

Nanogels are a group of three-dimensional cross-linked polymer networks, which are created

by the help of covalent linkages and can also be modified to gel networks with degradable

and biocompatible properties. The porosity among these cross-linked networks not only

serves as a perfect reservoir for loading drugs but also keeps them from environmental

degradation. The swelling property of nanogels is controlled in an aqueous environment by

using the polymer chemical structure, cross-linking degree and the polyelectrolyte gel’s

charge density and/or by pH level, ionic strength, as well as the chemical nature of the low

molecular mass (Yallapu et al. 2011). Besides, nanogels can be altered chemically to include

different ligands for delivery of the targeted drug, the release of the triggered drug, or


composite materials preparation. Nanogels are introduced as carriers for drug delivery and

can be managed to allow oral delivery and brain bioavailability of low-molecular-weight

drugs and biomacromolecules (Kabanov and Vinogradov 2009). An efficient nanogel carrier

with broad biomedical capabilities should obtain acceptable stability in biological fluids, to

prevent aggregation (Gonçalves et al. 2012). Different nanogel properties can be realised by

modifying the functional groups, cross-linking density, and surface-active and stimuli-

responsive elements. Nanogels show excellent potential for systemic drug delivery that

should have some common characteristics such as a small (10-200 nm) particle size,

biodegradability, biocompatibility, high stability, lengthy half-life, high levels of drug

loading and/or entrapment, and protection from the immune system (Yallapu et al. 2011).

Drug release from nanogels’ networks depends on the hydrophobic and hydrogen

interactions and also, the harmonisation of drug molecules with polymer chain networks.

Preclinical studies recommend that nanogels can be used for biopharmaceuticals delivery in

cells in an efficient way (Maya et al. 2013). They also recommend that the nanogels can be

used for increasing delivery of a drug across cellular barriers. Overall, the architecture of

nanogel-bioactive conjugates can increase drug bioavailability, stability, loading efficiency,

dispersibility, effective transdermal penetration, efficient targeting, and treatment ability

against drug-resistant cancer cells and multicellular spheroids (Maya et al. 2013).

1.2.10. Polymeric-based Particles

Polymeric based particles are more common in drug delivery compared to lipid-based

particles due to their tissue and organ targeting properties. They can also be classified as


synthetic, or natural polymers. The following are some of the common natural types of

polymer-based particles utilised in drug delivery:

1.2.10.1. Chitosan

Chitosan is a positively charged biopolymer in the form of a linear polysaccharide derived

from deacetylation of chitin by using sodium hydroxide. Chitosan is insoluble in water and

organic solvents however, it is soluble in aqueous acidic solutions (pH < 6.5) and at lower

pH values it forms a gel. Chitosan is commercially available in the form of powder, dry flakes

or solution and has no toxicity in humans. Other properties, including biodegradability and

good compatibility make it a unique substance to be used in the biomedical and

pharmaceutical industries. One of the pharmaceutical applications of chitosan is to be applied

as a carrier for specific drugs or components (Bernkop-Schnürch and Dünnhaupt 2012).

Another important usage of chitosan is in wound healing as a result of some properties such

as non-toxicity, anti-inflammatory effect, hemostatic action, biodegradability,

biocompatibility, antimicrobial effect and stimulation of human dermal fibroblast activities.

Figure 4 shows the structure of chitosan achieved by modification of chitin.


Figure 4. Structure of chitosan. Adapted from (Albuquerque et al. 2016) under creative common rules

1.2.10.2. Alginate

Alginate is a linear anionic biopolymer consisting of 1, 4-linked β-mannuronic and α-

guluronic acid residues, which are obtained from algae. Alginate is extensively used in

delivery systems because of its simple structure, biodegradability, and biocompatibility.

Alginate is able to absorb water and make three-dimensional gels in the presence of calcium

in aqueous solutions. Because of the mentioned properties, alginate can be used as a

component of wound dressings as it can absorb body fluids up to 20 times its weight. The

resulting hydrophilic gel provides a moist environment for the wound and helps the healing

process. Figure 5 shows the structure of alginate.


Figure 5. Structure of alginate, adapted from (Rinaudo 2014) under creative commons

rules

The following are a few studies that have been conducted on the application of several drug

delivery materials, and structures for overcoming the limitation of curcumin pharmacological

action.

In a recent study, Das et al. (2010), designed a nanoformulation based on alginate (ALG) and

chitosan (CS), two biocompatible biopolymers to deliver curcumin to cancer cells. They

conducted initial studies by delivering the ALG-CS nanoparticles to HeLa cell lines.

However, the results were not promising because of the hydrophilic nature of the polymers

that caused low solubility of nanoparticles. In order to overcome the problem, they added

pluronic F127 (PF127) and made a new composite. According to scanning electron

microscopic and atomic force analysis, the particles had a spherical shape (100 ± 20 nm).

Additionally, there was an increased efficiency in composite nanoparticles over the

nanoparticles without PF127. Also, according to the cytotoxicity assay at 500 µg/ml

concentration, the nanoparticles were non-toxic to the Hela cells. Furthermore, as curcumin

is fluorescent it could easily be found in the cells, and the fluorescence microscope images

proved internalisation of the curcumin loaded nanoparticles into the cells. Therefore, ALG-

CS-PF127 composite nanoparticles of a suitable size distribution, encapsulation efficiency,


and release kinetics had the potential to deliver hydrophobic drugs for treatment (Das et al.

2010).

In another study, Song et al. (2011), developed a new formulation for curcumin delivery by

applying a biodegradable and biocompatible poly (L-lactic acid) based poly (anhydride-

ester)-b-poly (ethylene glycol) (PAE-b-PEG) micelle. They actually had mixed a polyester-

based polymer with a polyanhydrides polymer in order to overcome the release problem of

the nanopolymers. Polyester based polymers generally undergo bulk erosion and therefore

degrade slowly, while polyanhydrides undergo surface erosion resulting in rapid degradation.

Therefore, in order to develop a formulation with proper and controlled release kinetics, Song

et al. (2011) applied a combination of both. The developed micelles were absolutely water-

dispersible, overcoming the bioavailability problem. In vitro experiments indicated that

curcumin micelles were taken up by endocytosis and showed increased cytotoxic activity on

cancer cells compared to free curcumin. Additionally, the micelles exhibited stronger

antitumor activity, increased anti-angiogenesis effects and apoptosis on the EMT6 breast

tumour model. Also, in vivo safety tests including haematology analysis showed no sub-acute

toxicity to major organs or haematological system in mice intravenous administration (Song

et al. 2011).

Sahu et al. (2010) investigated the potential of a nanoformulation based on the two common

Pluronic triblock copolymer micelles including Pluronic F127 and F68 for curcumin

encapsulation. The potential of the micelles was examined by the encapsulation efficiency,

in vitro cytotoxicity and in vitro drug release of the encapsulated samples. The encapsulated

formulations were freeze-dried and reconstituted without using cryoprotectants. PF127

exhibited better efficiency in encapsulation and acceptable stability when stored for longer


periods compared to PF68. Physical interaction between curcumin and Pluronic was

examined by XRD analysis, fluorescence, UV-visible and FT-IR spectroscopy. According to

the AFM studies, the micelles had a spherical shape, and the diameters were below 100 nm.

In addition, the in vitro cytotoxicity of the drug formulations was carried out via HeLa cancer

cells. Also, encapsulated curcumin showed significant anti-cancer activity compared to free

curcumin. Therefore, a formulation based on Pluronic is a favourable means of curcumin-

delivery for clinical applications (Sahu et al. 2010).

Mukerjee et al. (2009) developed a carrier made of PLGA using solid/oil/water emulsion by

a solvent evaporation method for encapsulating curcumin. The experiments included

physicochemical characterisation plus cell viability and cellular uptake of the nanoparticles

in prostate cancer therapy. Physicochemical experiments revealed that the nanoparticles had

a spherical smooth surface with an encapsulation efficiency of around 90%. The size of the

nanoparticles ranged from 35 to 100 nm, with the mean size of 45 nm. Studies using a

fluorescence microscope showed an intracellular uptake of the nanoparticles in which the

cells were exposed to 100 µg/ml concentrations of curcumin-loaded nanoparticles for up to

3 hours. Before that, the cells were labelled with Nile red and DAPI. The results of their

investigation showed robust uptake of nanoparticles in all 3 prostate cell lines (Mukerjee and

Vishwanatha 2009).

1.3. Wound classification

Wounds are generally classified as acute or chronic based on the time the wounds require

healing. An acute wound can be described as a wound which heals within a short time and

can be repaired on its own in a well processed, orderly and time organised procedure.

Sustained physiologic and anatomical restoration of the function and integrity of the damaged


skin can thus be achieved (Nicks et al. 2010). Acute wounds generally occur as a result of

traumatic injuries such as burns, abrasions and lacerations (Nicks et al. 2010). A chronic

wound does not heal in a predictable, timely and well-programmed manner and fails to

establish an absolute restoration of the skin damaged structure and function (Lazarus et al.

1994, Sen et al. 2009). Wounds that cannot heal in three months are generally regarded as

chronic wounds and include pressure ulcers, venous ulcers, ischemic ulcers and diabetic foot

ulcers (Sen et al. 2009, Posnett et al. 2009).

Pressure ulcers often happen in patients with insufficient mobility and extend the damage

from skin to other parts of the body such as muscles, bones and tendons. In progressed levels,

they can lead to infection, osteomyelitis, septicaemia, and in more advanced levels, they may

even lead to death (Posnett et al. 2009, Sen et al. 2009).

Venous ulcers are usually produced as a result of venous hypertension (Billiar et al. 2004).

Ischemic wounds, which are characterised by a pale base, are caused by a declined blood

supply to the damaged parts that can lead to necrosis (Billiar et al. 2004).

Diabetic foot ulcers are one of the most common and prevalent complications that might take

place in diabetic patients, and early diagnosis is a pivotal factor in avoiding these

complications (Sen et al. 2009).

1.4. Natural wound healing mechanism

Skin serves as a natural barrier between an organism and its environment, protecting the

organism from a variety of damages including physical, chemical and microbial. When the

integrity of the skin is compromised by any injury, the body starts a continuous, multi-step

complex process at the injured site as a response in order to restore the function of injured


tissues. The main principles of optimal wound healing are to minimise tissue injury and

provide sufficient tissue perfusion and a moist environment to restore the function of the

damaged site (Eming et al. 2007).

The above-described natural process of wound healing consists of four distinct but

overlapping phases including homoeostasis, inflammation, proliferation and remodelling.

Homoeostasis starts upon injury during which platelet aggregation and blood clot formation

occur, which provides an extracellular cell matrix (ECM) for cell migration.

Hemostasis is the most immediate response to the injury and happens as a result of damage

to the blood vessels. Therefore, it is imperative to stop haemorrhage in damaged sites. This

is achieved by activation and aggregation of platelets through damaged blood vessels, which

leads to the formation of a clot composed of a network of insoluble fibrin fibres. The

produced clot provides a bed for growth factors and migration of various cells (At 2008). The

activated platelets are a major source of growth factors such as fibroblast growth factor,

platelet-derived growth factor, vascular endothelial growth factor and transforming growth

factor-β (TGFβ). These boost the different perspectives of the healing process and play an

important role in inflammation, angiogenesis, migration of fibroblasts and keratinocytes

(Bahou and Gnatenko 2004). Furthermore, serum, the fluid portion of clotted blood, is

another effective factor in the repair process as it contains factors such as interleukins, tumour

necrosis factor-α, colony stimulating factors, interferon-γ, and a series of other components

that together lead to a serum response factor (SRF). SRF induces transcription of

corresponding genes and various reports have indicated that up or down regulation of a broad

spectrum of genes occurs as an early response to injury even during the first hour in a


wounded site (Chai and Tarnawski 2002, Grose et al. 2002, Cole et al. 2001, Deonarine et al.

2007, Roy et al. 2008).

The inflammatory response, as the second phase consists of the migration of the blood cells,

including macrophages and phagocytic neutrophils towards the wound site. At the outset, the

phagocytes remove foreign particles and towards the end of the phase they start to produce

cytokines, which enhance the migration of fibroblasts.

The inflammatory response is initiated upon the leakage of neutrophils through injured blood

vessels into the wounded area within minutes of injury (Kim et al. 2008). Neutrophils are

responsible for cleaning the wound from invading microorganisms via various strategies such

as recruitment of reactive oxygen species (ROS) (Dovi et al. 2004). Moreover, the induced

expression of some genes in neutrophils in damaged parts is an obvious indicator of

neutrophils effect in other aspects of wound repair such as the resolution of the formed clots,

the progression of angiogenesis and also re-epithelialisation (Theilgaard-Mönch et al. 2004).

Monocytes are dragged from the blood circulation later than neutrophils and transform into

matured macrophages upon their entrance to the wounded tissue where they play a pivotal

role in clearing up the matrix and cellular debris (Mori et al. 2008, Martinez et al. 2006).

Furthermore, the immune cells, which reside within the damaged site such as mast cells, T

cells and Langerhans cells, are also stimulated, which in turn start releasing chemokines and

cytokines (Noli and Miolo 2001, Jameson et al. 2004, Cumberbatch et al. 2000). Also,

inflammatory cells can influence the surrounding tissue by ROS and NO formation. Although

it is well known that NO and ROS drive definite aspects of tissue repair, the injured sites

should perform some detoxifying strategies as a response to these stressors (Schäfer and

Werner 2008, Sen and Roy 2008).


Therefore, the inflammatory response is started with the recruitment of neutrophils and

macrophages from surrounded vessels, and is continued by induction of growth factor signals

from the serum and inflammatory cells (Eming et al. 2007). These signals subsequently

trigger expression of selectins, which control the passage of leukocytes to the vessel wall,

and crossing of the endothelial hedge (T. J. Shaw and Martin 2009).

A large number of studies have been performed to compare the gene signatures of different

wounds with and without inflammation. Although there are still debates about whether the

presence of inflammatory cells is a vital requirement for tissue repair or not, studies have

suggested that these cells can affect all the other cells at the injured site, as well as in the

surrounding tissue (Martin and Leibovich 2005). For example, it has been well established

that one of the main functions that inflammatory cytokines exhibit together with signals from

other cells during the repair process is angiogenesis (Tonnesen et al. 2000).

The third phase of wound healing is characterised by the formation of new blood vessels

(angiogenesis or neovascularisation), which helps to sustain the new tissues and the

fabrication and deposition of ECM protein such as collagen and granulation tissue and

epithelialisation. Overall proliferation, migration and contraction, which involve the actions

required to a temporary closure of wounds and renewal of the lost tissue happen in this phase.

This process starts within hours of the injury and will terminate at different times depending

on the size and location of the damaged site, as well as the health condition and age of the

patient. Angiogenesis, which is imperative to a successful wound healing involves the

formation of blood vessels and initiates with an endothelial cell bud migrating through the

wound gap (Fisher et al. 1994). The endothelial cells then divide and develop tubular

structures, which connect with other buds and differentiate into capillaries, venules and


arterioles accordingly (Martin 1997). The capillaries start to form a microvascular network,

which will be evident within a few days of injury. The microvascular network supplies

oxygen and nutrients to the damaged tissues and assists in developing the provisional matrix

called granulation tissue (Tonnesen et al. 2000).

The dominant acting cells in this phase are keratinocytes and fibroblasts, which reside in the

epidermis and dermis respectively. Re-epithelialisation of the wound, which proceeds in

parallel to angiogenesis is achieved through migration together with the proliferation of

keratinocytes in the wound gap. Generally, keratinocytes migrate across the wound

collectively after experiencing a series of modifications to ease their movements.

Keratinocytes modify cell-cell and cell-matrix bonds to potentiate their development from a

collagen-IV-rich BM and laminin-V onto the provisional matrix layers (T. J. Shaw and

Martin 2009). Likewise, they induce the expression of proteinases such as matrix

metalloproteinases (MMPs) (Pilcher et al. 1999). Fibroblasts are the dominant cells in the

dermis that generate the new extracellular matrix called granulation tissue using collagen

fibres as building units, which are crucial for tissue consistency (Garlick and Taichman

1994). Fibroblasts are drawn from different sources including the healthy and intact dermis

at the wound site, circulating fibrocytes, bone marrow pioneer cells and the pericytes, which

are associated with adjacent blood vessels (Hinz 2007, Abe et al. 2001, T. J. Shaw and Martin

2009). Collagen is a protein essential to the wound healing process and is known as the most

crucial connective tissue protein. There are several types of collagens according to the

structures they form among which type I and III are the most common identified in wound

healing. Generally, collagen fibres are formed in a basketweave design in healthy tissues.

However, this systematised structure cannot be formed at the wound site as fibroblasts nearby


the wound area align collagen fibres in concert to the stress lines of damaged tissue (Singh

et al. 2013). The formed stress fibres, consisting of actin bundles, which are able to contract

weakly, enable connective tissue contraction and the wound starts to shrink by recruiting

nearby tissue and drawing it into the wound. Fibroblasts are modified in a way that not only

allows them to move across the wound and be mobile, but also simultaneously, can pull the

edges of the wound to the centre of the wound (Singh et al. 2013). In the extracellular matrix,

collagen helps in maintaining the cells in place and enhances the contraction process, and re-

epithelialisation happens as a part of this phase (Gilbertson et al. 2001). Hence, fibroblasts,

and more specifically myofibroblasts, play a crucial role in this phase by contributing to the

formation, bundling and arrangement of collagen fibres, the principal component of scar

tissue (Singh et al. 2013).

The last phase of wound repair, which is characterised by tissue remodelling, epithelialisation

and scar formation is essential for full restoration of the function and appearance of the

damaged site. Several changes occur, during this phase at both epidermis and dermis level.

Keratinocytes, which act through migration and proliferation in the previous phase, confront

one another at the wound margins as the wound closes, after which they stop acting (T. J.

Shaw and Martin 2009). The blood vessels of the scar get refined and modified to generate a

functional network. ECM, which was formed in the early stages is remodelled, which restores

the normal structure of the dermis. Later in this phase activation of some enzymes such as

MMPs occurs which results in degradation of ECM proteins at the injured site (T. J. Shaw

and Martin 2009). Endothelial cells and myofibroblasts undergo apoptosis and macrophages

are deactivated by cytokines or phagocytosis. Overall, the quantity of collagen declines

whereas tensile strength increases due to the physical alterations of the newly formed


collagen (Enoch et al. 2006). Figure 6 indicates the different phases of wound healing

process.

Figure 6. Schematic process of wound healing process

1.5. Diabetes

Diabetes mellitus is a chronic disease and metabolic disorder described by high blood glucose

levels. Based on the statistics, in 2010, 285 million people had diabetes and Shaw et al. (2010)

reported that the number would increase to 439 million by 2030, which represents a 54%

increase (J. E. Shaw et al. 2010). A variety of factors will contribute to this substantial


increase including decreased physical activity and exercise, obesity, ageing of the

populations and modernisation of lifestyle. The above-mentioned figure for the incidence of

diabetes is assessed to have a 42.4% rise in North America, which was the most dominant

part of the world for diabetes incidence in 2010, and 65.1% rise in South, and Central

America by 2030. Europe, Pacific, and South Asia are expected to experience a 20, 47, and

72% increment in diabetes prevalence, while Africa is predicted to have a drastic increase of

98 % in adults by 2030. According to the study by Shaw et al. (2010) the diabetic population

will be evidently higher in developing countries by 2030 as the average population age will

slightly rise (J. E. Shaw et al. 2010)

Figure 7, compares diabetes outbreaks in 2010 and 2013, in different parts of the world.

If diabetes is not properly controlled, it might lead to various problems such as obesity,

stroke, coronary heart disease, diabetic retinopathy and diabetic nephropathy. Also, diabetes

contributes to numerous complications of the foot including foot ulcer, osteomyelitis and

Charcot’s neuroarthropathy. According to the literature, Australia’s national health

expenditure on chronic wounds such as foot ulcers is estimated to be approximately 500

million dollars per year (McGuiness and Rice 2009). The chronicity of wounds has become

a major problem not only because of the economic burden of the clinical costs, but also its

impact on patients’ emotional well-being and quality of life (McGuiness and Rice 2009).

Hence, it is imperative to conduct further investigations in the field of chronic wounds and

their management in order to improve both economic and social aspects of patients’ lives.


Figure 7. Prevalence of diabetes in the world in 2010 and 2030. Adapted from (J. E. Shaw

et al. 2010)

1.6. Diabetic foot ulcer

Whilst the sequence of healing occurs orderly and in a well-orchestrated pattern in acute

wounds eventually leading to wound closure, in diabetic wounds this process is disrupted

and results in non-healing wounds that are arrested in one, or more of the aforementioned

phases (Falanga 2005). Diabetic neuropathy known as nerve tissue damage, which is a

common complication occurring in diabetic patients, is characterised by an enhanced loss of

peripheral nerve fibres as a result of diminished blood flow and increased glycemic levels

(Dyck et al. 1993, Bašić-Kes et al. 2011). Diabetic neuropathy can occur in both diabetes

type I as well as type II, and is more frequent in aged patients. Typically, the symptoms of

neuropathy emerge 10-20 years following diagnosis of diabetes and almost 50% of the

diabetic cases develop nerve damage to some extent (Rathur and Boulton 2005). While many

patients are prone to developing this complication even at early stages, others might never

experience it (Boulton et al. 2004). According to a wide range of studies, numerous factors


contribute to the emergence of foot ulcers. Vascular diseases and ischemia disrupt the healing

process because of the decreased levels of oxygen and nutrients conveyed to the damaged

tissue (Guo and DiPietro 2010). Also, enhanced nitric oxide levels, which lead to increased

formation of ROS, along with downregulation of glutathione and cysteine contribute to

impaired healing (Brem and Tomic-Canic 2007). Additionally, abnormal extension of the

inflammatory phase originating from impaired expression of growth factors such as TGF-β

contributes to this pathology (Tsunawaki et al. 1988). There are reports indicating decreased

production of cytokines and macrophage dysfunction, consequently leading to an impaired

capacity of macrophages to clear the wound site from necrotic materials, which is essential

for a proper and organised healing process (Khanna et al. 2010, Mirza and Koh 2011, Robert

Blakytny and Jude 2009, Liu et al. 1999).

The factors which contribute to impaired healing in diabetes during the angiogenesis phase

include downregulation of hypoxia inducible factor-1α (HIF-1α), which is responsible for

transcription of some growth factors such as vascular endothelial growth factor (VEGF),

thereby the levels of VEGF will be diminished (Catrina et al. 2004, Botusan et al. 2008). A

study by Pradhan et al. (2011) revealed decreased expression of several other angiogenesis

promoting growth factors, such as insulin like growth factor 1 (IGF-1), epidermal growth

factor (EGF), platelet derived growth factor (PDGF) and interleukin-8 (IL-8), which

stimulate cell proliferation, and migration (Pradhan et al. 2011). A synchronised formation

of ECM is of great importance in the process of wound healing, as different endothelial cells

migrate in this way and use ECM as a scaffold while creating new blood vessels during

neovascularization. Hence, finally and most significantly, excessive activity of matrix

metalloproteinases (MMPs), or downregulation of MMP inhibitors, represent the most


destructive factors in diabetic wound healing process. MMPs can disrupt the healing

procedure by breaking down growth factors, as well as ECM components such as collagen,

leading to poorly developed ECM and consequently impaired re-epithelialisation and

impaired wound healing (Natrajan et al. 2015, Stricklin et al. 1993, Han et al. 2001). Figure

8 illustrates the healing process in regular wound healing compared to the diabetic wound.

The clinical treatment of skin injury due to different severe burns or wounds continues to be

a significant problem. A prominent therapeutic agent should be able to augment one or more

phases of the wound healing process without making detrimental side effects.


Figure 8. Wound healing process in normal and diabetic wounds. Reproduced with

permission from (Moura et al. 2013)


1.7. Effects of curcumin on different phases of wound healing and mechanism of

action

According to the previous published studies, curcumin can influence different stages of

wound healing including inflammation, proliferation and contraction.

1.7.1. Effect of curcumin on inflammation

Inflammation is the second most serious stage in wound healing and is often known as the

first decisive step in optimum recovery. Having control over immediate acute inflammation

of skin injury is essential. Indeed, uncontrolled inflammatory reactions can lead to serious

and sometimes severe subsequent tissue damages which can be seen in rheumatoid arthritis

as an inflammatory disorder (Singer and Clark 1999). Joe et al. (2004) studied the mechanism

of action of curcumin on inflammation and summarised:

- Ability to inhibit the production of two main cytokines released from macrophages

and monocytes that have a major impact on the regulation of inflammatory responses

comprising tumour necrosis factor alpha (TNF-α), and (IL-1).

- Ability to inhibit activity of NF-(κ) B (nuclear factor kappa-light-chain-enhancer of

activated B cells), which has been considered as an oxidant-responsive transcription

factor that regulates many genes involved in the initiation of inflammatory reactions

(Frey and Malik 2004).

- Reducing oxidation (a major cause of inflammation) by exhibiting scavenging action

on reactive oxygen species (ROS) (Joe et al. 2004).


Curcumin has been shown to reduce inflammation which consequently leads to the

acceleration of the transition to the subsequent phases including proliferative and

remodelling.

1.7.2. Effect of curcumin on the proliferative phase of wound healing

This phase includes four stages of fibroblast proliferation, granulation tissue formation,

collagen deposition and, apoptosis. The effect of curcumin on all these stages has been

investigated as below:

1.7.2.1. Effects of curcumin on the fibroblast proliferation

The presence of fibroblasts in the wound and damaged sites is of essential importance for the

damaged tissue to be healed. According to the study by Blakytny and Jude (2006), cutaneous

wounds, which fail to be healed in an appropriate time period usually have impaired

fibroblast proliferation and migration in the damaged area. The infiltration of fibroblasts into

the damaged site is of significant importance for granulation tissue formation/remodelling,

collagen production and remodelling (R Blakytny and Jude 2006).

Several studies have shown the positive effect of curcumin on the infiltration of fibroblasts

into the wound area. Mohanty et al. (2012) investigated the formulation of a curcumin loaded

polymeric bandage in a rat model. The biochemical analysis revealed enhanced and faster

wound reduction and increased cell proliferation of the samples. The accelerated effect of the

bandage was due to early implementation of fibroblasts and their differentiation (Mohanty et

al. 2012). However, there are limitations in the application of curcumin. In contrast

Scharstuhl et al. (2009), revealed that higher concentrations of curcumin (25 µM) caused cell


apoptosis turning oxidative, but at lower concentrations positive results were achieved

without any indication of cell death (Scharstuhl et al. 2009).

1.7.2.2. Effects of curcumin on the stage of granulation tissue formation

Almost four days after skin injury, new stroma or granulation tissue starts to grow. This

process is characterised by the development of new small capillaries and the infiltration of

fibroblasts, which helps the formation of extracellular matrix (Singer and Clark 1999). The

newly formed tissue helps the migration of the epithelial cells and wound closure and

improves re-epithelialization. Mohanty et al. (2012), demonstrated that the wounds in rats,

which had been treated by COP (curcumin-loaded acid, oleic polymer) showed a better

alignment of granulation tissue 10 days post-treatment and just a marginal improvement four

days after injury (Mohanty et al. 2012).

1.7.2.3. Effect of curcumin on the stage of collagen deposition

Another main pre-requisite for the wounded site to be healed in a suitable period is the

remodelling of the extracellular matrix, which is composed of different proteins such as

collagen that mainly contributes to 70-80 % of the skin. The last purpose in the wound healing

process is the establishment of scar tissue, which mainly consists of collagenous fibres.

Therefore, the formation of adequate collagen and its deposition contributes to wound repair

to a large extent. Dai et al. (2009) demonstrated that the content of collagen increased in rat

models treated with curcumin sponge, and that they were thicker and had been organised in

more compact rows compared to controls (Dai et al. 2009). Similarly, another study by

Panchatcharam et al. (2006) showed not only increased amount of collagen in topically


curcumin treated wounds in rats, but also that collagens were able to mature faster. Another

result of their study was the increase in the amount of aldehyde in collagens, which is an

indicator of the high cross-linked structure of the produced fibres (Panchatcharam et al. 2006,

Mohanty et al. 2012). This finding also confirmed by Mohanty et al. (2012), demonstrated

an increase in collagen level as well as a higher content of aldehyde in a rat model wound,

treated with curcumin bandage compared to the control.

1.7.2.4. Effect of curcumin on the stage of apoptosis

During the wound healing process, apoptosis of a group of unwanted inflammatory cells is

as essential as the growth of some other cells, in order to pass the inflammatory stage and

achieve the next stage. Scharstuhl et al. (2009) showed that curcumin could cause apoptosis

due to its ability to induce ROS, even though there was little information on its mechanism

of action (Scharstuhl et al. 2009). Mohanty et al. (2012), using a rat model, reported the

apoptotic effect of curcumin at the four days post-treatment stage when treated with curcumin

bandages. They demonstrated that the rate of apoptosis was low at the early stage of wound

healing in control samples whereas in curcumin treated samples the transition from the early

stage to the proliferative stage had been accelerated (Mohanty et al. 2012).

1.7.3. Effect of curcumin on the stage of wound contraction

Wound contraction, which is the part of the ultimate stage of wound healing, occurs when

fibroblasts switch to myofibroblasts around two weeks after the injury. This stage is a

complex stage consisting of different interactions between extracellular matrix proteins, cells

and cytokines. Durgaprasad et al. (2011) reported that curcumin was able to augment wound


contraction, which leads to wound healing acceleration. By measuring the size of a wound

and tracking it during the post-wounding period, they showed that curcumin-treated samples

had a 20% increase in the wound contraction rate (Durgaprasad et al. 2011). Dai et al. (2009)

showed a 16% increase in wound contraction following curcumin sponge treatment

compared to controls (Dai et al. 2009).

1.8. Research gap

A review of the literature shows that there is extensive evidence that curcumin possesses a

number of therapeutic effects, most notably in wound healing. However, delivery and

bioavailability issues, arising from poor solubility, rapid metabolism in the body and

chemical instability, limited the practical development of curcumin as a promising drug

candidate. Curcumin is more suited as a wound healing agent for topical applications rather

than for oral administration. A number of delivery vehicles have been formulated with

curcumin in order to enhance its wound healing effects. These included collagen films

(Gopinath et al. 2004), chitosan-alginate sponges (Dai et al. 2009), alginate foams (Hegge et

al. 2011), polymeric bandages (Mohanty et al. 2012) and creams (Durgaprasad et al. 2011).

These formulations have been shown to increase the wound healing effect of curcumin

compared to the application of free curcumin itself. However, there is rather limited research

on the potential of curcumin delivered in the encapsulated form as a topical agent for wound

healing with the possibility of delivering better outcomes than the other formulations.

Optimisation of physicochemical characterisation of encapsulated curcumin as a wound

healing agent are yet to be established.


This thesis addresses this research gap by attempting to develop a biodegradable and

biocompatible polymeric composite for curcumin encapsulation, to establish the optimum

formulation conditions, to evaluate the drug release characteristics and to apply the

encapsulated curcumin in a wound healing cell line model.

1.9. Research hypotheses

The hypotheses addressed in this thesis were that:

1- Encapsulation of curcumin in a polymeric-based composite consisting of alginate,

chitosan and Pluronic F127 will improve solubility and stability compared to free

curcumin.

2- Pluronic F127 increases the encapsulation efficiency and release of curcumin with

alginate and chitosan.

3- Curcumin-loaded particles are taken up by human keratinocytes cells and display less

inhibitory effects on cell growth compared to free curcumin.

1.10. Research aims

In order to test the hypotheses, the aims of the thesis were to:

1- Establish optimal formulation parameters for the encapsulation of curcumin with

alginate and chitosan, with or without Pluronic F127.

2- Determine the physicochemical characteristics of the curcumin particles, which

would include

� Examination of particle shape by Transmission Electron Microscopy (TEM)


� Determination of zeta potential, polydispersity and size of the curcumin-loaded

particles

� Determination of curcumin encapsulation efficiency

� Studying in vitro release kinetics of curcumin from particles

3- Determine the effects of curcumin loaded particles on cell viability, cell cytotoxicity

and cellular uptake using a human keratinocyte cell line.

Chapter-2: Materials and methods Page 37

Chapter 2: Materials and methods

2.1. Materials

Sodium alginate (low viscosity), chitosan (low molecular weight, with 75-85% deacetylation

degree), Pluronic F127 (PF127), calcium chloride (CaCl2) and curcumin (≥ 94% curcuminoid

content, ≥ 80% curcumin) were all purchased from Sigma Aldrich. All the chemicals were

of analytical grade. MilliQ water was provided by the laboratory.

2.2. Equipment

UV-VIS spectrophotometer (LKB Navaspec Ⅱ, model 80-2088-64, serial no: 53652),

sonicator (Ultrasonic cleaner, Unisonics, type: FxF8M), centrifuge (Eppendorf, model:

Centrifuge 5415C), ultracentrifuge (Beckman Coulter, model Avanti JE, 875892) were the

major appliances used for this project. All the equipment were provided by Chemistry and

Microbiology Laboratory, Faculty of Veterinary and Agricultural Sciences (FVAS), The

University of Melbourne.

2.3. Preparation of blank alginate/chitosan/PF 127 particles

2.3.1. Preparation of stock solutions

Sodium alginate and calcium chloride solutions (0.63 and 2 mg/ml) were prepared by

dissolving 14.36 and 2.72 mg respectively in milliQ water. Chitosan solution (0.47 mg/ml)

was prepared by dissolving 2.24 mg in 1% (vol/vol) acetic acid. Curcumin solutions were


prepared in ethanol at a concentration of 1 and 2 mg/ml and pH values of alginate and

chitosan solution were adjusted to 5.2 and 4.9 respectively.

2.3.2. Preparation of the blank particles

Particles were prepared based on the ionotropic gelation at room temperature according to

Rajaonarivony et al. (1993), with some modifications. Briefly, PF127 was added at the

required concentrations (0, 0.1 and 1% w/v) to 1.36 ml calcium chloride (2 mg/ml), and the

solution was added to 22.8 ml sodium alginate solution (0.63 mg/ml) under mild magnetic

stirring for around 30 min, in a dropwise manner. After 15 min, 4.78 ml of chitosan solution

(0.47 mg/ml) was added in the same manner and stirred for 45 min. The resultant solution

was equilibrated overnight at room temperature to allow the particles to form (Das et al. 2010,

Rajaonarivony et al. 1993).

2.3.3. Preparation of curcumin loaded particles

CaCl2 solution (2 mg/ml) was added to curcumin solutions (1 and 2 mg/ml). After that PF127

solution, was added at the required concentrations (0, 0.1 and 1% w/v) to the resultant

curcumin-CaCl2 solution, and the next steps were followed as explained above for blank

particles.

Figure 9 schematises the particle formation process, and the ultracentrifuge utilised in

fabrication procedure.


Figure 9. Schematic process of curcumin particle fabrication


2.4. Optimisation of the procedure parameters

The characteristics of the fabricated particles were investigated by applying various

concentrations of curcumin solution (1 and 2 mg/ml), PF127 as a non-ionic surfactant (0, 0.1

and 1% w/v), and rinsing methanol/water solution (20/80, and 50/50). The optimum

formulation was chosen on the basis of the total physico-chemical characteristics including,

particle size, charge, encapsulation efficiency, and release studies.

2.5. Physicochemical characterisation of the particles

2.5.1. Particle size, zeta potential and polydispersity index

Characterisation of hydrodynamic size and zeta potential and PDI was performed by a

Zetasizer Nano-ZS Malvern. Particles size indicates the diameter of the particle and PDI is

an indicator of particle size distribution and stability. Zeta potential provides information

about the difference between the potential of the dispersion fluid and the attached layer of

the fluid to the particles.

All the measurements were performed using the 1:10 (v/v) diluted suspension of particles in

milliQ water with an equilibrium time of 120 second, and 20 runs. During the whole process

the temperature was kept at 25°C and 3 replicates were done for all 3 parameters.

2.5.2. Morphology determination by transmission electron microscopy (TEM)

In order to obtain precise morphological characterisation of the particles, transmission

electron microscopy (TEM) was applied to 4 sets of particles including alginate-chitosan-

PF127-curcumin at 0.1% concentration of PF127, and 0.1% curcumin, as the drug-loaded

particles and also the control particles without curcumin in the presence of 0%, and 0.1%


PF127. The particles were suspended in distilled water and a drop of each sample was placed

on a carbon-coated copper grid, which was left for a few minutes to be air dried at room

temperature. Then the samples were loaded into the TEM microscope model Philips CM120

BioTwin.

2.5.3. Encapsulation efficiency of loaded curcumin

To determine the encapsulation efficiency of the fabricated particles, the particle solution

was centrifuged at 20,000 rpm at 4°C for 20 min. The supernatant was removed, and the

pellet was washed twice with a mixture of methanol and distilled water based on the

experimental design, in order to remove any unbound curcumin. The curcumin in both

supernatant and the rinse washes were analysed and quantified spectrophotometrically at 425

nm, which corresponded to the maximum absorbance of curcumin.

Therefore, the amount of non-encapsulated curcumin (free curcumin) was calculated based

on a calibration curve equation:

y = 1586x- 0.1614 (R2 = 0.978)

y is the absorbance, and x is the amount of curcumin. Encapsulation efficiency was calculated

as follow:

Encapsulation efficiency (%) = (Total of curcumin-Total Free curcumin) / Total of curcumin

Total free curcumin is the amount of free curcumin in the first supernatant plus the amount

of free curcumin in rinse supernatants.

Figure 10 shows the standard curve of curcumin in methanol applied in determination of

encapsulation efficiency.


Figure 10. Standard curve of curcumin in methanol

2.5.4. In-vitro curcumin release studies

To perform the release study of encapsulated curcumin, dialysis methodology was applied.

Dialysis tubes were soaked in distilled water and then rinsed with phosphate buffered saline

(PBS) to remove preservatives. Then 2 ml of curcumin loaded particle suspension was placed

in the dialysis tube and sealed. For each experiment 8 tubes were prepared and dispersed in

600 ml PBS solution at pH 7.4. The solutions were placed in a shaking incubator under

stirring at 120 rpm at 37°C. At determined time intervals, one of the samples was withdrawn

and evaluated spectrophotometrically at 425 nm based on the standard curve of curcumin

explained above. The release was calculated as below:

Release (%) = Released curcumin / Total curcumin ×100

Figure 11 shows different solutions of curcumin, and the dialysis tube used in release kinetic

experiment.


a b c

Figure 11. a: curcumin particle solution, b: curcumin in distilled water, c: curcumin particle

in PBS in dialysis tube

2.6. Cell culture

The human FEP-188 keratinocyte cell line transformed by human papillomavirus, were

derived from neonatal foreskin (provided from Peter McCallum Cancer Institute, East

Melbourne), and cultured in serum-free growth medium (K-SFM) (Life Technologies),

containing L-glutamine, EGF and BPE, supplemented with 2.5 µg EGF human recombinant,

and 25 mg bovine pituitary extract. The cells were maintained in an incubator at 37°C in a

humidified atmosphere containing 5% CO2 up to the point when they reached 80% of

confluence.

2.7. Routine cell culture

Human FEP-188 keratinocyte cell line, which was cryopreserved using 10% DMSO, was

initially thawed and added to 10 ml of K-SFM media, then centrifuged at 1,200 rpm for 5


min, at room temperature using an Allegra X-22R centrifuge (Beckman Coulter, California,

USA). Following the removal of the supernatant, the cells were suspended in 10 ml of the

respective media, then transferred to a flask and incubated within a humidified atmosphere

including 5% CO2 at 37°C (Sanyo, Osaka, Japan). The cells were grown as a monolayer

within the flask and were visualised on a daily basis using an Olympus CKX41 phase contrast

microscope (Olympus, Tokyo, Japan) at 20X magnification. At appropriate time intervals,

the media was changed to supply the cells with enough nutrients for continued growth.

2.8. Preparation of cell culture for treatments

Once the cells reached about 80% confluence as a monolayer within the flask, the medium

was aspirated and washed two times with 10 ml of PBS. Subsequently, 5 ml of trypsin-

ethylenediaminetetraacetic (trypsin-EDTA) 0.05% (v/v) (Life Technologies) was used to

trypsinise the cells until the morphology of the cells switched from attached, elongated, dark

cells to detached, round, bold and bright cells. Then immediately 7 ml of supplemented

DMEM was used to inactivate the trypsin-EDTA, as K-SFM lacks FBS (fetal bovine serum),

which is necessary to inactivate the trypsin. This step was followed by pipetting the

suspension against the back wall and back corner of the flask, each for ten times, in order to

get a single cell suspension, and subsequently transferred to a 15 ml falcon tube and

centrifuged at 1,200 rpm, for 5 min at room temperature. The supernatant was discarded and

the cell pellet was suspended in 10 ml of the corresponding media, reverse pipetted in order

to achieve a uniform cell suspension, before being transferred to a T75 cm2 flask containing

medium.


In order to determine the number of cells required for the experiments, 10 µl of the above-

mentioned suspension was removed and the cells were counted using a haemocytometer in 4

fields of view and the number of the cells were estimated based on the mean of the mentioned

four fields.

2.9. Drug preparations for cell treatments

Free curcumin is not soluble in water, therefore first it was dissolved in DMSO and then

diluted with sterile PBS. The highest concentration of DMSO did not exceed 0.4% for cell

treatments. Encapsulated curcumin particles were directly suspended in sterile PBS and

administered to the cells.

2.10. Cell viability assay

In order to determine the susceptibility of FEP-188 keratinocyte cells to free and encapsulated

curcumin, a cell viability assay was performed using Cell Titre Blue® reagent (Promega,

Winchester, USA). For economy of resources, the FEP-188 keratinocyte cell line was applied

as a representative of skin cells in our study. FEP-188 keratinocyte cells were plated at a

density of 3 × 104 cells per well in a 96 well flat bottom black sterile culture plate (Promega)

and incubated to let the cells attach to the wells. After 24 h, the cells were treated with

different concentrations of free curcumin dissolved in DMSO, and curcumin particles (0,

0.156, 0.312, 0.625, 1.25, 2.5, 5, 10, 15, 20, and 25 µg/ml). This range was selected based on

the literature review (Scharstuhl et al. 2009, Phan et al. 2001) Untreated cells served as

controls. Then the cells were incubated, for another 24 h. After cell treatment with the drug,

20 µl of Cell Titre Blue® reagent was added to each well, which was followed by incubation


for 4 h. The procedure was terminated after 5 days, and cell proliferation was assessed by

fluorescence intensity at 550 nm excitation, and 615 nm for emission using the multilabel

plate reader (Perkin Elmer, Massachusetts, USA); (Boonkaew et al. 2014, Böhmert et al.

2012). All the experiments were performed in triplicates.

IC50 or half maximal inhibitory concentration is a specific concentration of the drug which

inhibits cell growth by 50%. Cell Titre Blue reagent is a buffered solution containing pure

resazurin dye, which is used to evaluate the metabolic activity of cells, and consequently,

indicates the cell viability. Viable cells retain the ability to reduce resazurin to resorufin, and

produce fluorescent signals, while nonviable cells lose their capacity to reduce resorufin and

thus cannot generate a fluorescent signal. Resazurin is dark blue in colour and produce low

intrinsic fluorescence until it is reduced to resorufin, which is pink in colour and strongly

fluorescent. Resazurin is reduced to resorufin as a result of various redox enzymes released

from mitochondrial, microsomal, and cytosolic sites.

2.11. Microscopic cell toxicity

Morphology analysis was carried out to verify the microscopic features of the FEP-188

keratinocyte cells treated with different concentrations of curcumin particles and free

curcumin dissolved in DMSO. FEP-188 keratinocyte cells were plated at a density of 2 × 105

cells per well, in 6- well culture plates and incubated at 37°C in a humidified atmosphere

containing 5% CO2 to let the cells become confluent. After 24 h, the cells were treated with

four different concentrations of free curcumin dissolved in DMSO and equivalent

concentrations of encapsulated curcumin, followed by incubation of the cells for another day.

Untreated cells served as controls. The procedure was terminated after 3 days.


2.12. Cellular uptake by confocal microscopy imaging

Having determined the physicochemical characteristics of different formulations, the ability

of the optimal formulations to internalise FEP-188 keratinocyte cells was investigated by

confocal microscopy. Curcumin is a fluorescent compound and can therefore be tracked

inside the cell by examining fluorescence without the need to tag a fluorescent dye to the

compound. Therefore, taking advantage of the intrinsic photochemical property of curcumin,

an investigation on curcumin intracellular uptake by FEP-188 keratinocyte cells was

conducted.

To prepare the samples for microscopic imaging, FEP-188 keratinocyte cells were cultured

in serum-free growth medium (K-SFM) containing L-glutamine, EGF, BPE, supplemented

with EGF human recombinant and bovine pituitary extract. Subsequently, cells were seeded

at a density of 105 cells on a coverslip in a 6-well tissue culture plate, followed by incubation

at 37°C in a humidified atmosphere containing 5% CO2. After 24 h the cells were exposed

to 20 µg/ml of free and encapsulated curcumin, and confocal scanning microscopic imaging

was carried out on the control cells, cells with free curcumin in PBS, cells with empty

particles and cells with curcumin particles over a time-dependent period. Fluorescent images

of the treated and untreated cells were monitored with a Nikon A1r confocal microscope

(Nikon, Tokyo, Japan).

2.13. Statistical analysis

SPSS software (version 23) was applied for the statistical analysis. All the data were

expressed as mean ± SD. One-way ANOVA was utilised for comparing the mean values, and


multiple comparisons were made using Tukey’s multiple comparison tests. Also, T-test was

used whenever required. Statistical significance was determined at P < 0.05.

Chapter-3: Results and discussion Page 49

Chapter 3: Results and discussion

3.1. Physicochemical experiments

3.1.1. Effects of methanol (%) rinse on particles

The concentration of methanol was optimised to enable separation of free from encapsulated

curcumin while retaining particle integrity. Higher concentrations of methanol (%) rinse,

resulted in significantly lower encapsulation efficiency at lower curcumin concentrations.

However, higher curcumin concentrations resisted methanol wash and the difference was not

significant (0.2%) (Figure 12). Also, the effect of different methanol (%) rinse concentrations

was not significant on other parameters including particle size, PDI, and zeta potential (p

value > 0.05) (The data are not shown).

a


b

Figure 12. Variation in curcumin encapsulation efficiency by variation of PF127

concentration, and methanol rinse (MR) at a: 0.1% curcumin concentration, and b: 0.2%

curcumin. All data represent mean ± SD (n =3) and *p value < 0.05

3.1.2. Effects of PF127

Up to 1% (w/v) PF127 was used and the results from previous reports were corroborated.

PF127, the non-ionic surfactant, decreased the surface tension and helped to increase the

solubility of curcumin in the hydrophilic fluid. The increase in the concentration of PF127

from 0% to 1% (w/v) enhanced the encapsulation efficiency in all formulations (Figure 13).

This could be attributed to the increased solubility of curcumin in the aqueous phase, which

consequently led to a marked enhancement in the encapsulation efficiency of the particles.

This finding is in accordance with the study by Rajath et al. (2013), which reported that

incorporation of PF127 to the formulation at an appropriate concentration resulted in a higher


encapsulation efficiency. Also, Das et al. (2010), found that addition of PF127 to the

formulation resulted in a 5-10 fold increase in the final encapsulation efficiency. Bhunchu et

al. (2015), demonstrated that an increase in the PF127 concentration from 0% to 1% (w/v)

resulted in an increase in the encapsulation loading capacity of the particles. However, further

increase in PF127 concentration to 1.5% (w/v) decreased the resulting encapsulation

efficiency.

A variety of surfactants are used in the pharmaceutical and cosmetic industries. Various

surfactants have been used in previous studies to decrease the surface tension between

hydrophobic and hydrophilic phases. The safety of non-ionic surfactants over anionic or

cationic ones is well established and satisfactory results have previously been reported with

PF127 (Raveendran et al. 2013, Sahu et al. 2010, Lippens et al. 2013).

The addition of 0.1% PF127 to the solution significantly decreased the particle size.

However, the size was increased at above 0.1% PF127 (Figure 13). This can be attributed to

the stabilisation effect of PF127, which inhibits the aggregation of the particles by steric

stabilisation (Bhunchu et al. 2015). Hence the size was decreased in the presence of PF127.

However, the increase in PF127 concentration beyond 0.1% raised the viscosity of the

aqueous phase, which resulted in the formation of larger particles. These results were further

supported by Bhunchu et al. (2015), who reported the increase in particle size at 1% PF127.


a

b

Figure 13. Variation in curcumin encapsulation efficiency, and particle size by variation of

PF127 concentration and methanol rinse (MR) at a: 0.1% curcumin concentration, and b:

0.2% curcumin. All data represent mean ± SD (n =3), and *p value < 0.05


PDI significantly increased at 1% PF127. However, no significant changes were seen at a

range of concentrations between 0% and 0.1% of PF127 (Figure 14).

The zeta potential of the particles was negative as alginate formed the core part of the

particles. The zeta potential became slightly less negative with a proportional increase in

PF127 concentration % (Figure 14). These phenomena may have been due to the micelle

forming behaviour of PF127 around curcumin, and the increased interaction of PF127 with

the polymeric system, by its polar head groups. Hence, the increase in PF127 concentration

from 0.1% to 1% can enhance the interaction of PF127 with the polymer network,

neutralising the negativity of the particle surface and resulting in the formation of larger

particles.

a


b

Figure 14. Variation in PDI, and zeta potential by variation of PF127 concentration and

methanol rinse (MR) at a: 0.1% curcumin concentration, and b: 0.2% curcumin. All data

represent mean ± SD (n =3), and *p value < 0.05

3.1.3. Effects of curcumin concentration on zeta potential

The zeta potential values were within the range of -6.60 to -17.50 mV for different curcumin

loaded particles, and -14.4 to -23.5 mV for the blank particles which were relatively lower

than the same polymeric matrix applied in previous studies conducted by Bhunchu et al.

(2015), and Fattahpour et al. (2015). A significant difference in zeta potential occurred

between the curcumin-loaded particles, and the blank particles without curcumin, indicating

more stability of the blank particles compared to curcumin loaded particles.

Zeta potential is a parameter which provides information on the stability of the dispersions

as well as information about the surface charge of the particles. Hence, zeta potential can

specify the interaction level between the particles. Generally, zeta potential values above +30


mV or below – 30 mV, is considered stable (Bhunchu et al. 2015). The mentioned range for

zeta potential is strong enough to maintain the repulsion between the particles, and to avoid

the aggregation of the particles. Higher rates of increase or decrease in zeta potential

intensifies the repulsion between particles ultimately resulting in the formation of more stable

dispersions, along with more uniform and narrow size distribution. Wu et al. (2011),

suggested that particle suspensions containing PF127 were stable within the zeta potential

range of -20 to -30 mV (Wu et al. 2011). Figure 15 depicts the zeta potential graphs of blank

particles, and optimised curcumin-alginate-chitosan-PF127 particles.

a


b

c

Figure 15. Zeta potential plot of curcumin particles a: empty particles (blank)

b: formulation with 20% methanol rinse, c: formulation with 50% methanol rinse


3.1.4. Effects of curcumin concentration on particle size

The addition of curcumin to the system produced significantly larger particles compared to

the blank particles without curcumin (P < 0.05) (Figure 16). However, no significant increase

in size was seen while different concentrations of curcumin were applied. There were no

significant changes in PDI, zeta potential and encapsulation efficiency of different

formulations.

Figure 16. Variation in particle size by variation of PF127, and methanol rinse

concentration at different concentrations of curcumin. All data represent mean ± SD (n =3),

a, b p value < 0.05 between different curcumin concentrations and * p value < 0.05 within

groups


The size of the particles was within the range of 382.2 to 1358 nm, and 956.77 to 2781.33

nm for the blank and curcumin loaded particles respectively. The size of the curcumin-loaded

particles was significantly increased (P < 0.05) over the blank particles in almost all

formulations. Figure 17 shows the size distribution (by intensity), graphs of blank particles,

and optimised curcumin-alginate-chitosan-PF127 particles.

PDI is an indicator of the dispersity of the particles and reveals if the particles were

monodisperse or not. The optimum and desirable particles are the monodispersed, which

have a narrow size distribution (Azevedo et al. 2014). The PDI values ranged from 0.38 to 1

with lower ones confirming more narrow distribution.

a


b

c

Figure 17. Size distribution of intensity for curcumin particles a: empty particles (blank) b:

formulation with 20% methanol rinse, c: formulation with 50% methanol rinse


Encapsulation efficiency is a crucial factor in the efficient characterisation of an encapsulated

drug. The highest level of encapsulation efficiency was 84.29%, which was reached at a

PF127 concentration of 1% (w/v). The encapsulation efficiencies in the optimal conditions

were 75.69% and 72.69%, being considerably better than the encapsulation efficiency

reported by Das et al. (2010), which was approximately 13%. Therefore, optimisation of

different formulations under different conditions resulted in a notably higher encapsulation

efficiency percentage..

The optimised formulations were selected based on the studied parameters, including

encapsulation efficiency, particle size, polydispersity and zeta potential. Since the results did

not reveal any significant changes in different concentrations of curcumin, the formulations

with the relatively minimum size, minimum PDI and a zeta potential in the appropriate range

were selected for further experiments.

From the 12 formulations, formulations 9 and 10 were selected as the optimal formulations

for further testing based on their relative lower PDI, higher encapsulation efficiency and a

reasonably smaller size over other formulations. However, the zeta potential was slightly

lower than the acceptable range.

3.1.5. Morphological characterisation of the particles

TEM images (Figure 18), confirmed rod-shaped morphology of the particles contrary to most

of the published reports, which have verified a spherical shape of the particles (Das et al.

2010, Bernela et al. 2014, Fattahpour et al. 2015, Bhunchu et al. 2015).

The particle size data obtained by TEM were in agreement with the data achieved from

particle size analyser and the expected slight differences can be attributed to the inability of


the particle size analyser in distinguishing between the sample and the impurities. In addition,

the images revealed that the overall shape of the particles was not affected by curcumin

incorporation. However, they became aggregated compared to the blank particles, which

supported previous zeta potential results.

a b c d

Figure 18. TEM images of a: curcumin+ NaAlg+CaCl2+chitosan+PF127, b: curcumin+

NaAlg+CaCl2+chitosan, c: NaAlg+CaCl2+ chitosan+PF127 (blank), d: NaAlg+CaCl2+

chitosan (blank)

3.1.6. Release kinetics

In vitro release studies were carried out on two formulations with different concentrations of

methanol rinse wash, which were selected based on their efficient level of encapsulation,

along with acceptable PDI, particle size and an acceptable zeta potential. The release results

for both formulated curcumin particles are given in Figure 19.

According to the observed curcumin release profiles from both formulations, curcumin

release occurred in a biphasic pattern over 5 days. The statistical comparison of the above-

mentioned curcumin cumulative release values achieved from different concentrations of

methanol rinse (20%, and 50%) at specific times, revealed significant differences in both


formulations. Interestingly during the first 5 hours, both formulations displayed very similar

release patterns with an initial burst of around 60%. However, after 7 hours the formulation

with 50% methanol rinse showed a significant increase in cumulative curcumin release. After

24 hours, the release percentage in formulations with 20% and 50% methanol rinse reached

78% and 65% respectively. By the end of 96 hours, both formulations reached a cumulative

curcumin release of over 90% with 50% methanol rinse formulation being dominant.

Therefore, these results, showed an initial burst corresponding to around 60% of curcumin

for both formulations, which was followed by a sustained curcumin release to a total of

approximately 91% and 95% for both formulations respectively. The 20% methanol rinse

formulation exhibited slower and more sustained release compared to 50% methanol rinse

formulation (Figure 19). The visualised rapid initial release of curcumin could be attributed

to the dissociation of curcumin, which was absorbed by the surface of the polymeric matrix.

However, the sustained release phase could be due to the release of curcumin entrapped

inside the particle matrix.

Figure 19. Release curves of curcumin particles. All values represent mean ± SD (n =3),

and *p value < 0.05


In order to interpret the mechanism of curcumin release, the achieved release profiles of

curcumin for both formulations were fitted to five classical models including Zero order,

First order, Hixon-Crowell, Higuchi, and Korsmeyer-Peppas models (Li et al. 2008,

Korsmeyer et al. 1983, Fattahpour et al. 2015, Subramanian et al. 2014). The mathematical

formulas for the mathematical models are as below:

1. Zero order model Mt/M∞ = Kt

2. First order model LogMt /M∞ = Kt

3. Higuchi model Mt /M∞ = Kt�/�

4. Korsmeyer-Peppas (Power law) model Log(Mt /M∞) = LogK + nLogt

5. Hixon-Crowell model (Mt /M∞)�/� = Kt

where, (Mt /M∞) refers to the fraction of released drug at time t. The value of K and n

represent the release constant and release exponent.

The observed fitted graphs are depicted in Figures 20 and 21.


a

b

c


d

e

Figure 20. Release kinetics of optimised 20% methanol rinse formulation


a

b

c


d

e

Figure 21. Release kinetics of optimised 50% methanol rinse formulation


Table 1 indicates the values for release constants (k), release exponents (n), and

determination coefficients (R2), which were calculated based on the release data fitted to the

corresponding models. The best-fitted model was determined based on the determination

coefficient (R2) obtained for the mentioned kinetic models.

Table 1. Estimated parameters for encapsulated curcumin obtained after fitting the release

experimental data to five kinetic models

Curcumin release kinetic parameters

Formulations

Zero order First order Higuchi Korsmeyer-Peppas Hixon -Crowell

K R2 K R2 K R2 K R2 n K R2

20% methanol wash 0.517 0.600 -0.008 0.904 6.4857 0.762 1.668 0.9515 0.141 0.018 0.82

50% methanol wash 0.573 0.602 -0.010 0.912 7.3109 0.794 1.595 0.942 0.199 0.0214 0.82

The first order profile represented the substantially higher R2 value of 0.904 and 0.912 for

20% and 50% methanol wash formulations respectively compared to the Zero order model

which gave values of 0.600 and 0.602 respectively for both formulations. This indicated that

curcumin release depended on the concentration of curcumin entrapped in the particles.

The values of R2 for Higuchi and Hixon-Crowell kinetic models were comparatively

moderate. The highest R2 value for both formulations corresponded to Korsmeyer-Peppas

model, representing 0.9515 and 0.942 for 20% and 50% methanol wash formulations. Hence

the achieved R2 values for the different models revealed that the Korsmeyer-Peppas model

fitted best with the curcumin release kinetic data. This finding was in agreement with the


study by Das et al. (2010). Furthermore, the value of n (release exponent) was 0.1414 and

0.1987 respectively for the mentioned formulations.

The value of n, which indicates the mechanism of release, can reveal whether the mechanism

is Fickian or non-Fickian. According to the previous reports, n ≤ 0.43 corresponds to the

Fickian behaviour (case I transport), while n ≥ 0.85 shows a non-Fickian case II transport

(relaxation-controlled behaviour) and 0.43 ≤ n ≤ 0.85 represents a non-Fickian release

(anolamous transport) (Takka et al. 1998, Siepmann and Peppas 2012, Peppas and Sahlin

1989, Ritger and Peppas 1987).

The obtained value for n, implied that the mechanism involved in curcumin release exhibited

Fickian kinetics, which was mainly the result of drug diffusion across a chemical gradient.

However, Das et al. (2010), reported an n value of 0.84, which implied a combination of

“diffusion controlled” and “swelling release” mechanisms. The K value indicated the release

speed, i.e. the higher K value corresponded to the faster release. Therefore, as can be seen in

Table 1, the studied formulations displayed K values of 1.668, and 1.595, which were almost

similar, with 20% methanol rinse formulation being relatively dominant.

3.2. Cellular experiments

These experiments were carried out to indicate the toxicity of curcumin particles, as well as

empty particles on FEP-188 keratinocyte cells, along with cellular internalisation of the

particles to the cells.


3.2.1. In vitro validation of particle cytotoxicity

3.2.1.1. Effect of particles on FEP-188 keratinocyte cell viability

The IC50 of free curcumin was 14, 11, and 7 µg/ml after 1, 3 and 5 days respectively. As

most of the released curcumin (90%) was already released by the end of the third day, IC50

of 11 µg/ml was considered as the IC50 for the cell toxicity assay (Figure 22). However,

encapsulated curcumin did not exhibit any significant decrease in cell viability up to 25 µg/ml

of the applied concentration. Two possible explanations exist for this finding. One possibility

was that polymeric alginate and chitosan still maintained interactions with released curcumin

on the particle surface, decreasing its toxicity to the cells compared to the free curcumin. A

second possibility was the failure of the curcumin particles to penetrate the cells, probably as

a result of the low zeta potential, which lead to aggregation of the particles. Therefore, the

particles could not be up taken efficiently by the cells. To further investigate the cytotoxic

effect of curcumin particles, a trial including micrograph imaging was conducted with

different concentrations of curcumin particles and free curcumin dissolved in DMSO on FEP-

188 keratinocyte cells.

Figure 22. Cell toxicity curve of curcumin in DMSO, curcumin particles, free particles,

and control. All values represent mean ± SD (n =3), and *p value < 0.05


3.2.1.2. Microscopic cell toxicity

An evident dose response was revealed in FEP-188 keratinocyte cells (Figure 23). The

concentrations of 5 and 10 µg/ml of curcumin particles resulted in similar morphologies of

the FEP-188 keratinocytes to that of the control cells. However, a slight decrease in density,

along with minor morphology changes, were seen in cells treated with 10 µg/ml of curcumin

in DMSO. By increasing the concentration up to 25 µg/ml, a prominent decrease in density

of the cells treated with curcumin in DMSO was observed. Likewise, obvious morphological

changes including cell aggregation, membrane shrinkage and detachment from the well

surface occurred. Nevertheless, considering the changes in morphology, the cytotoxic effect

of 25 µg/ml of curcumin particles was evidently less than the equivalent concentration of

curcumin in DMSO, (Figure 23) confirming the role of the polymeric matrix in decreasing

the cytotoxic effects of curcumin on FEP-188 keratinocyte cells. A substantial decrease in

cell viability following the application of 50 µg/ml as the highest concentration of both

curcumin dissolved in DMSO and curcumin particles was evident according to the

micrographs. Other changes including a drastic decrease in density of the cells, cell

detachment from the surface, jagged cell membrane, and more typically, rounding up the

cells were also observed in both samples.

According to the micrographs, slight cell death started to happen in FEP-188 keratinocyte

cells treated with 10 µg/ml curcumin in DMSO. However the initial morphology changes in

the cells treated with curcumin particles were revealed at the concentration of 25 µg/ml.

Thereby, the microscopic images were in agreement with the first hypothesis, indicating the

effect of the polymeric matrix in decreasing the toxic effects of curcumin on FEP-188

keratinocyte cells.


Control cells

5 µg/ml

10 µg/ml

25 µg/ml

50 µg/ml

curcumin particles curcumin dissolved in DMSO

Figure 23. Qualitative intracellular uptake of curcumin particles, and free curcumin in

0.4% DMSO by keratinocytes over 3 days


3.2.2. Cellular uptake by confocal microscopy imaging

The results of the qualitative cellular uptake indicated that curcumin particles had the ability

to penetrate the cells. Figure 24 demonstrates a panel of the monitored fluorescent images of

FEP-188 keratinocyte cells incubated with curcumin particles, empty particles (blank) and

free curcumin in PBS over a 10 hour period. The cells incubated with curcumin particles

exhibited strong green fluorescence following the internalisation and accumulation of

curcumin, whereas the control cells and the cells incubated with empty particles did not

display any detectable fluorescence signal as expected. No fluorescent signal appeared in the

cells treated with free curcumin in PBS and only minor fluorescence appeared from

crystalline curcumin outside the cells, being unable to enter the cells. Furthermore, the time-

dependent curcumin internalisation visualised from the microscopic images revealed low

fluorescence intensity (mostly on cell membranes), during the initial hours, which was

intensified with time and extended to the cell cytoplasm.

These results confirm that the polymer matrix played a major role in facilitating penetration

of the curcumin particles into the cells since free curcumin in PBS was not accumulated in

the cytosol and curcumin-cellular uptake was inefficient. However, more experiments are

needed to be performed in order to establish the mechanisms of the internalisation process,

which will be elucidated in future studies.


0.5 hours 2 hours 4 hours

6 hours 8 hours

Figure 24. Confocal images of keratinocytes over 8 hours treated by a - e: curcumin

particles, f: normal view of the keratinocytes, g: free curcumin in PBS, and h: empty

particles

Chapter-4: Conclusions and future works Page 75

Chapter 4: Conclusions and future

works

Biocompatible and biodegradable formulations for promising curcumin delivery were

successfully developed and optimised based on physicochemical studies. The optimum

formulations were selected to perform cellular experiments aimed at understanding the

toxicity of curcumin on cells in vitro and the uptake of curcumin formulations. The size of

the fabricated curcumin particles ranged from 1286 to 1485 nm, which were larger than the

empty particles. According to previous studies, the size of the particles employed in drug

delivery should be large enough to avoid their entrance into blood capillaries. However, if

the size exceeds a specific range, there is a possibility of drug failure in being properly taken

up by the cells (Cho et al. 2008).

The encapsulation efficiency of the optimised particles was enhanced to 75%, which was

quite remarkable compared to the study by Das et al. (2010), on the similar polymeric

structure (13%). The zeta potential exhibited values slightly below the stable range, which

lead to partial aggregation of the particles in some cellular studies. Physical characterisation

using TEM imaging demonstrated (i) the successful fabrication and encapsulation of

curcumin in the polymeric matrix and (ii) the formation of rod-shaped particles.

Release studies confirmed a biphasic release composed of an initial burst, followed by a

sustained release trend for curcumin particles. Among different mathematical models applied

for modelling the release kinetics of curcumin, the Korsmeyer-Peppas model showed the


highest correlation coefficient for both optimised formulations. Based on the obtained

mathematical values for the established models, the release kinetics of curcumin conformed

to a diffusion mechanism. In addition, in vitro sustained release was shown to have enhanced

efficacy compared to that of previously published reports as approximately 95% of curcumin

was efficiently released by the end of the experimental period (Das et al. 2010).

In vitro cytotoxicity assays using the Cell Titre Blue® reagent, were carried out to estimate

the systemic toxicity of both fabricated particles, as well as curcumin encapsulated particles.

This assay was further investigated in microscopic scale to confirm nontoxicity of the applied

concentration of curcumin particles below 25 µg/ml.

Furthermore, the results of the cellular uptake study validated the internalisation of curcumin

particles, although in some duplicates curcumin could not penetrate the cells efficiently,

which might have occurred as a result of the low zeta potential leading to aggregation, and

subsequently precipitation of the particles.

Thus, the developed biocompatible and biodegradable formulations have the potential to be

employed as drug delivery vehicles for curcumin. However, more experimental work is

required in order to improve the physicochemical properties and to overcome the problem of

low negativity of zeta potential which resulted in relative instability and precipitation of the

particles and also subsequent larger particle size in some trials. Furthermore, the storage

stability of the encapsulated curcumin particles needs to be evaluated since any physical

instability would affect the delivery of the drug. Also, a series of wound healing experiments

at cellular levels such as scratch migration assay, cell mito stress assay, and tube formation

(angiogenesis test), should be performed to substantiate the ability of the preparations to

accelerate wound healing and prepare these for in vivo animal trials. Further elucidation at


the cellular and molecular level to identify the precise mechanism of action of curcumin in

different wounds including diabetic wounds are of pivotal importance to establish suitable

therapies to improve wound healing. In later stages, extra validation using animal models is

required to be able to be implemented as a tool in drug delivery.

References Page 78

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:Shahnia, Maryam

Title:Formulating curcumin in a biodegradable polymeric composite material: a step towardswound healing applications

Date:2017

Persistent Link:http://hdl.handle.net/11343/197965

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