The effect of Polyethylene glycol (PEG) on the physicochemical properties of Polycaprolactone (PCL) nanoparticles

Table of Contents

Chapter One
1.1 Introduction
1.2 PCL and PEG
1.2.1 Physicochemical properties of PCL and PEG
1.2.2 Synthesis of PCL and PEG
1.2.3 PCL and PEG in drug delivery systems
1.3 Hypothesis
1.4 Specific aims
1.4.1 Specific aim 1
1.4.2 Specific aim 2

Chapter Two: Materials and Methods
2.1 Materials
2.2 Methods used for prepration and characterization of the nanoparticles
2.2.1 Preparation of the nanoparticles by double emulsion/solvent evaporation (DE/SE) technique
2.2.2 Determination of nanoparticle size by photon correlation spectroscopy (PCS)
2.2.3 Measurement of zeta-potential of nanoparticles
2.2.4 Determination of bovine serum albumin amounts in nanoparticles by bicinchoninic acid (BCA) protein assay method
2.2.5 Determination of protein release from polymeric nanoparticles
2.2.6 Determination of protein integrity by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
2.2.7 Scanning electron microscopy (SEM) for the determination of nanoparticles morphology

Chapter Three: Results
3.1 PCL(s) Nanoparticles
3.2 PCL/PEG Nanoparticles

Chapter Four: Discussion
4.1 PCL(s) Nanoparticles
4.2 PCL/PEG Nanoparticles

Chapter Five: Conclusion

List of references

A dissertation submitted in partial fulfillment of the requirement for the award of Master in Pharmacy to department of Pharmacy & Pharmaceutical Sciences, University of Ulster.

Acknowledgment

First and foremost, I like to thank God for helping me in this project and giving me the strength and patience needed to achieve it. I like to acknowledge the efforts of my supervisor Dr Ahmed Faheem, who have supervised my project. I would sincerely like to thank all the academic and technical staff for their efforts during the MSc course.

I would like to thank my dear parent who have always been encouraging and motivating me to perform better.

Finally, kind thanks are given to my Dell laptop for not giving up at such a crucial time, after accidentally spilling tea on it during one of the long write up nights.

MSc student: Samuel Girgis

List of Figures

Fig.1 Project outline

Fig.2 Publications using PCL in the field of Biomaterials or Tissue Engineering during the last 20 years, until April 2010

Fig.3 Structures made from PCL: Nanospheres (a,b). Nanofibres (c,d). Foams (e,f). Knitted textiles (g,h,i). Selective laser sintered scaffold (j-o). Fused deposition modelled scaffolds (p–u).

Fig.4 Graphical illustration of the PCl controlled degradation kinetics over time. Initial hydration (0–6 months), through degradation and mass loss (6-12 months), resorption (post 12 months) and metabolisation (post 18 months

Fig.5 Structure of copolymers

Fig.6 Chemical structures of some representative polyethylene glycol (PEG) molecules

Fig.7 Various pathways to produce PCL

Fig.8 Anionic ring-opening polymerization of EO initiated by hydroxide

Fig.9 Degradation modes for degradable polymers: a) Surface erosion b) Bulk degradation c) Bulk degradation with autocatalysis

Fig.10 Schematic diagram for preparation of dry nanoparticles b DE/SE

Fig.11 Schematic presentation of the changes in potential with distance from the particle surface

Fig.12 Chemical reactions of the BCA protein assay

Fig.13 Bovine serum albumin (BSA) standard curve adapted from the current project

Fig.14 Protein before and after SDS-PAGE

Fig.15 Methods used for preparation and characterization of PCL(s) nanoparticles

Fig.16 Actual loadings of PCL(s) nanoparticles

Fig.17 % loading efficiency of PCL(s) nanoparticles

Fig.18 Z-average of PCL(s) nanoparticles

Fig.19 Polydispersity index of PCL(s) nanoparticles

Fig.20 SEM image of 5% BSA loaded SPCL at magnification 8000.

Fig.21 SEM image of 5% BSA loaded SPCL at magnification 6000.

Fig.22 Zeta potential of PCL(s) nanoparticles

Fig.23 Release of BSA from 2% BSA loaded PCL(s) nanoparticles

Fig.24 Release of BSA from 5% BSA loaded PCL(s) nanoparticles

Fig.25 Release of BSA from 10% BSA loaded PCL(s) nanoparticles

Fig.26 Methods used for preparation and characterization of LPCL/PEG(s) nanoparticles

Fig.27 Actual loading of LPCL/PEG(s) nanoparticles

Fig.28 % loading efficiency of LPCL/PEG(s) nanoparticles

Fig.29 Z-average of LPCL/PEG(s) nanoparticles

Fig.30 PDI(s) of LPCL/PEG(s) nanoparticles

Fig.31 zeta potential of LPCL/PEG(s) nanoparticles

Fig.32 Release of BSA from 5% BSA loaded LPCL/SPEG(s) nanoparticles

Fig.33 Release of BSA from 5% BSA loaded LPCL/LPEG(s) nanoparticles

Fig.34 The order of BSA bands from left to right: BSA standard, 5% BSA loaded (SPCL, MPCL, LPCL), 5% BSA loaded (LPCL/SPEG2.5%, LPCL/SPEG5%, LPCL/LPEG5% LPCL/LPEG10%) & BSA standard

List of Tables

Table 1 The structure, names, abbreviation & molecular formula of Polycaprolactone

Table 2 Names and molecular formula of PEG

Table 3 The ratios of PCL(s) amounts and BSA loading

Table 4 Characterization of PCL nanoparticles

Table 5 The ratios of PCL(s) amounts, PEG amounts, and BSA loading

Table 6 Characterization of LPCL/PEG(s) nanoparticles

Abbreviations

PCL: Polycaprolactone.

SPCL: Small polycaprolactone, MWt (10000).

MPCL: Medium polycaprolactone, MWt (45000).

LPCL: Large polycaprolactone, MWt (80000).

PEG: Polyethylene glycol.

SPEG: small polyethylene glycol,l MWt (200).

LPEG: large polyethylene glycol, MWt(2000).

PVA: Polyvinyl alcohol.

BSA: Bovine serum albumin.

DCM: Dichloromethane.

SDS: Sodium dodecyl sulphate.

NaN3 : Sodium Azide.

PBS: Phosphate buffer saline.

NaOH: Sodium Hydroxide.

BCA: Bicinchoninic acid (reagent A).

CuSo4: Cupper sulphate (reagent B).

KCl: Potassium chloride.

Abstract

Oral route is the best route for drug administration because it avoids pain and infections associated with injections. Biodegradable polymeric nanoparticles are considered an attractive alternative to injections as a controlled oral release protein delivery system because they are highly stable in the gastrointestinal tract (GIT) and able to protect the loaded proteins from destruction in GIT.

Polycaprolactone (PCL) is a hydrophobic polymer, compatible with various hydrophilic and amphiphilic polymers, and a good candidate from the pharmaceutical point of view to form nanoparticles because its physical, chemical, and mechanical properties can be easily modified by copolymerization or blending or grafting with other polymers.

The aim of the current study is to design amphiphilic PCL/PEG nanoparticles having better physicochemical properties than the PCL nanoparticles. Here we show that increasing the MWt of PCL can increase the size, zeta potential, protein loading efficiency and protein release rate of the PCL nanoparticles while blending of PEG with PCL can increase the protein loading efficiency, increase the stability, decrease the size and enhance the protein release rate of PCL nanoparticles. The BSA loaded LPCL nanoparticles have higher protein loading efficiency, protein release rate, size and zeta potential than the SPCL and MPCL nanoparticles. All the LPCL/PEG blends produce nanoparticles with smaller sizes, narrower PDI(s), and higher protein release rates than the BSA loaded LPCL nanoparticles. LPCL/SPEG2.5% and LPCL/SPEG5% nanoparticles have higher protein loading efficiencies, smaller sizes, narrower PDI(s), and higher protein release rates than the BSA loaded LPCL nanoparticles.

Our results demonstrate how blending of different amounts of PEG(s) with PCL are likely to design amphiphilic PCL/PEG nanoparticles with better physicochemical properties than the PCL nanoparticles. These newly formed amphiphilic PCL/PEG nanoparticles system will be a good delivery system not only for proteins but also for any hydrophilic or hydrophobic drug. By increasing drug loading efficiency, large single dose can be administered instead of small frequent doses and by increasing the nanoparticles stability, the dose duration increases decreasing the administration frequency.

Keywords: PCL, PEG, BSA, nanoparticles

Chapter One

Introduction

Oral delivery route is the route of choice for drug administration because it avoids pain, contaminations and infections associated with injections. Biodegradable polymeric nanoparticles are of great importance from the pharmaceutical point of view for the oral protein delivery because they are highly stable in the gastrointestinal tract (GIT) and able to protect the loaded proteins from destruction in GIT (des Rieux et al. 2006). The use of different polymers allows the manipulation of the nanoparticles’ physicochemical properties like stability and loading efficiency.

Polycaprolactone (PCL) is a good candidate to design nanoparticles because its physical, chemical, and mechanical properties can be easily modified by copolymerization or blending or grafting with other polymers (Dash, Konkimalla 2012). PCL is a hydrophobic polymer and reported to be compatible with various hydrophilic polymers like polyvinyl alcohol (PVA) and amphiphilic polymers like polyethylene glycol (PEG) (Woodruff, Hutmacher 2010). The previous studies of PCL over the past 5-6 years were more dominant for the scaffold materials than formulations for drug delivery. The majority of previous PCL formulation studies included only its copolymerization with various polymers (Dash, Konkimalla 2012).

Fig.1 shows the outline of the current project which compares physicochemical properties of the different molecular weight PCL(s) like protein loading efficiency, protein release rate, zeta potential, size, and stability. PCL showing best physicochemical properties is blended with various PEG(s) to form many amphiphilic PCL/PEG blends whose physicochemical characteristics are identified. It is expected that these amphiphilic blends can be used for several formulations development.

Abbildung in dieser Leseprobe nicht enthalten

Fig.1 Project outline.

PCL and PEG

Physicochemical properties of PCL and PEG

PCL (table 1) is a hydrophobic biodegradable polymer, early synthesized and used in 1930, commonly used in the field of Biomaterials or Tissue Engineering during the last 20 years (fig.2), and has a semicrystalline property whose crystallinity is inversely proportional to its molecular weight (Woodruff, Hutmacher 2010).

Abbildung in dieser Leseprobe nicht enthalten

Table 1 The structure, names, abbreviation & molecular formula of Polycaprolactone adapted from (Budavari et al. 1989).

Abbildung in dieser Leseprobe nicht enthalten

Fig.2 Publications using PCL in the field of Biomaterials or Tissue Engineering during the last 20 years, until April 2010 (adapted from Web of Science).

PCL is characterized by having good solubility, low melting point (59–64°C) which enables easy formability at relatively low temperatures and blend-compatibility which enables it to be used in many biomedical applications (fig.3) e.g. nanoparticles, textiles, nano-fibres, scaffolds and foams (Nair, Laurencin 2007, Okada 2002, Chandra, Rustgi 1998). Consideration should be given to the multiple advantages of PCL over other polymers in use like mechanical properties, controlled degradation kinetics (fig.4), ease of shaping and manufacture.

Abbildung in dieser Leseprobe nicht enthalten

Fig.3 Structures made from PCL: Nanospheres (a,b). Nanofibres (c,d). Foams (e,f). Knitted textiles (g,h,i). Selective laser sintered scaffold (j-o). Fused deposition modelled scaffolds (p–u). [Adapted from (Woodruff, Hutmacher 2010)].

Abbildung in dieser Leseprobe nicht enthalten

Fig.4 Graphical illustration of the PCl controlled degradation kinetics over time. Initial hydration (0–6 months), through degradation and mass loss (6-12 months), resorption (post 12 months) and metabolisation (post 18 months). [Adapted from (Hutmacher 2001)].

PCL is highly soluble in dichloromethane (DCM) at room temperature (Coulembier et al. 2006) and can be blended with other polymers to improve stress, crack resistance, dyeability, adhesion and has been used in combination with polymers such as polylactic acid-co-glycolic acid for manipulating the drug release rate from microcapsules (Chandra, Rustgi 1998).

Polymer blends of PCL with different polymers like polylactic acid-co-glycolic acid were categorized with three types of compatibility; firstly having only a single glass transition temperature (Tg); secondly as mechanically compatible, having the Tg values of each component but with superior mechanical properties and thirdly as incompatible, having the enhanced properties of phase-separated material (Sinha et al. 2004). Compatibility of PCL with other polymers depends on the ratios included in the blend and is generally useful to control the rate of drug release and the permeability of the delivery systems. Fig.5 shows the copolymers (block and random) of PCL that can be formed using many monomers e.g. substituted caprolactones, (Okada 2002).

Abbildung in dieser Leseprobe nicht enthalten

Fig.5 Structure of copolymers.

PEGs are amphiphilic polymers (table 2, fig.6) , soluble in water and dichloromethane, and commercially available over a wide range of molecular weights from 300 g/mol to 10,000,000 g/mol (Bailon, Berthold 1998, J. Milton Harris 1992).

Abbildung in dieser Leseprobe nicht enthalten

Table.2 Names and molecular formula of PEG adapted from (Budavari et al. 1989)].

Abbildung in dieser Leseprobe nicht enthalten

Fig.6 Chemical structures of some representative polyethylene glycol (PEG) molecules adapted from (Abuchowski et al. 1977) .

Synthesis of PCL and PEG

Two main pathways to produce polycaprolactone are commonly used (fig.7) (Labet, Thielemans 2009): the polycondensation of 6-hydroxyhexanoic acid, and the ring-opening polymerisation (ROP) of the lactone ε-caprolactone (ε-CL). Catalysts such as stannous octoate are used to catalyze the polymerization and low molecular weight alcohols can be used to control the molecular weight of the polymer (Storey, Taylor 1996).

Abbildung in dieser Leseprobe nicht enthalten

Fig.7 Various pathways to produce PCL adapted from (Storey, Taylor 1996).

Polyethylene glycol is easily produced by the reaction (fig.8.) of ethylene oxide (EO) with water that is catalysed by acidic or basic catalysts (Thompson et al. 2008).

Abbildung in dieser Leseprobe nicht enthalten

Fig.8 Anionic ring-opening polymerization of EO initiated by hydroxide.

PCL and PEG in drug delivery systems

Generally, microparticles and nanoparticles can be either spheres (matrix type) or capsules (reservoir type). Drug is released by erosion from matrix type and by diffusion from both matrix and reservoir (Dash, Konkimalla 2012). Several researches have been focused on degradable polymeric micro/nanoparticles for drug delivery because these systems can be ingested or injected, tailored for the required release profiles and can even achieve specific organs-targeted release (Freiberg, Zhu 2004). Drugs or proteins loaded within polymers are shown to have many advantages, such as improving the therapeutic efficacy, decreasing the side effects, prolonging the biological activity, controlling the drug release rate and decreasing the administration frequency (des Rieux et al. 2006).

PCL is a good candidate for controlled drug delivery due to its high permeability to many drugs, good biocompatibility and its complete excretion from the body after its bioresorption. PCL can be used to form compatible blends with other polymers and can easily form various copolymers to modify the degradation modes (fig.9) and kinetics, enabling better tailoring for the required release profiles (Sinha et al. 2004, Freiberg, Zhu 2004, Merkli et al. 1998).

Abbildung in dieser Leseprobe nicht enthalten

Fig.9 Degradation modes for degradable polymers: a) Surface erosion b) Bulk degradation c) Bulk degradation with autocatalysis. [Adapted from (Woodruff, Hutmacher 2010)].

PCL copolymers can act as surfactants to form micelles that can solubilize hydrophobic molecules because they are characterized by having a hydrophobic core and a hydrophilic coat (Gaucher et al. 2005). Cryoprotective agents like sucrose or trehalose should be used to stabilize the PCL nanoparticles and preserve their properties regardless of the freezing procedure (Saez et al. 2000). Within the last decades, PCL polymers had the major area of interest to develop controlled delivery systems especially for peptides and proteins (Sinha et al. 2004).

Colloidal PCL nanoparticle suspension containing cartelol provides better cartelol entrapment and more obvious effect on intraocular pressure compared with cartelol eye drops (Marchal-Heussler et al. 1993). PCL nanosphere/nanocapsules have been investigated for oral delivery of antihypertensive agents, such as isradipine to prolong the antihypertensive effect and decrease the initial hypotensive peak (Leroueil-Le Verger et al. 1998). PCL/Pluronic F68 compounds loaded with levonorgestral powder are used to design a two year contraceptive which is approved to enter phase II human clinical trials in China (Sun et al. 2006).

On blending or copolymerizing of the hydrophilic PEG polymer to the hydrophobic PCL polymer, the resulting blend or copolymer is amphiphilic as it comprises hydrophobic and hydrophilic polymers and has new physicochemical properties (Jia et al. 2008). Melting transition temperature of PCL/PEG copolymer is lower than that of the homopolymers (Zhang, Zhuo 2005). Particle size decreases with increasing molecular weight of PEG in the copolymer and the amphiphilic copolymers with longer PEG can diffuse more easily in the aqueous medium (Zhang, Zhuo 2005).

PEG in PEG/PCL microspheres increases drug loading and encapsulation efficiency of hydrophobic drugs as 4-DMEP (4-demethyl-epipodophyllotoxin) (Zhang, Zhuo 2005). PEG/PCL copolymer improves the encapsulation of poorly water soluble drugs like ketoprofen and increases the solubility of fatty acid prodrugs like geldanamycin (Wei et al. 2009). Drug diffusion and release of rifampicin from polyethylene glycol–poly(ɛ- caprolactone) networks is enhanced by the presence of PEG in a concentration dependent manner (Jones, McCoy & Andrews 2011). PEG has been used as the hydrophilic block of amphiphilic block copolymers used to create some polymersomes (Rameez, Alosta & Palmer 2008), modifies the properties of proteins to enhance their therapeutic potential (Katre 1993) and decreases the zeta potential of the protein loaded polyesters nanoparticles (Tobio et al. 1998).

Hypothesis

The main objective of this project is to formulate amphiphilic PCL/PEG nanoparticles having better physicochemical properties than PCL nanoparticles.

Specific aims

For better clearing of the above hypothesis, specific aims were detailed as follow:

Specific aim 1

Use three MWt PCL(s) [10000, 45000, 80000] to prepare nanoparticles loaded with a model protein [bovine serum albumin (BSA)] at three loading levels (2, 5, 10 %) by DE/SE technique then Identify for the various PCL(s) nanoparticles their physicochemical properties like protein loading efficiency, release profile, size, zeta potential and stability by various methods stated in the experimental part to determine the PCL having the best physicochemical properties.

Specific aim 2

Use two different MWt PEG(s) [200, 2000] in different amounts to form various amphiphilic blends with the selected P CL having the highest protein loading efficiency to prepare amphiphilic nanoparticles loaded with the model protein then identify and compare the physicochemical properties of the various newly formed PCL/PEG nanoparticles.

Chapter Two: Materials and Methods

2.1. Materials

All the chemicals used were of analytical grade and used as received from source without any further purification. PCL(s) [10000, 45000, 80000], PEG(s) [200, 2000], PVA, BSA, DCM, SDS, NaN3, NaOH, KCL, Tris base, HCL, Bromophenol blue, 2-Mercapto ethanol, Glycine, Methanol, Glacial acetic acid and Glycerol were purchased from (Aldrich, UK). CuSo4 and BCA were purchased from (Thermo-scientific, UK). Coomassie Brilliant Blue solution and Ready-made gels were purchased from (Biorad, UK). PBS is purchased from (Oxoid, UK).

Methods used for prepration and characterization of the nanoparticles

Preparation of the nanoparticles by double emulsion/solvent evaporation (DE/SE) technique.

PCL or PCL/PEG blend nanoparticles were prepared by a w/o/w solvent evaporation technique which comprised the drop wise addition of 1ml of 2.5% m/v aqueous solution of polyvinyl alcohol (PVA) containing theoretical bovine serum albumin (BSA) loading of 2, 5, or 10% m/m based on PCL OR PCL/PEG blend, to 4ml of dichloromethane (DCM) containing 200 mg of the polymer and different amounts of PEG (2.5, 5, 10 %) m/m, according to the experiment, based on the polymer, then homogenisation was done using the hand-held homogenizer (VWR VDI 25 Homogenizer, VWR international, UK) at a speed of 10000 revolutions per minute (rpm) for 2 minutes. The resulting w/o emulsion was subsequently added drop wise to 50ml of 1.25% m/v aqueous PVA solution followed by homogenising at a speed of 10000 rpm using Silverson L4R homogeniser (Silverson, UK) for 6 minutes. The homogenisation process was performed over ice and the resulting w/o/w emulsion was stirred continuously and allowed to evaporate overnight. The particles were then collected by centrifugation (sigma centrifugation apparatus, Sigma, UK) at 4○C at 10,000 rpm for 30 minutes and washed with water a further 3 times using the same centrifugation parameters. The sediment obtained was resuspended in water and freeze dried at -85 0c & 0.012 mbar (Labconco freeze dryer, Freezone 4.5 Plus, Labconco, UK) for 48h (Dash, Konkimalla 2012).

Freeze drying of polymeric particles included the addition of diluted solutions of washed polymeric particles to 20 mL clear freeze-drying glass containers, covered with Parafilm M film perforated with 20 needle width holes, and frozen at -16οC for 12 hours. After that, the samples were lyophilised for 48 hours using the freeze dryer, under vacuum drawn by a high vacuum pump. Dried polymeric particles loaded with protein were stored at room temperature in desiccator (Dash, Konkimalla 2012). Fig 10 shows a schematic representation of the technique (DE/SE) used for preparation of the nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.10 Schematic diagram for preparation of dry nanoparticles b DE/SE.

Determination of nanoparticle size by photon correlation spectroscopy (PCS)

The size of nanoparticles was determined by PCS technique using the Malvern Zetasizer nanoseries (Malvern Instruments, UK) which is a useful for determination of the particle sizes of submicron particles in the range of 3 to 1000 nm. The theory of PCS technique is based on the use of dynamic light scattering for determination of the shape and size of particulate systems. The electric field of the incident light induces an oscillating polarisation on the particles in the sample, and then these particles whose polarity differs from the surroundings scatter the incident light. The particles in the sample are in constant motion which is called Brownian motion and this motion makes fluctuations in the detected intensity signal that can be measured digitally by PCS. The duration of the fluctuations provides information about the particles, including size and polydispersity (Akbari, Tavandashti & Zandrahimi 2011).

Nanoparticles suspension was prepared by dispersing 5 mg of the nanoparticles in 1 to 2 ml distilled ddH2O, then 0.1 ml nanoparticles suspension were dispersed in 2 to 3 ml of 0.22 µm filtered ddH2O, and then measured by Malvern instruments with regard to polydispersity and Z-average diameter (Akbari, Tavandashti & Zandrahimi 2011).

Measurement of zeta-potential of nanoparticles

Determination of zeta-potential was measured by laser anemometry in millivolts (mV) by using a Malvern zetasizer nanoseries. Zeta-potential is the electrical potential existing at the stern plane of a particle (fig.11), which is an imaginary plane separating the thin layer of liquid bound to the particle surface from the rest of liquid and showing elastic and viscous behaviours. Colloidal particles are electrically charged due to their ionic characteristics and consequently, the distribution of ions in the neighbouring interfacial region will be affected by the resultant particle surface charge, and the counter ions (fixed layer) concentration will increase. A cloud-like area containing ions of opposite charges is formed outside the fixed layer. The net result is the formation of an electrical double layer in the region of the particle/liquid interface, with an inner region formed of ions strongly bound to the surface, and an outer diffuse ionic region. The potential in this region declines with the distance from the surface until at a certain distance it reaches zero (fig.11) (Ravi Kumar, Bakowsky & Lehr 2004, Konan et al. 2003). When a voltage is applied to the solution in which particles are suspended, particles are attracted to the electrode of the opposite polarity, associated by the fixed layer and part of the diffuse double layer.

Nanoparticles suspension was prepared by dispersing 5 mg of the nanoparticles in 1 to 2 ml distilled H20, and then 0.5 ml of the nanoparticle suspension was diluted in 0.001 M KCL solution, which acts as a weak electrolyte, to get a sample of appropriate concentration for the measurement.

Abbildung in dieser Leseprobe nicht enthalten

Fig.11 Schematic presentation of the changes in potential with distance from the particle surface adapted from http://nition.com.

Determination of bovine serum albumin amounts in nanoparticles by bicinchoninic acid (BCA) protein assay method

The loading efficiency of protein was calculated by dividing the actual loading over the theoretical loading of the protein used in the preparation of the nanoparticles, as follows: % Protein loading efficiency = (actual loading/theoretical loading) x 100

The amount of actual protein loaded per unit weight of particles was determined using the BCA assay (Bainor et al. 2011). The BCA protein assay includes the reduction of cupric (Cu2+) to cuprous (Cu1+) by protein in an alkaline medium followed by a highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) by bicinchoninic acid.

Abbildung in dieser Leseprobe nicht enthalten

Fig.12 Chemical reactions of the BCA protein assay

The first step (fig.12) is the protein chelation for the copper in an alkaline medium to form a blue coloured complex (biuret reaction). The second step (fig.12.) is the colour development reaction, in which BCA; a selective and sensitive colorimetric detection reagent reacts with the cuprous cation to form a purple coloured product by the chelation of one cuprous cation with two molecules of BCA. This purple coloured product is water soluble and can be measured spectrophotometrically at 562 nm. The Cu2+ reduction leading to BCA colour formation is highly affected by the presence of any of the four amino acid residues (tyrosine, tryptophan, cysteine and cystin) in the protein amino acid sequence. The peptide bond only is responsible for colour development at high temperatures and thus, the reaction is done at 60oC to increase the sensitivity. This technique has the advantages of compatibility with non-ionic and ionic detergents, minimum protein-to-protein variation and limited interactions with most copper chelators and reducing agents.

A 5mg nanoparticles sample was suspended in 1 ml of 1 M NaOH at 37°C overnight for digesting the polymer. The suspension was then centrifuged and 25 µL of the supernatant was added to 3 wells. A series of calibrated BSA protein standards were prepared in distilled water and 25 µL of each standard were added to 3 wells. 200 μL of BCA reagent were added to each well. Precision and accuracy of protein absorption were determined by using of a minimum of three absorption determinations for each standard, blank and test samples. After the purple colour development, the absorbance of the contents of each well was measured at 562 nm using a plate reader. The following diagram (fig.13) shows one of the BSA standard curves obtained during BCA assays.

Abbildung in dieser Leseprobe nicht enthalten

Fig.13 Bovine serum albumin (BSA) standard curve adapted from the current project.

Determination of protein release from polymeric nanoparticles

In vitro protein release from particles was performed to determine its release rate by incubation of 5mg of particles in 1 ml of phosphate buffered saline (PBS) having pH 7.4, containing 0.01 % sodium azide as a bacteriostatic agent and 5 mM SDS in 1.5 ml eppendorf tubes. The particles were incubated at 37°C, shaken, and then samples were withdrawn at appropriate time intervals, centrifuged and the amount of BSA in the samples was analysed using a BCA assay.

Determination of protein integrity by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Protein stability can be detected by SDS-PAGE which is the process of charged molecules movement in solution by applying an electrical field across the solution. The charged molecules move in an electrical field with a velocity depending on their sizes, shapes, and charges. Electrophoresis is a simple and rapid analytical tool, used for analysis and purification of large molecules such as proteins and can also be applied to simple charged molecules like peptides, amino acids and simple ions (Gupta, Shepherd 1990, Hartinger et al. 1996). Electrophoresis is carried out by applying a small volume of a sample to a solution stabilised by a porous matrix and the different molecules in the sample move through the matrix at different velocities under the effect of the applied voltage showing visualized bands at different positions in the matrix. The matrix comprises a number of different materials, including gels made of polyacrylamide and the separated molecules can be easily visualized in position in the gel by staining and the gel is dried for permanent storage.

In SDS-PAGE, movement is not determined by intrinsic charge of proteins but by molecular weight. The sample is heated with Laemmli buffer which comprises 50 mM tris- HcL pH (6.8), 2% SDS, 10% glycerol, 2-mercaptoethanol, 0.02 % bromophenol blue SDS, and 2-mercaptoethanol. Sodium dodecylsulfate (SDS) is an anionic detergent that can denature proteins by wrapping around the polypeptide chain producing a net negative charge to the polypeptide proportionally to its length. In addition to SDS, a reducing agent, such as 2-mercaptoethanol (beta-mercaptoethanol), further denatures the proteins by reducing disulphide bonds overcoming the structures of tertiary protein folding and breaking up the quaternary protein structure. Bromophenol blue is a common tracking dye of a known electrophoretic mobility and helps to easily follow the progress through the gel during electrophoresis. When treating the protein sample with Laemmli buffer, the protein becomes a rod of negative charges with equal charge densities (charge per unit length) which is shown in (fig.14.). The position of a protein along the lane gives a good indication of its stability after staining with Coomassie Brilliant Blue solution.

A 5mg nanoparticles sample was suspended in 1 ml of 1 M NaOH at 37°C overnight for digesting the polymer. The suspension was centrifuged and then the supernatant was mixed with Laemmli buffer in the ratios (1:3), boiled over boling water bath for 10 minutes, left to cool by the room temperature for 30 minutes, and then applied to the ready-made Biorad gels. Electrophoresis of samples was carried out at a constant voltage mode at 200 volts using a Bio-Rad power supply in a Tris/glycine/SDS buffer. Each gel was stained with 100 mls Coomassie Brilliant Blue solution for 30 minutes, destained with 100 mls aqueous solution of 40% methanol and 10% glacial acetic acid for 12 hours, immersed in 40 mls aqueous solution of 30% methanol and 5% glycerol for 20 minutes, and left to dry in the room temperature for 24 hours. The protein standard can be used as a reference.

Abbildung in dieser Leseprobe nicht enthalten

Fig.14 Protein before and after SDS-PAGE.

Scanning electron microscopy (SEM) for the determination of nanoparticles morphology

SEM microscopy is used to characterize the morphology and size of the nano/microparticles. A thin layer of nano/microparticles was spread on a circular aluminium plate using a carbon disc and the surface was then coated with a gold film using a sputter coater under an Argon atmosphere. Nano/microparticles were identified by magnification with a scanning electron microscope (Cambridge Instruments Stereoscan 90B, 25 kV, Cambridge, UK) (Todokoro, Otaka 1995).

Chapter Three Results

All the nanoparticles were prepared by the DE/SE and characterized by the same methods under the same conditions. Laser anemometry was used for zeta potential measurment, PCS was used for size and PDI measurment, BCA asssay was used for protein loading efficiency and release rate measurements, SEM was used for morphological identification of the nanoparticles and SDS-PAGE was used for protein stability identification. The results are presented in two sections : section (1) for the PCL(s) nanoparticles and section (2) for the PCL/PEG nanoparticles.

3.1 PCL(s) Nanoparticles

The ratios of PCL(s) amounts and theoretical BSA loadings used for preparation of the various PCL(s) nanoparticles are shown in table 3.

Abbildung in dieser Leseprobe nicht enthalten

Table 3 The ratios of PCL(s) amounts and BSA loading

The following diagram (fig.15) shows the methods used for preparation and characterization of PCL (s) nanoparticles. Table 4 shows the results of the PCL(s) nanoparticles characterization which includes size (Z-average & PDI), zeta potential, actual loading and loading eficiency.

Abbildung in dieser Leseprobe nicht enthalten

Fig.15 Methods used for preparation and characterization of PCL(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Table 4 Characterization of PCL nanoparticles.

On comparing the three PCL(s) according to their MWt in (fig.16), LPCL shows the highest the % actual loadings (0.53, 1.79, 1.2) at all the theoretical loading levels (2, 5, 10 %) followed by the MPCL (0.51, 1.4, 0.47), and the SPCL (0.47, 0.71, 0.36) and consequently will give the same order for the % loading efficiency.

Abbildung in dieser Leseprobe nicht enthalten

Fig.16 Actual loadings of PCL(s) nanoparticles.

The % loading efficiency can be calculated by the following equation: % Loading efficiency = (Actual loading/Theoretical loading) × 100

It is clear that increasing the PCL MWt can increase the loading efficiency for each theoretical loading. The % loading efficiency has the highest value at 5 % theoretical loading and decreases when increasing theoretical loading above 5 % (fig.17).

Abbildung in dieser Leseprobe nicht enthalten

Fig.17 % loading efficiency of PCL(s) nanoparticles.

By identifying the sizes of the nanoparticles according the MWt of the PCl(s) (fig.18), it is obeserved that LPCL shows the largest sizes at all the theoretical loading levels (373.63, 408, 393.43) but the sizes are reduced for the MPCL (333.4, 366.97, 341.67) and show the lowest values for the SPCL (294.67, 335.5, 307.33). It is also clear that the size of the nanopaticles increases by increasing the theoretical loading from 2 to 5 %, then declines after further rise of the theortical loading to 10 %. However, the PDI values are not dependent on the nanoparticle size or the PCL MWt (fig.19). PDI starts at low values for 2 % loading of the MPCL (0.37) and LPCL (0.39), then increases by increasing the loading while it starts at high value for 2 % loading of the SPCL (0.54) then decreases at the 5 % loading (0.37) and increases again at the 10 % loading (0.39).

Abbildung in dieser Leseprobe nicht enthalten

Fig.18 Z-average of PCL(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.19 Polydispersity index of PCL(s) nanoparticles.

The following SEM images prove that increasing the MWt of PCL increases the the size of the PCL nanoparticles. The 5% BSA loaded SPCL nanoparticles (fig.20) are much smaller than the 5% BSA loaded LPCL nanoparticles (fig.21).

Abbildung in dieser Leseprobe nicht enthalten

Fig.20 SEM image of 5% BSA loaded SPCL at magnification 8000.

Abbildung in dieser Leseprobe nicht enthalten

Fig.21 SEM image of 5% BSA loaded LPCL at magnification 6000.

It is obvious that LPCL has the highest zeta potential values at 2, 5 and 10 % loadings (- 9.19, -10.02, -6.06) followed by the MPCL (-7.04, -7.57, -5.98) and SPCL (-6.01, -6.12, - 5.62) (fig.22). Additionally, zeta potential values increase on increasing the loading from 2 to 5 %, then decreases on increasing the loading to 10 %.

Abbildung in dieser Leseprobe nicht enthalten

Fig.22 Zeta potential of PCL(s) nanoparticles.

Concerning the protein release rates (figures 23, 24, 25), it is obvious that they are obeying the order of the % loading efficiency such that the LPCL having the highest loading efficiency shows the highest release followed by the MPCL and SPCL. Since the BSA loading efficiency is higher at the 5% loading followed by the 2 % and 10%, the BSA release rate is higher at the 5% loading followed by the 2 % and 10%.

Abbildung in dieser Leseprobe nicht enthalten

Fig.23 Release of BSA from 2% BSA loaded PCL(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.24 Release of BSA from 5% BSA loaded PCL(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.25 Release of BSA from 10% BSA loaded PCL(s) nanoparticles.

It is concluded that increasing the MWt of the PCL at each BSA loading can increase loading efficiency which in turn increases the nanoparticle size and release rate. According to the stated aims, the PCL having the best physicochemical properties must be selected for blending with different PEG(s) and thus, LPCL with 5 % BSA loading is the one of choice because it has the highest protein loading efficiency, highest protein release rate and highest zeta potential.

3.2 PCL/PEG Nanoparticles

The ratios of PCL(s) amounts, PEG amounts and theoretical BSA loadings used for preparation of the various LPCL/PEG(s) nanoparticles are shown in table 5.

Abbildung in dieser Leseprobe nicht enthalten

Table 5 The ratios of PCL(s) amounts, PEG amounts, and BSA loading.

The following diagram (fig.26) shows the methods used for preparation and characterization of LPCL/PEG(s) nanoparticles. Table 6 shows the results of the LPCL/PEG(s) nanoparticles characterization which includes size (Z-average & PDI), zeta potential, actual loading and loading eficiency.

Abbildung in dieser Leseprobe nicht enthalten

Fig.26 Methods used for preparation and characterization of LPCL/PEG(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Table 6 Characterization of LPCL/PEG(s) nanoparticles.

The SPEG/LPCL nanoparticles show higher actual loading (2.71, 1.97, 1.49) than the LPEG/LPCL nanoparticles (1.43, 1.38, 1.08) (fig.27). On increasing the SPEG or LPEG in their blends, the actual loading decreases. It is important to notice that SPEG2.5%/LPCL and SPEG5%/LPCL nanoparticles have higher actual loadings than the 5% BSA loaded LPCL nanoparticles (1.79).

Abbildung in dieser Leseprobe nicht enthalten

Fig.27 Actual loading of LPCL/PEG(s) nanoparticles.

Since the loading efficiency value is directly proportional to the actual loading, the SPEG/LPCL nanoparticles have higher loading efficiency (54.27, 39.47, 29.87) than the LPEG/LPCL (28.53, 27.67, 21.67) and increasing the SPEG or LPEG in their blends decreases the loading efficiency (fig.28). It is also obvious that SPEG2.5%/LPCL and SPEG5%/LPCL nanoparticles have higher loading efficiencies (54.27, 39.47) than the 5% BSA loaded LPCL nanoparticles (35.73).

Abbildung in dieser Leseprobe nicht enthalten

Fig.28 % loading efficiency of LPCL/PEG(s) nanoparticles.

On comparing the effect of the two MWt PEG(s), it is observed that SPEG/LPCL blends produce nanoparticles with smaller sizes (242.47, 254.67, 269.2) and narrower PDI(s) (0.14, 0.18, 0.16) than LPEG/LPCL [sizes: (262.8, 299.43, 332.67), PDI(s): (0.18 0.21, 0.3) (figures 29, 30). Moreover, increasing either the content of SPEG or LPEG in their blends increases the size and PDI of the nanoparticles. It is also clear that all the PEG/LPCL blends produce nanoparticles with smaller sizes and narrower PDI(s) than the 5% BSA loaded LPCL nanoparticles (408, 0.51).

Abbildung in dieser Leseprobe nicht enthalten

Fig.29 Z-average of LPCL/PEG(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.30 PDI(s) of LPCL/PEG(s) nanoparticles.

The SPEG is more effective in decreasing the zeta potential values than the LPEG (fig. 31). SPEG blends have lower zeta potentials (-5.78, -4.34, -1.32) than LPEG blends (- 7.31, --4.86, -3.38). Increasing the amount of either the SPEG or LPEG in their blends enhances the reduction of the zeta potential. It is also obvious that all the LPCL/PEG nanoparticles have lower zeta potentials than the 5% BSA loaded LPCL nanoparticles (- 10.02).

Abbildung in dieser Leseprobe nicht enthalten

Fig.31 Zeta potential of LPCL/PEG(s) nanoparticles.

Regarding the protein release rates (figures 32, 33), LPEG/LPCL nanoparticles have higher protein release rates than SPEG/LPCL nanoparticles. Increasing the amounts of SPEG or LPEG in their blends enhances the release rate. It is also clear that all the LPCL/PEG nanoparticles have higher protein release rates than the 5% BSA loaded LPCL nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.32 Release of BSA from 5% BSA loaded LPCL/SPEG(s) nanoparticles.

Abbildung in dieser Leseprobe nicht enthalten

Fig.33 Release of BSA from 5% BSA loaded LPCL/LPEG(s) nanoparticles

The following figure (fig.34) shows the protein bands in the PCL nanoparticles [5% BSA loaded SPCL, MPCL, LPCL] and the PEG/LPCL nanoparticles [SPEG2.5%/LPCL, SPEG5%/ LPCL, LPEG5%/ LPCL, LPEG10%/LPCL]. BSA stability is similar in the various PCL nanoparticles regardless the MWt of the PCL. The BSA stability is similar in the various amphiphilic LPCL/PEG nanoparticles regardless the MWt and amount of the PEG. Additionally, it is clear that the PEG(s) don’t provide more BSA stability than LPCL. There are no aggregates or fragments below or above the BSA bands.

Abbildung in dieser Leseprobe nicht enthalten

Fig.34 The order of BSA bands from left to right: BSA standard, 5% BSA loaded (SPCL, MPCL, LPCL), 5% BSA loaded (LPCL/SPEG2.5%, LPCL/SPEG5%, LPCL/LPEG5% LPCL/LPEG10%) & BSA standard.

It is concluded that all the LPCL/PEG blends produce nanoparticles with similar protein stability, smaller size, narrower PDI, lower zeta potentials and higher release rates than the 5% BSA loaded LPCL nanoparticles. Moreover, SPEG/LPCL nanoparticles are characterized by having higher protein loading efficiencies, smaller sizes, narrower PDI(s), lower zeta potentials and lower protein release rates than the LPEG/LPCL nanoparticles. LPCL/SPEG2.5% and LPCL/SPEG5% nanoparticles have higher protein loading efficiencies than the 5% BSA loaded LPCL nanoparticles.

Chapter Four Discussion

Physicochemical properties of different MWt PCL(s) nanoparticles loaded with BSA were compared to select the PCL showing the best physicochemical properties. All the parameters of the preparation and characterization procedures were fixed to identify the effect of the MWt on the physicochemical properties of the PCL nanoparticles.

The selected PCL was blended with different amounts of different MWt PEG(s) and the physicochemical properties of the amphiphilic PCL/PEG nanoparticles were compared. All the parameters of the preparation and characterization procedures were fixed to accurately compare the physicochemical properties of the PCL/PEG nanoparticles.

The discussion is divided into two sections: the first section explains the results of the PCL(s) nanoparticles and the second section clarifies the results of the PEG/LPCL nanoparticles.

PCL(s) Nanoparticles

BSA is characterized by having tensoactive properties and high affinity to the thermoplastic aliphatic polyesters which are considered the major determinants of the protein loading (Woodruff, Hutmacher 2010, Blanco, Alonso 1998). These aliphatic polyesters have good mechanical strengths to carry a variety of drug classes such as micromolecules, vaccines, peptides and proteins (Woodruff, Hutmacher 2010). The loading efficiency is affected by several factors like the nature of the polymer, the MWt of the polymer, the surfactant type, polymer/surfactant ratio and BSA concentration. The amount and type of surfactant are responsible for the interaction between the surfactant and the protein, which protect BSA from the solvent and inhibit its dissolution in the external aqueous phase (Coccoli et al. 2008). The polymer concentration in the organic phase controls the emulsion viscosity which can increase BSA loading efficiency and prevent BSA diffusion towards the external aqueous (Coccoli et al. 2008, Youan et al. 1999). BSA has limits of loading for the constant amounts of polymer and surfactant, above which any increase in the BSA content will decrease the BSA interaction with the polymer and the surfactant which in turn will decrease the BSA loading efficiency (Feczkó et al. 2011).

For the current study of the PCL(s) nanoparticles, the amount of the PCL polymer is constant, PVA is used in fixed amount as a surfactant and the BSA theoretical loading is constant, so that the effect of the PCL MWt can be easily identified. It is clear that for each BSA theoretical loading, LPCL had the highest loading efficiency, SPCL has the lowest protein loading efficiency, and the MPCL has an intermediate value. This proves the validity of the concept which reveals that increasing the MWt of the polyester can increase the mechanical strength of the polyester that enhances the loading efficiency (Blanco, Alonso 1998, Wu et al. 2012). Additionally increasing the MWt of the PCL leads to an increase in the viscosity of organic phase which reduces the BSA diffusion to the external aqueous phase before the nanoparticles hardening. Nevertheless, the results show that accepted limits of the BSA theoretical loading to enhance protein loading efficiency are from 2 to 5 %, and any increment of the BSA content above these limits sharply reduces the Loading efficiency because the BSA interactions with the available amounts of PCL and PVA decrease.

The size of many polyester nanoparticles is affected by several factors like amount of the loaded drug, polymer/surfactant ratio, MWt of the polymer, and method of nanoparticles’ preparation (Youan et al. 1999, Feczkó et al. 2011, Wu et al. 2012). Since all the parameters are fixed except the MWt of the polymer, it is clear that increasing the molecular weight of PCL increases the nanoparticles sizes at all the BSA loading levels. Increment of the protein loading efficiency by increasing the PCL MWt is the major determinant for increasing the nanoparticles size. The PDI is mainly depending on the method of nanoparticles preparation and type of surfactant used e.g. sonication gives cyclosporine PLGA nanoparticles with better PDI than homogenization (Jain et al. 2010) and PCL-Tween 80 copolymer gives nanoparticles with narrower PDI than PCL alone (Ma et al. 2011). This explains why the PDI of the PCL nanoparticles is slightly affected by the PCL MWt showing low values for 2% BSA loaded LPCL and 2% BSA loaded MPCL, one high value for the 10% BSA loaded SPCL and intermediate close values for the remaining of the PCL(s) nanoparticles.

It has been reported that the zeta potential of the polyesters nanoparticles e.g. PCL, PLGA without any surfactant like PVA is high due to the presence of uncapped end carboxylic groups of the polyester at the particle nanoparticle surface which provide high negative charge (Krishnamachari 2011, Sahoo et al. 2002). On adding PVA to the polyester, it forms a layer at the surface of the polyester nanoparticles that shields the negative surface charge of the polyesters in an amount dependent manner (Sahoo et al. 2002). All the PCL(s) nanoparticles are prepared using the PVA as a surfactant in constant amount, so it is strongly concluded that increasing the molecular weight of PCL can reduce the shielding effect of the PVA for the surface negative charges of the PCL end carboxylic groups allowing the LPCL to achieve the highest negative charge of zeta potential. The high zeta potential either by negative or positive is beneficial to the stabilization of particles suspension and prevention of the particles aggregation as the charged particles can repel one another (Vandervoort, Ludwig 2002, Dubey et al. 2012), therefore LPCL nanoparticles suspension achieves the highest stability.

PCL is characterized by acting as a reservoir in which the drug molecules can be loaded through physical, chemical or electrostatic interactions according to their physicochemical properties. Additionally, PCL has a good permeability for the release of different drugs and the drugs release rates from PCL are affected by several factors like technique of preparation, PCL type, PCL content, and loading efficiency of the drug loaded within the the micro/nanoparticles (Woodruff, Hutmacher 2010). On using the same amount of PCL to prepare PCL nanoparticles by DE/SE under the same conditions but increasing the MWt of the PCL, the protein loading efficiency at all the theoretical loadings increases which in turn increases the protein release rates in the same order showing the highest protein release rates for the LPCL followed by the MPCL and SPCL.

The loading of proteins into polyesters nanoparticles presents some problems as instability problems. For example, in the first step of the w/o/w emulsion formulation procedure, the protein dissolved in the aqueous phase may be denatured at the water/organic interface or aggregate or unfold because of the shear stress used for the formation of the primary emulsion (Danhier et al. 2012). However, the use of stabilizers likes PVA can increase the protein stability (Danhier et al. 2012). The stability of the loaded proteins within the polyesters polymeric nanoparticles relies on several factors like the procedure used for the nanoparticles formulation, properties of the surfactants used, and the properties of the polymer. The physical cross-links of the polymers through stereocomplex formation are highly responsible for keeping the protein stability (Kang et al. 2005). The hydrophobic interactions of the polyesters are very useful for the stabilization of the protein with the polyesters nanoparticles (Akagi, Baba & Akashi 2012). Increasing the molecular weight of the polyester can slightly improve the protein stability (Kang et al. 2005). The bands of the BSA in the various 5% BSA loaded PCL(s) nanoparticles are similar to each other and consequently, it can be concluded that the physical cross-links through stereocomplex formation and the hydrophobic interactions of the various PCL(s) are quiet similar to each other. It is clear that increasing the MWt of the PCL doesn’t have any effect on the protein stability.

In conclusion, it is clear that 5% BSA loaded LPCL nanoparticles have the advantages of the highest loading efficiency, highest release, and highest zeta potential.

PCL/PEG Nanoparticles

LPCL with 5% BSA loading is selected for blending with (2.5, 5, 10%) of SPEG (200) and LPEG (2000) because the 5% BSA loaded LPCL nanoparticles show the highest loading efficiency over the other PCL(s) nanoparticles.

PEG is an amphiphilic polymer having hydrophobic and hydrophilic properties but the polyesters like PCL are hydrophobic having a limited water-uptake. Proteins like BSA have a hydrophilic nature and are highly attached to hydrophilic moieties like PEG (Buske et al. 2012). PEG can act as surfactant by accumulating at the inner interface of the particle consisting of the polymer mixture of polyesters and PEG to help protein spread throughout the inner phase (Buske et al. 2012). However, the protein loading efficiency within the amphiphilic blend or copolymer of polyester and PEG is affected by the PEG MWt and amount. On blending small MWt PEG with polyester, water-uptake of the blend is low regardless of the polyester MWt but on increasing the PEG MWt, the water-uptake increases. The ‘‘anti-water-uptake’’ effect of the polyester becomes more pronounced with large MWt PEG due to the higher water uptake resulting in significant decrease of the protein loading (Tran et al. 2012). Additionally, as the MWt of PEG(s) increases, the hydrophilicity power increases leading to protein release and diffusion through the matrix of the particles during the particles preparation resulting in decrease of the loading efficiency (Bouillot et al. 1999). PEG can enhance protein loading within certain limits but on increasing the PEG content above these limits, water uptake by the matrix increases facilitating the protein diffusion towards the external aqueous phase during the particles preparation (Essa, Rabanel & Hildgen 2011). LPEG (2000) shows lower protein loading than SPEG (200) due to two reasons. Firstly, the LPEG has higher water uptake than the SPEG which enhances the prominence of the anti-water-uptake effect of the LPCL to decrease the BSA loading. Secondly, LPEG is more hydrophilic than the SPEG and consequently enhances the BSA diffusion and release to the external aqueous phase during the DE/SE of LPCL/PEG nanoparticles.

Within the blends set of the SPEG/LPCL, SPEG has the highest loading at 2.5% as this amount is the optimum to promote the BSA diffusion towards the inner interface of the SPEG/LPCL nanoparticles but on increasing the SPEG content above 2.5%, water uptake by the SPEG/LPCL blend increases enhancing the partition and diffusion of the BSA towards the external aqueous phase. Regarding the LPEG/LPCL blends, they share the SPEG/LPCL blends the same phenomenon for the PEG content; as the LPEG content increases, the BSA loading decreases due to the same reason. It is important to note that each of the SPEG2.5%/LPCL and SPEG5%/LPCL nanoparticles achieve higher BSA loading than the 5% BSA loaded LPCL nanoparticles whereas the SPEG2.5% /LPCL nanoparticles have the highest loading efficiency than the other blends nanoparticles .

It was proved before that using surfactants like tween or PEG decreased the size and narrowed the PDI of the nanoparticles by producing uniform finer emulsion droplets through lowering the interfacial tension of the emulsion during the particles preparation (Jain et al. 2010, Bouillot et al. 1999, Essa, Rabanel & Hildgen 2011). This fact can describe why all the LPCL/PEG nanoparticles have smaller sizes and narrower PDI(s) than the original 5% BSA loaded LPCL nanoparticles. It was also reported that increasing the PEG MWt or content increases the water uptake and the polymeric aggregation to produce a more compact steric arrangement giving larger nanoparticles (Bouillot et al. 1999, Adami et al. 2012, Zamani, Khoee 2012). The LPEG produces larger nanoparticles with wider PDI than the SPEG due to its higher water uptake and entanglement than the SPEG. Nevertheless, increasing the PEG content in SPEG/LPCL and LPEG/LPCL blends increases the nanoparticles sizes and PDI(s) due to the enhancement of water uptake and PEG aggregation. According to the aforementioned fact that nanoparticles with lower sizes and narrow PDI(s) are preferred because these two factors increase the stability of nanoparticles in the emulsion (Sahoo et al. 2002, Essa, Rabanel & Hildgen 2011, Santander-Ortega et al. 2007), therefore SPEG2.5%/LPCL nanoparticles are more stable in emulsion than the other blends nanoparticles due to their lowest sizes and narrowest PDI(s).

Coating of nano/microparticles with many surfactants like amiphiphilic polymers normally reduces the zeta potential because the coating layers can mask the surface charge and move the shear plane outwards from the particle surface (Sahoo et al. 2002). This proves that the surfactant molecules, at least in part are located on the surface of the particles, which consequently displace the shear plane of the diffuse layer producing lower zeta potential values (Santander-Ortega et al. 2007). Zeta potential of the particles is also affected by the amount the hydrophilic polymers used; increasing content of the hydrophilic polymers leads to more reduction of the zeta potential due to enhancement of the shielding effect of the hydrophilic polymers at the nanoparticle surface (Dubey et al. 2012). All the LPCL/PEG nanoparticles are negatively charged due to the presence of charged carboxyl groups on the surface of the nanoparticles and presence of PEG in LPCL/PEG nanoparticles can be indicated by the lower zeta potential compared to LPCL nanoparticles. SPEG can be easier and more located on the surfaces of LPCL nanoparticle than LPEG due to its lower MWt, so the shielding effect of the SPEG on the nanoparticle surface is more pronounced than LPEG and as a result, SPEG/LPCL nanoparticles have lower zeta potential than LPEG/LPCL nanoparticles. Increasing the content of SPEG or LPEG in their blends increases the shielding effect at the nanoparticle surface that can obviously decreases the zeta potential. The SPEG2.5%/LPCL and LPEG2.5%/LPCL can have the best stability in suspensions because they have the highest zeta potentials.

PEG can increase the protein release rate when used as graft or copolymer or in blend with the polyesters by many mechanisms. The most important mechanism is increasing water absorption by its hydrophilic property that helps diffusion of the protein resulting in high release rate (Buske et al. 2012, Bouillot et al. 1999). PEG may lead to pores formation at the surface of the polyester particles that enhances water penetration into the particles producing higher release of the protein (Buske et al. 2012, Tran et al. 2012). On increasing the MWt or the content of the PEG, the particles acquire more and more hydrophilic property which consequently increases water absorption leading to higher protein release (Buske et al. 2012, Tran et al. 2012, Bouillot et al. 1999). Since the LPEG has more oxyethlene content than the SPEG, therefore the LPEG blends have higher hydrophilic power for more water absorption that results to more protein diffusion and release than the SPEG blends. Increasing the amount of SPEG or LPEG in their blends also raises the hydrophilic property for more water absorption producing higher protein release.

The PEG in the amphiphilic nanoparticles is not only oriented towards the external aqueous phase but also towards the inner aqueous phase and thus, the protein reservoir can be surrounded by a PEG barrier which protects the protein during the emulsification procedure and keeps its stability (Li et al. 2001). It is obvious that the BSA in the various PEG/LPCL nanoparticles has the same stability level regardless the MWt and amount of the PEG used. This confirms that the PEG barrier can keep the protein stability without being affected by the MWt or amount of PEG. All the amphiphilic nanoparticles don’t provide more BSA stability than the LPCL nanoparticles because the BSA protection by the hydrophobic interactions and physical cross links of LPCL is sufficient without the PEG barrier.

Finally, it can be observed that the various PCL/PEG blends formulate nanoparticles having better physicochemical properties than LPCL nanoparticles. All the LPCL/SPEG and LPCL/LPEG blends produce nanoparticles with smaller size, narrower PDI, lower zeta potentials and higher protein release rates than the 5% BSA loaded LPCL nanoparticles. SPEG2.5%/LPCL and SPEG5%LPCL nanoparticles have higher protein loading efficiencies than the 5% BSA loaded LPCL nanoparticles.

Chapter Five Conclusion

We successfully demonstrated the formulation and characterization of various PCL and amphiphilic PCL/PEG nanoparticles. LPCL nanoparticles have higher protein loading efficiencies, higher protein release rates, larger sizes and higher zeta potentials than MPCL and SPCL nanoparticles. Blending of PEG with PCL produces nanoparticles having better physicochemical properties than LPCL nanoparticles e.g. higher protein loading efficiencies, smaller sizes, narrower PDI(s), lower zeta potentials and higher protein release rates. All the PEG/LPCL nanoparticles have smaller sizes, narrower PDI(s), lower zeta potentials and higher protein release rates than the LPCL nanoparticles. The LPCL/SPEG2.5% and LPCL/SPEG5% nanoparticles achieve higher protein loading efficiencies than the LPCL nanoparticles.

These newly formed amphiphilic PCL/PEG nanoparticles system can be used as a good delivery system not only for proteins and peptides but also for hydrophilic and hydrophobic drugs. By increasing drug loading efficiency, large single dose can be administered instead of small frequent doses and by increasing the nanoparticles stability, the dose duration increases decreasing the administration frequency.

For better clearing of the benefits of this study, animal and human serum studies are recommended to be done on these various newly formed nanoparticles on loading different proteins like insulin, hydrophilic drugs like ranitidine and hydrophobic drugs like griseofulvin. Oral administration of certain proteins like insulin instead of injection will be more comfortable for the patients because it avoids infections, pain, contamination and other injection problems. This application will be highly valuable for treatment of diabetes because more than 347 million people worldwide have diabetes and a diabetic person has to pay direct costs for supplies and medication ranging from £ 621.5 to £ 9308 GBP a year. Moreover, it can enable the diabetic patients in the third world countries to avoid severe blood transmitted infections that occur on reusing of syringes e.g. AIDS and hepatitis c.

Sharing of the University of Ulster Researches to manage problems in the pharmaceutical industrial sector will effectively encourage the cooperation between industrial and academic sectors for research funding and training.

References

Abuchowski, A., Van Es, T., Palczuk, N.C. & Davis, F.F. (1977) Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. Journal of Biological Chemistry, 252(11), 3578-3581.

Adami, R., Liparoti, S., Izzo, L., Pappalardo, D. & Reverchon, E. (2012) PLA-PEG copolymers micronization by Supercritical Assisted Atomization. The Journal of Supercritical Fluids,.

Akagi, T., Baba, M. & Akashi, M. 2012, "Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine" in P olymers in Nanomedicine Springer, , pp. 31- 64.

Akbari, B., Tavandashti, M.P. & Zandrahimi, M. (2011) PARTICLE SIZE CHARACTERIZATION OF NANOPARTICLES–A PRACTICALAPPROACH. Iranian Journal of Materials Science and Engineering, 8(2), 48-56.

Bailon, P. & Berthold, W. (1998) Polyethylene glycol-conjugated pharmaceutical proteins. Pharmaceutical science & technology today, 1(8), 352-356.

Bainor, A., Chang, L., McQuade, T.J., Webb, B. & Gestwicki, J.E. (2011) Bicinchoninic acid (BCA) assay in low volume. Analytical Biochemistry, 410(2), 310-312.

Blanco, D. & Alonso, M.J. (1998) Protein encapsulation and release from poly (lactide-co-glycolide) microspheres: effect of the protein and polymer properties and of the co-encapsulation of surfactants. European Journal of Pharmaceutics and Biopharmaceutics, 45(3), 285-294.

Bouillot, P., Ubrich, N., Sommer, F., Minh Duc, T., Loeffler, J. & Dellacherie, E. (1999) Protein encapsulation in biodegradable amphiphilic microspheres. International journal of pharmaceutics, 181(2), 159-172.

Budavari, S., O'Neil, M.J., Smith, A. & Heckelman, P.E. (1989) The Merck Index, Merck & Co. Inc., Rahway, NJ, 1606.

Buske, J., König, C., Bassarab, S., Lamprecht, A., Mühlau, S. & Wagner, K.G. (2012) Influence of PEG in PEG–PLGA microspheres on particle properties and protein release. European Journal of Pharmaceutics and Biopharmaceutics, 81(1), 57-63.

Chandra, R. & Rustgi, R. (1998) Biodegradable polymers. Progress in Polymer Science (Oxford), 23(7), 1273-1335.

Coccoli, V., Luciani, A., Orsi, S., Guarino, V., Causa, F. & Netti, P.A. (2008) Engineering of poly (ε-caprolactone) microcarriers to modulate protein encapsulation capability and release kinetic. Journal of Materials Science: Materials in Medicine, 19(4), 1703-1711.

Coulembier, O., Degée, P., Hedrick, J.L. & Dubois, P. (2006) From controlled ring- opening polymerization to biodegradable aliphatic polyester: Especially poly(ß-malic acid) derivatives. Progress in Polymer Science (Oxford), 31(8), 723-747.

Danhier, F., Ansorena, E., Silva, J.M., Coco, R., Le Breton, A. & Préat, V. (2012) PLGA-based nanoparticles: an overview of biomedical applications. Journal of Controlled Release, 161(2), 505-522.

Dash, T.K. & Konkimalla, V.B. (2012) Poly-є-caprolactone based formulations for drug delivery and tissue engineering: A review. Journal of Controlled Release, 158(1), 15-33. des Rieux, A., Fievez, V., Garinot, M., Schneider, Y. & Préat, V. (2006) Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach. Journal of Controlled Release, 116(1), 1-27.

Dubey, N., Varshney, R., Shukla, J., Ganeshpurkar, A., Hazari, P.P., Bandopadhaya, G.P., Mishra, A.K. & Trivedi, P. (2012) Synthesis and evaluation of biodegradable PCL/PEG nanoparticles for neuroendocrine tumor targeted delivery of somatostatin analog. Drug delivery, 19(3), 132-142.

Essa, S., Rabanel, J.M. & Hildgen, P. (2011) Characterization of rhodamine loaded PEG-g-PLA nanoparticles (NPs): Effect of poly(ethylene glycol) grafting density. International journal of pharmaceutics, 411(1–2), 178-187.

Feczkó, T., Tóth, J., Dósa, G. & Gyenis, J. (2011) Optimization of protein encapsulation in PLGA nanoparticles. Chemical Engineering and Processing: Process Intensification, 50(8), 757-765.

Freiberg, S. & Zhu, X.X. (2004) Polymer microspheres for controlled drug release. International journal of pharmaceutics, 282(1-2), 1-18.

Gaucher, G., Dufresne, M.-., Sant, V.P., Kang, N., Maysinger, D. & Leroux, J.-. (2005) Block copolymer micelles: Preparation, characterization and application in drug delivery. Journal of Controlled Release, 109(1-3), 169-188.

Gupta, R. & Shepherd, K. (1990) Two-step one-dimensional SDS-PAGE analysis of LMW subunits of glutelin. Theoretical and Applied Genetics, 80(1), 65-74.

Hartinger, J., Stenius, K., Högemann, D. & Jahn, R. (1996) 16-BAC/SDS–PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. A nalytical Biochemistry, 240(1), 126-133.

Hutmacher, D.W. (2001) Scaffold design and fabrication technologies for engineering tissues - State of the art and future perspectives. Journal of Biomaterials Science, Polymer Edition, 12(1), 107-124.

J. Milton Harris (1992) Poly (ethylene glycol) chemistry: biotechnical and biomedical applications. Plenum Publishing Corporation.

Jain, S., Mittal, A., K Jain, A., R Mahajan, R. & Singh, D. (2010) Cyclosporin A loaded PLGA nanoparticle: preparation, optimization, in-vitro characterization and stability studies. C urrent Nanoscience, 6(4), 422-431.

Jia, W.J., Gu, Y.C., Gou, M.L., Dai, M., Li, X.Y., Kan, B., Yang, J.L., Song, Q.F., Wei, Y.Q. & Qian, Z.Y. (2008) Preparation of biodegradable polycaprolactone/poly (ethylene glycol)/polycaprolactone (PCEC) nanoparticles. Drug delivery, 15(7), 409-416.

Jones, D.S., McCoy, C.P. & Andrews, G.P. (2011) Physicochemical and drug diffusion analysis of rifampicin containing polyethylene glycol–poly(ɛ- caprolactone) networks designed for medical device applications. Chemical Engineering Journal, 172(2–3), 1088-1095.

Kang, N., Perron, M., Prud'Homme, R.E., Zhang, Y., Gaucher, G. & Leroux, J. (2005) Stereocomplex block copolymer micelles: core-shell nanostructures with enhanced stability. Nano letters, 5(2), 315-319.

Katre, N.V. (1993) The conjugation of proteins with polyethylene glycol and other polymers: Altering properties of proteins to enhance their therapeutic potential. Advanced Drug Delivery Reviews, 10(1), 91-114.

Konan, Y.N., Cerny, R., Favet, J., Berton, M., Gurny, R. & Allémann, E. (2003) Preparation and characterization of sterile sub-200 nm meso-tetra(4- hydroxylphenyl)porphyrin-loaded nanoparticles for photodynamic therapy. European Journal of Pharmaceutics and Biopharmaceutics, 55(1), 115-124.

Krishnamachari, Y. (2011) PLGA microparticle based vaccine carriers for an improved and efficacious tumor therapy.

Labet, M. & Thielemans, W. (2009) Synthesis of polycaprolactone: a review. Chemical Society Reviews, 38(12), 3484-3504.

Leroueil-Le Verger, M., Fluckiger, L., Kim, Y., Hoffman, M. & Maincent, P. (1998) Preparation and characterization of nanoparticles containing an antihypertensive agent. European journal of pharmaceutics and biopharmaceutics, 46(2), 137-143.

Li, Y., Pei, Y., Zhang, X., Gu, Z., Zhou, Z., Yuan, W., Zhou, J., Zhu, J. & Gao, X. (2001) PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats. Journal of Controlled Release, 71(2), 203-211.

Ma, Y., Zheng, Y., Zeng, X., Jiang, L., Chen, H., Liu, R., Huang, L. & Mei, L. (2011) Novel docetaxel-loaded nanoparticles based on PCL-Tween 80 copolymer for cancer treatment. International journal of nanomedicine, 6, 2679.

Marchal-Heussler, L., Sirbat, D., Hoffman, M. & Maincent, P. (1993) Poly(e- caprolactone) nanocapsules in carteolol ophthalmic delivery. Pharmaceutical research, 10(3), 386-390.

Merkli, A., Tabatabay, C., Gurny, R. & Heller, J. (1998) Biodegradable polymers for the controlled release of ocular drugs. Progress in Polymer Science (Oxford), 23(3), 563-580.

Nair, L.S. & Laurencin, C.T. (2007) Biodegradable polymers as biomaterials. Progress in Polymer Science (Oxford), 32(8-9), 762-798.

Okada, M. (2002) Chemical syntheses of biodegradable polymers. Progress in Polymer Science (Oxford), 27(1), 87-133.

Rameez, S., Alosta, H. & Palmer, A.F. (2008) Biocompatible and biodegradable polymersome encapsulated hemoglobin: a potential oxygen carrier. Bioconjugate chemistry, 19(5), 1025-1032.

Ravi Kumar, M.N.V., Bakowsky, U. & Lehr, C.M. (2004) Preparation and characterization of cationic PLGA nanospheres as DNA carriers. Biomaterials, 25(10), 1771-1777.

Saez, A., Guzmán, M., Molpeceres, J. & Aberturas, M.R. (2000) Freeze-drying of polycaprolactone and poly(D,L-lactic-glycolic) nanoparticles induce minor particle size changes affecting the oral pharmacokinetics of loaded drugs. European Journal of Pharmaceutics and Biopharmaceutics, 50(3), 379-387.

Sahoo, S.K., Panyam, J., Prabha, S. & Labhasetwar, V. (2002) Residual polyvinyl alcohol associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. Journal of Controlled Release, 82(1), 105-114.

Santander-Ortega, M., Csaba, N., Alonso, M., Ortega-Vinuesa, J. & Bastos- González, D. (2007) Stability and physicochemical characteristics of PLGA, PLGA: poloxamer and PLGA: poloxamine blend nanoparticles: a comparative study. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 296(1), 132-140.

Sinha, V.R., Bansal, K., Kaushik, R., Kumria, R. & Trehan, A. (2004) Poly-ϵ- caprolactone microspheres and nanospheres: an overview. International journal of pharmaceutics, 278(1), 1-23.

Storey, R. & Taylor, A. 1996, "Effect of stannous octoate concentration on the ethylene glycol-initiated polymerization of epsilon-caprolactone", ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY, pp. 114.

Sun, H., Mei, L., Song, C., Cui, X. & Wang, P. (2006) The in vivo degradation, absorption and excretion of PCL-based implant. Biomaterials, 27(9), 1735- 1740.

Thompson, M.S., Vadala, T.P., Vadala, M.L., Lin, Y. & Riffle, J.S. (2008) Synthesis and applications of heterobifunctional poly(ethylene oxide) oligomers. Polymer, 49(2), 345-373.

Tobio, M., Gref, R., Sanchez, A., Langer, R. & Alonso, M. (1998) Stealth PLA-PEG nanoparticles as protein carriers for nasal administration. Pharmaceutical research, 15(2), 270-275.

Todokoro, H. & Otaka, T. (1995) Scanning electron microscope,.

Tran, V., Karam, J., Garric, X., Coudane, J., Benoît, J., Montero-Menei, C.N. & Venier-Julienne, M. (2012) Protein-loaded PLGA–PEG–PLGA microspheres: A tool for cell therapy. European Journal of Pharmaceutical Sciences, 45(1), 128-137.

Vandervoort, J. & Ludwig, A. (2002) Biocompatible stabilizers in the preparation of PLGA nanoparticles: a factorial design study. International journal of pharmaceutics, 238(1–2), 77-92.

Wei, X., Gong, C., Gou, M., Fu, S., Guo, Q., Shi, S., Luo, F., Guo, G., Qiu, L. & Qian, Z. (2009) Biodegradable poly (ɛ-caprolactone)–poly (ethylene glycol) copolymers as drug delivery system. International journal of pharmaceutics, 381(1), 1-18.

Woodruff, M.A. & Hutmacher, D.W. (2010) The return of a forgotten polymer— Polycaprolactone in the 21st century. Progress in Polymer Science, 35(10), 1217-1256.

Wu, J.M., Li, Z., Zhao, Y., Guan, J., Huang, S., Li, R. & Zhang, X. (2012) Effect of the Lactide/Glycolide Ratio and Molecular Weight of PLGA on the Bovine BMPs Microspheres Systems. Advanced Materials Research, 466, 405-410.

Youan, B., Benoit, M., Baras, B. & Gillard, J. (1999) Protein-loaded poly (epsilon- caprolactone) microparticles. I. Optimization of the preparation by (water-in- oil)-in water emulsion solvent evaporation. Journal of microencapsulation, 16(5), 587-599.

Zamani, S. & Khoee, S. (2012) Preparation of core-shell chitosan/PCL-PEG triblock copolymer nanoparticles with ABA and BAB morphologies: Effect of intraparticle interactions on physicochemical properties. Polymer,.

Zhang, Y. & Zhuo, R. (2005) Synthesis and in vitro drug release behavior of amphiphilic triblock copolymer nanoparticles based on poly (ethylene glycol) and polycaprolactone. Biomaterials, 26(33), 6736-6742.

[...]