STERIC STABILIZATION OF POLYLACTIDE PARTICLES ...622327/...————— Purpose of the study...

STERIC STABILIZATION OF POLYLACTIDE PARTICLES ACHIEVED BY

COVALENT ‘GRAFTING-FROM’ WITH HYDROPHILIC POLYMERS

Robertus Wahyu N. Nugroho

AKADEMISK AVHANDLING Som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknisk licentiatexamen fredagen den 10 juni 2013, kl. 10.00 i sal K2, Teknikringen 28, KTH, Stockholm. Avhandlingen försvaras på engelska.

ABSTRACT

Despite numerous advantages of using particles in a wide range of applications, they have one drawback that is their tendency to agglomerate. One way to overcome this problem is to sterically stabilize the particles by introducing polymeric chains covalently attached to the surface. Surface modification by covalently attaching polymer chains to the particle surface can be achieved by e.g. a ‘grafting-from’ technique under UV irradiation. In this thesis, polylactide (PLA) particles were surface modified, under UV irradiation, with the hydrophilic monomers: acrylamide (AAm), acrylic acid (AA), and maleic anhydride (MAH). The developed ‘grafting-from’ technique was shown to be nondestructive method for surface modification of PLA particles of two different geometries. The change in surface chemistry of the PLA particles was confirmed by FTIR and XPS, indicating the success of the surface grafting technique. Force interaction between two surface grafted PLA substrates was measured by colloidal probe AFM in different salt concentrations. In order to understand the repulsive force, the AFM force profiles were compared to the DLVO theory and AdG model. Long range repulsive interactions were mainly observed when hydrophilic polymers were covalently attached to the surface of PLA particles, leading to steric interaction. Attractive force dominated the interaction when neat PLA particle was approaching each other, resulting in particle aggregation, even though short range repulsion was observed at small separation distance, i.e. approximately 10 nm. Attractive interaction was also observed when neat PLA was approaching to PAA-grafted PLA substrate. This attractive interaction was much greater than force interaction between two neat PLA substrates. The surface grafted particles can be used in biomedical application where secondary interactions are important to overcome particle agglomeration such as particle-based drug delivery. Keywords: Steric stabilization, AFM, hydrophilicity, surface modification, poly(lactide), acrylamide, acrylic acid, maleic anhydride

SAMMANFATTNING

Trots de många fördelarna med att använda partiklar i ett flertal applikationer finns en stor nackdel; deras benägenhet att agglomerera. Ett sätt att undvika detta är att steriskt stabilisera partiklarna genom att kovalent ympa kedjor på ytan. Ytmodifiering genom att kovalent ympa polymerkedjor på partiklar kan göras genom s.k. ”ympning-från” under UV-ljus. I denna avhandling presenteras UV-ljus initierad ytmodifiering av polylaktid (PLA)-partiklar med akrylamid (AAm), akrylsyra (AA), and maleinsyraanhydrid (MAH). Den utvecklade “ympning-från”-tekniken visades vara icke-förstörande vid ytmodifiering av PLA-partiklar med två olika geometrier. Förändringen i de ytkemiska egenskaperna hos PLA-partiklarna efter ympning bekräftades av FTIR och XPS. Detta visade på en fungerande ytmodifieringsteknik. Kraftinteraktionen mellan två ytmodifierade PLA-substrat mättes därefter genom kolloidal probe AFM i saltlösningar med olika koncentrationer. För att förstå karaktären av de uppmätta krafterna jämfördes AFM-kraftprofilerna med DLVO-teorin och AdG-modellen. Huvudsakligen observerades långtgående repulsiva interaktioner när de hydrofila polymererna hade kovalent ympats på ytan. Detta ledde till sterisk stabilisering. Attraktiva krafter dominerade när två rena PLA-partiklar närmade sig varandra. Detta ledde till att partiklarna agglomererade även om en kortgående repulsion observerades vid ett kort avstånd (ca 10 nm). Attraktiva interaktioner iakttogs också när en ren PLA-partikel närmade sig ett PAA-ympat PLA-substrat. Denna attraktiva interaktion var mycket större än kraftinteraktionen mellan två rena PLA-substrat. De ytmodifierade partiklarna kan användas i biomedicinska applikationer där sekundära interaktioner är viktiga för att undvika agglomerering, t.ex. partikelbaserad kontrollerad läkemedelsfrisättning. Nyckelord: Sterisk stabilisering, AFM, hydrofilictet, ytmodifiering, polylaktid, akrylamid, akrylsyra, maleinsyraanhydrid

LIST OF PAPERS

This thesis is a summary of the following papers: I “Nondestructive Covalent ‘Grafting-from’ of Poly(lactide) Particles of

Different Geometries”, Nugroho, R.W.N., Odelius, K., Höglund, A., Albertsson, A.-C., ACS Applied Materials and Interfaces 2012, 4, 2978-2984

II “Force Interactions of Non-agglomerating Polylactide Particles Obtained

through Covalent Surface Grafting with Hydrophilic Polymers”, Nugroho, R.W.N., Pettersson, T., Odelius, K., Höglund, A., Albertsson, A.-C., Langmuir, accepted

Paper not included in the thesis:

III “Crosslinked PVAL Nanofibers with Enhanced Long-term Stability Prepared by Single-step Electrospining”, Nugroho, R.W.N., Roy, P.K., Odelius, K, and Albertsson, A.-C., Polymer for Advanced Technologies, 2012, 24, 421-429

TABLE OF CONTENTS

ABBREVIATIONS..................................................................................................... 3

1 PURPOSE OF THE STUDY .............................................................................. 1

2 INTRODUCTION ............................................................................................... 2

2.1 STERIC STABILIZATION ........................................................................ 2 2.1.1 The DLVO and AdG model ................................................................... 3

2.2 FORCE MEASUREMENT ......................................................................... 6 2.3 SURFACE MODIFICATION .................................................................... 8

2.3.1 ‘Grafting-From’ polymerization ........................................................... 9 2.4 POLYLACTIDE .......................................................................................... 9 2.5 HYDROPHILIC MONOMERS ............................................................... 10

3 EXPERIMENTAL ............................................................................................ 12

3.1 MATERIALS .................................................................................................. 12 3.2 PARTICLE AND FILM FABRICATION ............................................................... 12 3.3 ‘GRAFTING-FROM’ POLYMERIZATION .......................................................... 13 3.4 CHARACTERIZATION METHODS ................................................................... 14

3.4.1 Size Exclusion Chromatography (SEC) .............................................. 14 3.4.2 Fourier Transform Infrared (FTIR) Spectroscopy ............................ 14 3.4.3 X-ray Photoelectron Spectroscopy (XPS) ........................................... 14 3.4.4 Scanning Electron Microscopy (SEM) ............................................... 14 3.4.5 Atomic Force Microscopy (AFM) ....................................................... 14

4 RESULTS AND DISCUSSION........................................................................ 16

4.1 PARTICLE FABRICATION .............................................................................. 16 4.2 NONDESTRUCTIVE ‘GRAFTING-FROM’ METHOD .......................................... 17 4.3 PARTICLE SURFACE CHEMISTRY .................................................................. 19

4.4 PARTICLE SURFACE MORPHOLOGY .............................................................. 23 4.5 PARTICLE SURFACE TOPOGRAPHY ............................................................... 24 4.6 FORCE INTERACTIONS OF DIFFERENT PLA SUBSTRATE SURFACES.............. 27

5 CONCLUSIONS ............................................................................................... 33

6 FUTURE WORK .............................................................................................. 34

7 ACKNOWLEDGEMENTS .............................................................................. 35

8 REFERENCES .................................................................................................. 37

ABBREVIATIONS

A Hamaker constant AA Acrylic Acid AAm Acrylamide AdG Alexander de Gennes AFM Atomic Force Microscopy ATR Attenuated Total Reflectance BP Benzophenone D Distance DCM Dichloromethane DLVO Derjaguin-Landau-Verwey-Overbeek EDL Electrical Double Layer F Force FTIR Fourier Transform Infra Red kB Boltzmann constant Lo Thickness of the grafted chains MAH Maleic Anhydride Mn Number-average molecular weight NaCl Natrium Chloride O/W Oil-in-Water PAA Poly(acrylic acid) PAAm Poly(acrylamide) PDI Polydispersity Index PLA Poly(lactide) PLA-g-PAA PLA that was grafted with PAA PLA-g-PAAm PLA that was grafted with PAAm PMAH Poly(maleic anhydride) R Radius RO Reverse Osmosis

ROP Ring Opening Polymerization rpm revolutions per minute s Average distance of grafting site SEC Size Exclusion Chromatography SEM Scanning Electron Microscopy SFA Surface Force Aparatus T Temperature UV Ultraviolet VAc Vinyl Acetate Vtot Total interaction force Velec Repulsive electrostatic force VvdW van der Waals force W Interaction energy XPS X-Ray Photoelectron Spectroscopy Zc Cantilever deflection Zp Piezo position 1/κ Debye length ΔG Gibbs energy ΔH Entalphy difference ΔS Entropy difference ρ∞ Ionic concentrations 𝜑𝑜 Surface potential Γ Grafting density

————— Purpose of the study —————

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1 PURPOSE OF THE STUDY

Steric stabilization of particles is one of the promising ways to overcome particle aggregation. The aggregation of particulate materials limits their potential use in a broad range of applications and the effect is sometimes negative when the aggregated particles lack repulsive interaction. The particles have a tendency to aggregate to reduce their high surface energy. Thus, stability issues in particulate materials have been the focus of attempts to inhibit agglomeration. The most common strategy is to attach stabilizers to the substrate physically or chemically. Surface active agents, i.e. surfactant, are often employed and anchored on the surface by means of physical adsorption. The main drawback of this technique is their temporary attachment on the substrate surface so that, the stability is not permanent. A number of surface grafting techniques have been demonstrated on planar surfaces and particles, and a ‘grafting-from’ appproach is commonly used to achieve high-density end-grafted polymers by covalent attachment with a mild process at low cost. This is, however, a two-step process where the polymer surface is first activated with a photosensitive initiator, benzophenone (BP), under UV irradiation, after which an unsaturated monomer is ‘grafted-from’ the polymer surface via photo-induced free-radical polymerization in the second step. In this work, hydrophobic PLA was selected as polymeric substrate because of its biodegradability, renewability and inertness. The chemical inertness makes it difficult to modify the surface properties, thus surface modification would increase to improve the physicochemical properties of PLA indented for biomedical applications where secondary interactions are important. Three hydrophilic monomers were used in this work: acrylamide (AAm), acrylic Acid (AA), and maleic Anhydride (MAH). The specific objective of this thesis is therefore to tailor sterically-stabilized systems by covalent surface grafting of PLA with hydrophilic polymers. Steric stabilization is required to overcome agglomeration which commonly occurs, for example in particle-based drug delivery.

————— Introduction —————

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2 INTRODUCTION

2.1 STERIC STABILIZATION Steric stabilization is related to all aspects of the stabilization of nonionic polymer-grafted particles that are dispersed in good solvents. The term ‘steric’ is directed more to repulsion in general thermodynamics than in organic chemistry that has also similar term.1,2 The presence of a repulsive interaction was first observed when poly(ethylene oxide) chains were used to stabilize certain insoluble polymer in water.3 When interpenetration of end-grafted chains attached to the particle surface occurs, the Gibbs energy, given by the Gibbs-Helmholtz relationships ΔGR = ΔHR- TΔSR, changes. If ΔGR is negative, particle aggregation has a tendency to occur. If ΔGR is equal to zero, aggregation proceeds as if the end-grafted polymers did not exist. Stabilization of particles is observed if ΔGR has a positive value. Particle aggregation can also be explained in terms of the interaction energy (W) (Figure 1). The depth of the potential energy minimum (Wo) depends on two main factors, the thickness of the adsorbed layer at the surface and the magnitude of the van der Waals interaction energy. The former parameter depends on polymer-surface interaction, polymer-solvent interaction and the dimension of the polymer coils, while the later depends on the nature of the particle and medium, i.e. the Hamaker constant and the particle dimensions.4,5 The use of hydrophilic substances in stabilizing hydrophobic dispersions has been known since the last century. Steric repulsion operates to overcome particle aggregation at a distance where attraction due to van der Waals forces dominates. In practice, steric and electrostatic mechanisms may operate simultaneously, particularly in aqueous systems. Steric interaction is advantageous in aqueous systems as follows: (a) intolerance to the electrolyte medium (b) the electro-viscous effect due to the particle charge can be reduced by an adding electrolyte to the medium without coagulation (c) stable dispersions may be prepared at much higher


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solid concentrations. Several practical examples such as pharmaceuticals, cosmetics, food processing, paint and paper are fields where the end-grafted polymers are used to stabilize dispersions.6,7

2.1.1 The DLVO and AdG model

There are two mechanisms for stabilizing colloids, electrostatic and steric, that are based on two colloidal concepts, the Derjaguin-Landau-Verwey-Overbeek (DLVO) and the Alexander de Gennes (AdG) model. The former explains stabilization that is caused by electrostatic interaction, while the latter provides a fundamental approach to steric stabilization to overcome particle aggregation. The DLVO theory8,9 predicts that highly charged colloidal particles exert a purely repulsive columbic interaction at large separation.10 The theory further provides an approximate solution to the Poisson-Boltzmann equation describing total coupling11 between repulsive electrostatic forces and attractive van der Waals forces according to the equation: Vtot = Velectrostatic + VvdW (1)

𝑉𝑡𝑜𝑡 = (64𝜋𝑘𝐵𝑇𝑅𝜌∞𝛾2

𝜅2)𝑒−𝜅𝐷 + −𝐴𝑅

6𝐷 (2)

where kB is the Boltzmann constant, T is the temperature, R is the radius, ρ∞ is the ionic concentration of ions in the bulk, γ can be calculated from γ = tan h [𝜑𝑜(mV)/103]; 𝜑𝑜 is the surface potential, 1/κ is the Debye length, D is the separation distance, and A is the Hamaker constant. Vvdw is dependent on the Hamaker constant, particle size, and distance between particles, while Velectrostatic is dependent on particle size, distance between particles, zeta potential, ion concentration and dielectric constant of the medium. As the ionic strength is increased in the medium, the electrical double layer (EDL) shows a decrease due to the screening of the surface charge. This causes a decrease in Velectrostatic, and increases the tendency for particle agglomeration to occur. The repulsive force due to electrostatic interaction originates from the presence of overlapping of EDL surrounding the particles in the electrolyte. As a consequence, particle agglomeration is overcome. EDL involves two main layers: (1) the Stern layer that is filled with counter ions surrounding the particle surface to maintain electroneutrality in the system and (2) the Gouy layer that consists of a diffusion layer of ions.12 Depending on the electrolyte concentration and the surface charge density, the following interactions may occur:13


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a) For surfaces that are highly charged in a dilute electrolyte (i.e. long Debye length), the long-range repulsion appears at some distance with a certain energy barrier (Figure 1a).

b) In electrolyte solutions of higher concentration, the secondary minimum peak is commonly beyond 3 nm prior to the energy barrier. The minimum of potential energy at contact refers to the primary minimum (Figure 1b).

c) For surfaces that have a low charge density, the energy barrier is much lower (Figure 1c), and aggregation or flocculation occurs. When the energy barrier falls below W=0, the particles rapidly agglomerate, and they are referred to as being unstable (Figure 1d).

d) If the surface charge or potential has a zero value, the interaction curve shows an approach to the pure van der Waals curve and as a consequence of the zero surface charge, the two surfaces give attractive interaction each other strongly at all separations (Figure 1e).

Figure 1. A typical energy profile versus distance of DLVO interaction (a) two surfaces repel each other; the colloidal particles maintain their stability, (b) two surfaces located at stable equilibrium at secondary minimum; the colloidal particles are still stable, (c) two surfaces are in a secondary minimum; the colloidal particles slowly aggregate (d) two surfaces may remain in secondary minimum or adhere; aggregation of the colloidal particles occurs, and (e) the colloidal particles rapidly coalesce.


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Another model that can be used to explain steric stabilization is the Alexander-de Gennes (AdG) model,14 which assumes that all chains are uncharged, and that there is no adsorption and no interpenetration between the end-grafted polymers interacting with each other.

Figure 2. The AdG model of a polymer brush. The size of the polymer blob (ξ) is equal to the average distance between two grafting sites of the end-grafted chain.

According to this model, the so-called box-model, the chains in the brush-shaped polymer, i.e. the conformation of the end-grafted polymer, are described as a blob or scaling model (Figure 2). It can be assumed that the polymers are stretched out uniformly and all chain ends are positioned at the edge of the polymer brush. The confinement of the end-grafted chains is caused by the influence of other neighboring chains that results in the formation of a number of chain blobs, as displayed in Figure 2. A blob is thus described as a string of monomers, the conformations of which are not perturbed by any intermolecular interactions.15,16 de Gennes later extended these concepts to the case of the interaction force versus area between two parallel flat plates covered by the end-grafted layers according to the equation:17 𝑓(𝐷) = 𝑘𝐵𝑇

𝑠3[(2𝐿𝑜

𝐷)94 − ( 𝐷

2𝐿𝑜)34] 𝐷 < 2𝐿𝑜 (3)

𝛤 = 1𝑠2

(4)

where f(D), kB, s, Lo, D and Γ the are interaction force per unit area, Boltzmann constant, the average distance of grafting site, the thickness of the grafted chain, the distance between neat substrates, and the grafting density, respectively.


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2.2 FORCE MEASUREMENT The measurement of surface forces, such as the force of an end-grafted polymer attached to the surface, has attracted much interest in colloidal and surface science. The surface force apparatus (SFA) was previously used to investigate the interaction between two smooth mica surfaces at a molecular scale.18-20 The instrument has given a valuable contribution to the understanding of surface forces at the nanometer level. The atomic force microscopy (AFM) (Figure 3), invented by Binnig et al. in the 1980s21 and belonging to the scanning probe microscope,22 has made it possible in colloidal and surface science to investigate all kinds of surface and not only smooth ones.20

Figure 3. The elements of an atomic force microscope.

AFM has been developed as a powerful tool in imaging, measuring forces at the molecular level between hydrophobic,23,24 hydrophilic/hydrophobic25-27 and hydrophilic substrates,28-30 evaluating changes in surface topography after periods of degradation,31 and monitoring several processes across the length from nanometers to hundreds of micrometers.32 In the AFM, the sample surface is scanned by a micrometer-sized cantilever to which a silicon-based tip is attached. While the probe scans the surface topography, the force occurring between the tip and the substrate surface is measured from the deflection of the cantilever. A topographic image can


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then be achieved by plotting the deflection of the cantilever,estimated by Hooke’s law, versus its location on the sample surface: F=-kcδc (5) where kc and δc are the cantilever spring constant and the cantilever displacement (deflection), respectively. AFM feedback loop controls the height position of the sample that gives a force constantly interacting between tip and substrate. The contrast of the topographic image arises because the force between the tip and substrate constitutes a function of tip and sample separation, the materials properties of the tip, and the sample. Low spring constants, i.e. <10 N/m, will not damage the sample surface during cantilever deflection.22

Figure 4. A typical scheme of cantilever deflection versus piezo height during force measurement; (A) cantilever starts at distance far above the surface, no force; (B) a repulsive force deflects the cantilever; (C) a jump into contact due to the gradient of the tip-sample force is greater than the spring force of the cantileverl; (D) and (E) are the condition where the tip is in contact with the sample; (F) the cantilever stores the energy greater than the adhesive energy; (G) the cantilever returns to its initial position. In force measurements, one important step towards understanding force interactions at molecular surfaces was the introduction of colloidal probe AFM method,33,34 where a spherical particle having a diameter of approximately 2-20 µm is glued on the free-end of cantilever for use as the sensor during force interaction. The force between


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this single spherical particle and a flat surface is thereafter measured, where the interaction between the probe and the substrate depend on the influences of functional groups attached to the surface. By means of AFM colloidal probe, interacting forces on the surface can be quantitatively measured because the radius of a single sphere glued onto the end-cantilever can be determined. The sample surface goes up and down as a voltage is applied and connected to the piezoelectric translator onto which the sample is located, while the cantilever deflection is measured, as seen in Figure 4. The force measurement provides a result in terms of the cantilever deflection (Zc) versus the position of the piezo (Zp) normal to the surface (Figure 4). In order to obtain a force curve, i.e. Force (F) versus distance (D), Zp and Zc are converted to force and distance. The force is calculated from the cantilever deflection multiplied by the spring constant according to the Hooke’s law (Eq.5). The separation between the micrometer-sized probe and the sample surface is obtained by adding the value of deflection to the position; D = Zc + Zp. It is then commonly called as the separation distance in the force curve.22

2.3 SURFACE MODIFICATION In order to obtain steric stabilization, the surface of a material is often modified. In general, a polymer-based material is selected for a specific application due to its favorable bulk properties for instance solvent resistance, thermal stability, and mechanical strength. The chosen polymer sometimes has insufficient surface characteristics for the intended applications, i.e. a hydrophobic, chemically inert, non-polar surface. To alter this, different surface modification strategies have been prepared and implemented. Surface modification can be divided into two broad groups:

• physical adsorption leading to alterations in the atoms or molecules present on the surface.

• covalent attachment to the surface with a material yielding a new surface composition (e.g. UV irradiation induced grafting, plasma modification).

Several technologies have been addressed to the attachment of new functionalities to the polymer surfaces, including surface hydrophilicity, hardness, roughness, conductivity, lubrication and adhesion. Theoretically, there is a significant difference in properties between the bulk and the surface of a material and only the outermost surface needs to be concerned when the surface properties are taken into consideration. However, this is not the case for a polymer surface, as the surface is not fixed in general, but changes with time due to microscopic Brownian motion of


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polymer segments.35-38 As has been stated earlier, the surface of the object can be modified physically or chemically. Numerous chemical modification techniques have been used to modify the surface of aliphatic polyesters including alkaline hydrolysis reactions,39 ozone treatment,40 plasma treatment,41 high-energy irradiation,42-44 vapor phase grafting,45-50 and UV irradiation or photo-grafting.51,52 A variety of new technologies have been demonstrated to improve surface characteristics, and surface grafting offers versatility for introducing new functionalities into the existing polymers.36

2.3.1 ‘Grafting-From’ polymerization

The polymer chains covalently attached to modify a surface can be performed by two distinct approaches: ‘grafting-from’ and ‘grafting-to’. The former involves the synthesis of polymer chains from the substrate surface which is initially activated by initiator to allow for chain growth mechanism, whereas the latter involves the attachment of the preformed polymer that has a functional group able to bond with the surface.37 The former technique gives more promising result in producing a polymer film with a larger thickness and higher grafting density than the latter due to diffusion and less steric hindrance.20 The net result of these both cases is the end-grafted polymers covalently attached to the substrate surface. A versatile method to attach unsaturated monomers, i.e.vinyl or acrylic monomers, is the free-radical polymerization reaction. For this type of polymerization, unpaired electrons need to be created at the substrate surface in order to make the surface chemically reactive. Care must be taken to produce the reactive sites only on the surface and to leave the bulk unperturbed. Unpaired electrons can be produced on the surface in several ways and one of these ways is by means of UV irradiation.37 Ultraviolet (UV) radiation appears to be excellent for graft polymerization due to its simplicity, cleanliness, low cost energy, and permanent covalent attachment.36

2.4 POLYLACTIDE Polylactide (PLA) (Figure 6) has been known for several decades, but interest in this material is swiftly increasing. Its monomer, lactide, is used in the production of high molar mass PLA by ring-opening polymerization (ROP) and is the most important industrial process to produce high molar mass PLA.53,54 Specific chemical functionalities, hydrophilicity, surface roughness and topography are needed to improve its physicochemical properties.55 PLA has been surface-modified by several techniques. For example, surface alkali hydrolysis constitutes a simple chemical modification method. After chemical mechanism of hydrolysis occurring on the


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surface of aliphatic polyester, hydrophilic carboxyls and hydroxyls can be created by cleaving the ester bond.56 Since PLA has inertness on the surface, photo-grafting is another useful method for PLA surface modification. As has been previously noted, this method is classified into two different approaches, ‘grafting-from’ and ‘grafting-to’.55 Janorkar et al. have demonstrated the ‘grafting-from’ technique on PLA surfaces to which they were first attached with amine groups,-NH2. They have also demonstrated that the same substrate could be photo-grafted with three different hydrophilic monomers, vinyl acetate (VAc), acrylamide (AAm) and acrylic acid (AA) to tailor the wettability of PLA surface.57,58

Figure 6. The chemical structure of PLA.

2.5 HYDROPHILIC MONOMERS Several different monomers can be used for covalent surface grafting as described below:

Figure 7. The chemical structure of AAm.

Acrylamide (AAm) (Figure 7) is a colorless white, odorless crystalline solid. It is soluble in various polar solvents such as water, methanol and ethanol, but poorly solubility in chloroform, benzene and hexane. AAm is radically polymerized to PAAm on melting or exposure to UV irradition.59 Several studies have shown the potential use of AAm to modify membrane surfaces due to its high hydrophilicity.60-

62 The conformation of PAAm end-grafted onto the mica surface has been


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investigated. AFM measurements have enabled that the loop length distribution of the grafted polymer to be observed.63 Acrylic acid (AA) (Figure 8) is a clear, colorless liquid which forms crystalline needles in the solid state. It is highly soluble in water, alcohol and esters. Acrylic acid and its esters can be easily polymerized by heat, UV radiation or peroxides.64 PAA can be categorized as a weak polyacid, meaning that it can dissociate only at certain pH values. In addition, its charge also depends on the equilibrium between the charged and uncharged species. The interesting behavior of PAA can be found in its dependence on the pH, the concentration of the surrounding medium, and the grafting density of the polymer chains.65,66 PAA has been widely used to stabilize particulate materials. Its steric and bridging forces have also been investigated using AFM.67 In addition, PAA was covalently attached to the PLA surface to accelerate the degradation process.68

Figure 8. The chemical structure of AA.

Maleic anhydride (MAH) (Figure 9) forms orthorhombic needles when crystallized. It reacts readily with water and alcohol. Compared to previous hydrophilic monomers, MAH is sometimes difficult to polymerize by free radicals69,70 because of steric hindrance by 1,2 disubsitution.71

Figure 9. The chemical structure of MAH.

————— Experimental —————

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3 EXPERIMENTAL

3.1 MATERIALS PLA was used in this study (Nature Works Co. Ltd. USA, grade 5200D, Mn = 150,000 g/mol, PDI=1.50). Benzophenone (BP) (99 %, Sigma Aldrich), acrylamide (AAm) (98.5 %, Acros), maleic anhydride (MAH) (> 99%, Fluka) were used as received. Acrylic acid (AA) (90%, Alfa Aesar) was purified by vacuum distillation at 40o C prior to use. Dichloromethane (DCM) (> 99%, Fisher Scientific), ethanol (96% v/v, VWR), polysorbate 80 (commonly known as Tween 80) (Fluka) and sodium chloride (NaCl) (p.a, Merck) were used as received. Three different concentrations of NaCl solution, 0.1, 1 and 10 mM were prepared using water pre-treated and connected to a Milli-RO unit. A purification step was then carried out with a Milli-Q Plus 185 system. The water resistivity was 18.2 MΩ cm and the pH of the Milli-Q water was measured and estimated to be approximately 5.6.

3.2 PARTICLE AND FILM FABRICATION Oil-in-Water Emulsion. The Oil-in-Water (O/W) technique was used to create spherical PLA particles. 0.2 g of PLA was dissolved in 100 mL of DCM (organic phase). 10 mL of this organic phase was poured into an aqueous solution containing 0.05 % w/v of Tween 80 (continuous phase). These two immiscible phases were stirred continuously at room temperature overnight until DCM evaporated. Finally, spherical PLA particles settled at the bottom of the beaker ranging between 10 and 60 µm in size. In order to recover the particles, they were filtered, rinsed with deionized water to remove surfactant and dried overnight. Cryogenic Milling. 10 g of PLA in pellet form was frozen in a cylindrical thermos previously filled with liquid nitrogen to reduce overheating which sometimes occurs during the grinding process. Milling was performed using a ZM 200 Retschmill,


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equipped with a stainless steel bath, and adjusted to a fixed rotational velocity of 12 000 rpm. Irregular PLA particles were then recovered in the range of Feret’s diameter of 600-1200 µm from the bath. The grinding process was run for a maximum time of 1 min in order to avoid thermal degradation during the process. Solvent Casting. 4 g of PLA was dissolved in 100 mL chloroform. The solution was then poured into a Petri dish from which the solvent was allowed to evaporate slowly. Rectangular pieces with a width of 2 cm and a height of 1 cm were cut from the circular film and used as sample substrates with 500 µm thick.

3.3 ‘GRAFTING-FROM’ POLYMERIZATION Photoactivation. In order to activate the inert surface of the PLA particles, 5 % w/v BP in ethanol was added to a Pyrex tube containing the particles. The solution containting the PLA particles was then placed on a stirring plate and irradiated for 20 min with a UV lamp with a wavelength range of 280-320 nm. After the surface activation was carried out, the particles were centrifuged using a Hettich Universal 30 at a rotational velocity of 5000 rpm for 3 min to remove the unattached initiator. Ethanol was then used to rinse the activated particles several times. They were then placed in a closed Petri dish, dried overnight, and finally characterized by FTIR spectroscopy. Control particles were prepared using the same procedure without any BP. PLA films were also activated in the same way as the particles. Photoinduced Polymerization of the activated PLA particles and PLA films. The BP-activated particles were then polymerized with 20 % w/v of acrylamide (AAm), 20 % v/v of maleic anhydride (MAH), or 20 % w/v of acrylic acid (AA). The particles were placed in Pyrex tubes using ethanol as solvent and later exposed to the UV irradiation for periods of 15 min, 30 min, 45 min, 1 h, and 1.5 h. The surface-grafted particles were then centrifuged at a rotational velocity of 5000 rpm for 3 min to remove all ungrafted polymer chains, rinsed with ethanol, dried overnight, and characterized by FTIR spectroscopy. Control particles were also prepared using the same procedure without monomer. For PLA films used for force measurements, two monomers, 20 % w/v acrylamide (AAm) and 20 % v/v acrylic acid were used and the particles were exposed to UV irradiation for periods of 1.5 h.


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3.4 CHARACTERIZATION METHODS

3.4.1 Size Exclusion Chromatography (SEC)

The molecular weights of the neat, control, activated, and grafted PLA particles were determined using a Waters 717 plus autosampler and a Waters model 510 apparatus equipped with two PL gel 10 µm mixed B columns, 300 x 7.5 mm (Polymer Laboratories, U.K). Chloroform was used as eluent at a flow rate of 0.1 mL/min. The instrument was calibrated with polystyrene standards with molecular weight distributions in the range from 580 to 400 000 g/mol.

3.4.2 Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectra of the surface-grafted PLA particles were recorded in the range of 4000-600 cm-1 on a Spectrum 2000 Perkin-Elmers spectrometer equipped with an attenuated total reflectance (ATR) accessory (Golden Gate) providing an analysis of the sample surface to a depth of approximately 1 µm. All FTIR spectra were obtained as means of 5 samples and 16 individual scans at 4 cm-1 resolution.

3.4.3 X-ray Photoelectron Spectroscopy (XPS)

The surface compositions of the irregular PLA particles were verified using XPS. The XPS measurements were run on a Phi Electronic Quantum 2000, using a monochromatic Al X-ray source (hυ=1486.86 eV). For X-ray surface analysis, a 45o angle monochromatic Al Kα X-ray source operating at 21.1 W and a pass energy of 58.7 eV was used.

3.4.4 Scanning Electron Microscopy (SEM)

The spherical PLA particles, fabricated using the O/W emulsification technique, were examined using a Hitachi S-4800 scanning electron microscope (SEM) at an accelerating voltage of 0.7 kV. The particles were mounted on adhesive carbon black and sputter coated.

3.4.5 Atomic Force Microscopy (AFM)

The surface topography of the PLA particles and of the PLA substrate films before and after surface grafting was determined in the tapping mode using a Nanoscope IIIa


15

multimode AFM (Digital Instruments, Santa Barbara, CA). The same instrument was also used to measure force interactions between the different substrate film/probe systems. In the tapping mode, a silicon tip (NSC14/no Al, Mikromasch, Estonia) with a normal spring constant (k) of 5.7 N/m and a resonant frequency (fo) of 110-220 kHz was used together with an EV scanner. Force measurements were carried out using a Nanoscope IIIa AFM with a picoforce extension (Veeco Instruments, Santa Barbara, CA) in liquid. Tipless cantilevers 250 µm in length and 35 µm in width and with a normal spring constant in the range of 0.35-1.2 N/m (NSC12/no Al, Mikromasch, Estonia) were previously calibrated to determine the accurate values of the spring constant using AFM Tune IT v2.5 software (ForceIT, Sweden), which is based on a thermal noise method.72,73 After cantilever calibration, a spherical PLA particle was then glued to the free-end of the cantilever with the aid of manual micromanipulator and a reflected light microscope. Before the force measurements, all the PLA colloidal probes were cleaned by exposure to UV plasma treatment for approximately 2 min at medium power (Harrick Plasma Inc, U.S). The AFM liquid cell and all other parts related to the liquid were cleaned by soaking in Hellmanex II (Helma GmbH & Co, Germany) for at least 1 h, followed by rinsing continuously with Milli-Q water and drying in a flow of filtered nitrogen gas. Colloidal probe placed in the liquid cell was inserted into the AFM for force measurement. The probe was then brought into close proximity to the sample surface. The lowest concentration of NaCl, 0.1 mM, was then injected into the liquid cell using a 10 mL syringe and the system was allowed to equilibrate for at least 10 min. The approach rate of the colloidal probe towards the solid substrate used throughout this measurement was 250 nm/s. Force measurements were also performed in two other salt solutions, i.e.1 mM and 10 mM NaCl. The force profiles, obtained from AFM measurement, were therafter fitted with the DLVO and the AdG model (Eq. 3).

————— Results and discussion —————

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4 RESULTS AND DISCUSSION

Steric stabilization of grafted polymeric particles obtained by a covalent ‘grafting-from’ technique with hydrophilic polymers has been studied. Steric stabilization from which long-range repulsive force operates is required to prevent particle agglomeration. Before surface modification, particles were made by two different methods, oil-in-water emulsion (O/W emulsion) and cryogenic milling. The former needs two different phases, an organic phase containing the dissolved polymer and continuous phase, where water and surfactant exist. Solvent evaporation then leads to dried micro- or nanospheres. In the milling method, the PLA pellets were mechanically grinded, resulting in large and irregularly-shaped particles. Three hydrophilic monomers, acrylamide (AAm), acrylic acid (AA), and maleic anhydride (MAH), were then attached on the substrate surface under UV irradiation. Colloidal probe AFM technique was used to study force interactions at the molecular level.

4.1 PARTICLE FABRICATION Particles of different geometries were made using two familiar techniques, O/W emulsion and cryogenic milling. The number average molecular weight (Mn) measured by SEC showed no significant difference between these two techniques, although the Mn of particles produced by these techniques was slightly lower than that of the pellet form of PLA (Table 1). The cryogenic milling technique resulted in a higher yield and was more efficient. These particles were used to develop the surface-grafting method to change their surface chemistry. Alterations in surface morphology, topography and chemistry were also achieved using the spherical particles produced by the O/W emulsion technique.


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Table 1. The number average molecular weight (Mn) and PDI of PLA before and after particle fabrication. Sample Mn

a PDIa

PLA 166 000 ± 3800 1.46 ± 0.00 Cryogenic milled PLA 145 000 ± 200 1.62 ± 0.01 Emulsified PLA particles 146 000 ± 10 300 1.62 ± 0.05 aMeasured by SEC using PS standards.

4.2 NONDESTRUCTIVE ‘GRAFTING-FROM’ METHOD Under UV irradiation, BP absorbs photon energy and electrons in the molecule jump from the ground to the excited state. BP then abstracts hydrogen from the substrate, resulting in a radical on the substrate surface. The radical created on the surface will then react with the hydrophilic monomer to initiate polymerization in the second step. To show the nondestructive ‘grafting-from’ method, the Mn of the PLA particles after surface activation and after grafting were determined (Table 2). The activation and the polymerization initiated by UV irradiation had no significant influence on the molecular weights. Co- and graft polymers commonly demonstrate an increase or decrease in molecular weight, due to the different hydrodynamic volumes in the organic solvent during SEC measurement. The molecular weights of the end-grafted chains have previously been measured and depending on the thickness of the grafting layer, their behavior in common SEC solvent will differ.46,52 A thin grafted film with a low grafting yield is commonly soluble in SEC solvent, while a thicker film is insoluble in this solvent.


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Table 2.The number average molecular weight (Mn) and PDI of PLA particle after activation and surface grafting

Sample Mnb PDIb

BP activated irregular particlesa 144 000 ± 3300 1.64 ± 0.02 BP activated irregular particlesa 142 000 ± 13000 1.64 ± 0.17

BP activated irregular particles and UV irradiated for 1.5 h 158 000 ± 3600 1.54 ± 0.02

BP activated spherical particles and UV irradiated for 1.5 h 131 000 ± 5423 1.72 ± 0.14

MAH grafted PLA irregular particles for 1.5 h 126 000 ± 3600 1.46 ± 0.02 MAH grafted PLA spherical particles for 1.5 hc - -

aThe PLA particles were surface-activated in ethanol for 20 min. bMeasured by SEC using PS standards. cInsoluble in chloroform.


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4.3 PARTICLE SURFACE CHEMISTRY The surface chemistries of the grafted-irregular PLA particles were examined by FTIR and XPS, confirming the success of the ‘grafting-from’ technique. With FTIR, the neat PLA particles showed a characteristic band at 1745 cm-1, corresponding to an ester C=O band. Upon AAm grafting, the PLA particles showed a shoulder after 30 min of grafting, giving characteristic C=O band at 1653 cm-1 (Figure 1). This corresponds to the stretching vibrations caused by primary amides typically in the 1670 – 1650 cm-1 region (amide I band). The amide II band was captured at 1615 cm-

1 after 1 h grafting,46,52,74 confirming the success of surface grafting of AAm on the PLA surface. The peak absorbance of the C-N stretching bands is typically observed at approximately 1440-1200 cm-1. They are however difficult to identify due to overlapping bands with the PLA substrate.

Figure 1. ATR-FTIR spectra of irregular PLA particles before and after surface grafting with acrylamide for different times.


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Figure 2. ATR-FTIR spectra of irregular PLA particles before and after surface grafting with acrylic acid for different times. For PAA-grafted PLA, a shoulder was observed at 1747 cm-1, corresponding to the stretching of the carboxylic acid of PAA and this shoulder was significantly wider57 after UV irradiation times exceeding 1 h (Figure 2). For MAH-grafted PLA, the extent of the grafting obtained was low and the anhydride peaks could not be identified in the 1840-1720 cm-1 region.57 The C=O stretching absorption peak at approximately 1635 cm-1 was indeed seen and this may indicate that MAH was covalently bound to the surface of the PLA particles but that the amount of MAH was low (Figure 3). A monolayer of anhydride is commonly formed on the substrate surface45 and this is difficult to analyze with FTIR due to the high analysis depth of FTIR, i.e. approximately 1 µm.


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Figure 3. ATR-FTIR spectra of irregular PLA particles before and after surface grafting with maleic anhydride for different times. A test was also performed to ascertain that no grafting layers were physically adsorbed on the surface, but that was a permanent covalent attachment of the grafted chains. Water was chosen as a medium due to the solubility of both the monomer and the polymer. The grafted particles were immersed in water for 24 h, withdrawn, and analyzed using FTIR. No change in the surface characteristics of the PLA-g-PAAm was observed before and after water immersion. AAm and MAH were two-selected monomers with which spherically-shaped particles were surface grafted in order to examine the versatility of the surface grafting technique for other geometries. After 1.5 h, the surface chemistry of the grafted-spherical particles was analyzed by FTIR and the spectra were similar to those observed for the grafted irregular particles (Figure 4). The slight difference in the absorption bands of the grafted AAm and MAH between the two different geometries could be due to the larger surface area of the spherical particles than that of the irregular ones. Hence, a longer reaction time is thus needed to synchronize the degree of grafting.


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Figure 4. ATR-FTIR spectra of spherical particles of neat PLA, PLA-g-PMAH, and PLA-g-PAAm, respectively. The grafted layers were further analyzed with XPS to determine the surface compositions of the irregular particles (Table 2). XPS measures the surface compositions of the AAm-grafted particles as atomic % of the elements present, with the exception of hydrogen. The nitrogen content was lower than the theoretical value for pure AAm, possibly because the substrate surface was not totally covered with the grafting layer or because of penetration of the photo-emitted electrons deeper to the substrate than the thickness of the grafted layer so that the elemental composition of the layer of the bulk material was thus included in the XPS analysis. For the AA- and MAH-grafted particles, no significant deviations from the theoretical values of PAA and MAH were found (with the C: O ratio is 61.1: 38.9 for PAA and 61.4:38.6 for PMAH) since carbon and oxygen are present in these three polymers, PLA, PAA, and PMAH, but there is no nitrogen. The XPS results are then difficult to interpret. The oxygen content in the grafted particles was higher than that of the pristine PLA particles, indicating the success of the surface grafting of two hydrophilic monomers, AA and MAH, onto the PLA particle surface. The theoretical and measured amounts of carbon and oxygen on the surfaces did not agree. Small amounts of impurities


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adsorbed onto the PLA surface or contamination of the XPS instrument might explain the discrepancy in these values. Table 3. Theoretical(*) and measured values of the atomic compositions of PLA particles during particle fabrication, before and after surface grafting.

Sample C 1s atomic % O 1s atomic % N 1s atomic %

PLA(*) 60.0 40.0 - irregular PLA particles 63.9 36.1 -

AAm(*) 60.0 20.0 20.0

PLA-AAm 61.5 36.3 2.1

MAH(*) 57.1 42.9 - PLA-MAH 61.4 38.6 -

AA(*) 60.0 40.0 -

PLA-AA 61.1 38.9 -

4.4 PARTICLE SURFACE MORPHOLOGY The spherical PLA particles were analyzed with SEM before and after surface grafting to see how surface grafting affected the morphology. SEM images show random holes in the pristine PLA spheres which had an average diameter of 36 ± 13 µm, the holes probably being due to rapid solvent evaporation and the air humidity during the particle fabrication (Figure 5).75 Because of the many holes formed on the surface, these neat PLA particles were selected as an indicator to confirm complete surface coverage. Holes were also seen after 1.5 h UV irradiation in ethanol in the absence of monomers. No change in the surface morphology was observed after ethanol immersion, indicating that the solvent had no influence on the morphology of PLA particles. In contrast, no holes were found when acrylamide was surface grafted on the surface of PLA particles with an average diameter of 63 ± 28 µm, indicating complete surface coverage. This result supports the FTIR spectra which indicated that a thick layer of acrylamide was grafted onto the particles. For PLA-g-PMAH, the surface morphology was similar to that of the pristine PLA particles, leading to the low degree of grafting as previously indicated by FTIR.


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Figure 5. Surface morphology of (A) neat PLA particles, (B) PLA particles after UV irradiation for 1.5 h in ethanol, (C) PLA-g-PAAm after UV irradiation for 1.5 h, and (D) PLA-g-PMAH after UV irradiation for 1.5 h.

4.5 PARTICLE SURFACE TOPOGRAPHY The topography of the neat and grafted spherical PLA particles and PLA films were studied with AFM. A relatively smooth surface with an rms surface roughness of 3.0 ± 2.0 nm (Table 4) was observed on the surface of a neat PLA particle (Figure 6A). Like the surface of PLA particles, the PLA film fabricated by solvent casting was achieved with an rms surface roughness of 4.0 ± 1.0 nm in air.


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Table 4. Surface roughness (Rq) of PLA, PLA-g-PAAm, and PLA-g-PAA in air and salt solutions.

Sample Surface roughness (nm)

PLA sphere PLA film substrate

Air NaCl solution

0.1 mM 1 mM 10 mM

PLA 3.0 ± 2.0 4.0 ± 1.0 8.0 ± 5.0 6.0 ± 2.0 5.3 ± 2.0

PLA-g-PAAm 20.0 ± 2.0 18.0 ± 1.0 29.0 ± 2.0 34.0±10.0 37.0 ± 2.0

PLA-g-PAA 34.0 ± 1.6 39.0 ± 5.0 28.0 ± 5.4 31.0 ± 12.0 26.0 ± 0.1

PLA-g-PAA (pH=7.4) - - 31.0 ± 5.0 31.0 ± 5.0 43.0 ± 13.0 AFM height images also showed an rms surface roughness of PLA-g-PAAm particles with an rms surface roughness of 20.0 ± 2.0 nm, while a rougher surface was observed for PLA-g-PAA particles with an average rms surface roughness of 34.0 ± 1.6 nm. The rms roughness of the PLA particles UV-irradiated for 1.5 h in ethanol in the absence of monomer was closer to that of the PLA-g-PAA particles, 37.2 nm, showing that the influence of ethanol on the surface topography of the spherical particles. Maleic anhydride showed the roughest surface, 42.8 nm, with an incomplete surface coverage of PLA-g-PMAH. In contrast to PLA-g-PMAH, a smoother and thicker grafting layer was obtained when acrylamide was surface grafted onto the PLA surface. The AFM images have demonstrated that the monomer-carrying solvent influenced the surface topography, although the molecular weights before and after surface grafting were not significantly different.


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Figure 6. Surface topography of the spherical particles of (A and E) neat PLA, (B and F) PLA-g-PAAm, (C and G) PLA-g-PAA, and (D and H) PLA-g-PMAH in air. The upper images show the height and the lower images show the phase. All phase images of AFM were scanned over 5 x 5 um. There was no significant difference in surface roughness between neat PLA, PLA-g-PAAm and PLA-g-PAA particles and films in air (Table 4), showing that these PLA-based films could be used to replace PLA-based particles substrates for the force measurement. A patterned structure formed on the surface of the neat PLA film when it was immersed in 0.1 mM NaCl at pH 5.6 (Figure 7A and D), but no patterned structures were found on the neat PLA substrate in air (Figure 6A and E). Finally, no significant surface roughness was observed for the neat PLA in air or in the salt solutions (Table 4).


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Figure 7. Surface topography of the spherical particles of (A and D) neat PLA, (B and E) PLA-g-PAAm, and (C and F) PLA-g-PAA in 0.1 mM of salt solution. The upper images show the height and the lower images show the phase. All phase images of AFM were scanned over 5 x 5 um. In contrast to the hydrophobic surface, no patterned structures were seen on the hydrophilic surfaces at pH 5.6 (Figure 8B and E) and (Figure 8C and F). AFM phase images shows an alteration of the chain conformation of PLA-g-PAAm in salt solutions (Figure 8E), leading to a rougher surface than in air. Increasing salt concentration had no significant influence on surface roughness. PLA-g-PAA did not exhibit any conformational change in the wet state (Figure 8C and F) since the surface roughness of the grafted chains in a salt solution was similar to that in air. The conformation of PAA chains was the same at two lower salt concentrations, i.e. 0.1 mM and 1 mM, at pH 5.6, but at pH 7.4, PAA chains showed a change in swelling behavior and the surface was rougher at the highest salt concentration, i.e. 10 mM.

4.6 FORCE INTERACTIONS OF DIFFERENT PLA SUBSTRATE SURFACES

The force interactions of the neat PLA substrates and the end-grafted PLA substrates, i.e. PLA-g-PAAm and PLA-g-PAA were studied (Table 5), in different salt concentrations at pH 5.6 unless otherwise stated.


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Table 5. The colloidal probe and the film substrates, type of interaction and the result of the force measurement.

System Colloidal probe Film

substrate Type of interaction Result 1 PLA PLA hydrophobic/hydrophobic Attractive 2 PLA-g-PAAm PLA-g-PAAm hydrophilic/hydrophilic Repulsive 3 PLA-g-PAA PLA-g-PAA hydrophilic/hydrophilic Repulsive 4 PLA-g-PAA PLA-g-PAAm hydrophilic/hydrophilic Repulsive 5 PLA PLA-g-PAAm hydrophobic/hydrophilic Repulsive 6 PLA PLA-g-PAA hydrophobic/hydrophilic Attractive

In the case of hydrophobic systems, at salt concentration of 0.1 mM, a short-range repulsion was observed before the colloidal probe experienced a jump into contact (Figure 8A). Repulsive interaction at the lowest salt concentration agreed well with the DLVO theory, resulting in a surface potential of PLA (𝜑𝑃𝐿𝐴) of approximately - 12 mV (Figure 8C). This short-range repulsion may also occur due to the adsorption of negative Cl- ions (ion specific adsorption)77 which charge the hydrophobic surface temporarily and lead to a weak repulsive double layer pressure. The screening effect increases with increasing salt concentration and the repulsive is reduced. Attractive interactions were observed at a separation distance of 20 nm when the symmetrical PLA/PLA system was immersed at the two higher salt concentrations, 1 and 10 mM (Figure 8B and C). Attractive interactions were also observed both on the approach and retraction curve of hydrophobic/hydrophilic PLA/PLA-g-PAA system in 0.1, 1 and 10 mM salt concentrations (Table 5). The attractive forces were seen at large separation distances of 30 to 60 nm, while the retraction curves gave adhesion, resulting in pull-off forces ranging between 1.3 and 2.7 mN/m. In general, attractive interactions dominate between two interacting hydrophobic surfaces that are separated at larger distance (D≥15-20 nm).78 Agglomeration cannot be overcome when colloidal PLA particles are dispersed in suspension due to the dominating of attractive force. Thus, a better system should be modified to eliminate of the particle agglomeration.


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Figure 8. Normalized force profiles between a PLA sphere and neat PLA film. (A) Five different locations, approach (green) and retraction curves (blue) in 0.1 mM of salt solution, (B) typical approach force curves at different salt concentrations, 0.1 mM (green), 1 mM (red), and 10 mM (blue), (C) the solid, dashed, and dotted grey lines represent the DLVO calculated force in 0.1, 1, and 10 mM salt solutions. All hydrophilic/hydrophilic and one hydrophobic/hydrophilic force profiles, i.e. PLA/PLA-g-PAAm, agreed well with the AdG model. These force profiles were not influenced by the different salt concentration. The DLVO theory, where the double layer is present, did not match the force curves of these systems at pH 5.6. For PLA-g-PAA/PLA-g-PAA, one of the force profiles was well-fitted to the DLVO theory at the highest salt concentration at pH 7.4. Long-range repulsive interactions were observed for both the approach and retraction curves of the PLA-g-PAAm/PLA-g-PAAm system (Figure 9) and one hydrophobic/hydrophilic system, i.e PLA/PLA-g-PAAm (Table 5). The steric repulsion could be described using the AdG model that


30

assumes that the polymers are end-grafted and that no chain interpenetration occurs. The AdG model of polymer brushes exhibited a good fit to the measured force profiles (Figure 9-10). It is possible to overcome aggregation or flocculation of colloidal particles in suspension and these designed systems can remain dispersed immersion in water. After force curve fitting to the AdG model (Figure 9B), this symmetrical system demonstrated the thickness of the grafting layer (Lo), 250 nm, and the grafting density (Г) and the average of grafting site (s) were estimated to be 0.14 ± 0.07 chains/nm2 and 2.91 ± 0.8 nm, respectively.

Figure 9. Normalized force profiles between a PLA-g-PAAm sphere and PLA-g-PAAm film. (A) Five different locations, approach (green) and retraction curves (blue) in 0.1 mM of salt solution, (B) typical approach curves at different salt concentrations, 0.1 mM (green), 1 mM (red), and 10 mM (blue). The solid, dashed, and dotted grey lines represent the DLVO calculated force in 0.1, 1 and 10 mM of salt solutions, the solid black line demonstrates the AdG fitting curve of 0.1 mM of salt solution. The approach and retraction curves of the PLA-g-PAA/PLA-g-PAA system demonstrated purely repulsive forces at pH 5.6 (Figure 10) and, unlike the previous hydrophilic system, PLA-g-PAAm/PLA-g-PAAm, the repulsive forces varied from location to location on the substrate surface (Figure 10A). As in the case of PLA-g-PAAm/PLA-g-PAAm, the experimental data showed a good fit to the AdG model, leading to a Lo of 140 nm, г of 0.06 ± 0.03 chains/nm2 and s of 3.74 ± 1.65 nm (Figure 10B). The grafted chains of PAA exhibited a different behavior when the pH was increased to 7.4. Steric factors dominated the system at two lower salt concentrations, i.e. 0.1 and 1 mM, whereas the DLVO theory was well-fitted to the system at the highest salt concentration, 10 mM, resulting in surface potential of PLA-g-PAA/PLA-g-PAA (𝜑(𝑃𝐴𝐴−𝑃𝐴𝐴)) equivalent to - 45 mV. This phenomenon indicates the contribution of electro-steric forces between the PLA- g-PAA/PLA-g-


31

PAA. The AdG model agreed well with the force profiles at the two lower salt concentrations, 0.1 mM and 1 mM, with a Lo of 149 and 107 nm, respectively (Figure 10C). In other words, Lo of the system decreased when the salt concentration was increased from 0.1 to 1 mM NaCl at pH 7.4. Lo also decreased to approximately 109 nm when the two substrates with different grafted chains, AAm and AA, were interacting, although the long-range repulsion was still dominant. However, the approach and retraction curves of the PLA-g-PAA/PLA-g-PAAm were different due to plastic deformation when the soft grafted samples were indented.

Figure 10. Normalized force profiles between a PLA-g-PAA sphere and PLA-g-PAA film. (A) Five different locations of approach (green) and retraction curves (blue) in 0.1 mM salt solution, (B) the approach curves at different salt concentrations, 0.1 mM (green), 1 mM (red), and 10 mM (blue). The solid, dashed, and dotted grey lines represent the DLVO calculated force of 0.1 mM, 1 mM, and 10 mM salt solutions, the solid black line demonstrates the AdG fitting curve of 0.1 mM salt solution at pH 5.6. (C) The approach curves at different salt concentrations, 0.1 mM (green),1 mM (red), and 10 mM (blue). The solid, dashed, and dotted grey lines represent the DLVO calculated force of 0.1 mM, 1 mM, and 10 mM salt solutions. The black solid line demonstrates the AdG fitting curves of 0.1 mM and 1 mM of salt solutions at pH 7.4.


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The mechanical interaction between weak polyacids is a complex process that is dependent on factors such as the salt concentration, pH, and density of the grafting layer.66,79 Different grafted polymer conformations from flat to mushroom or to an extended brush could be observed by altering these parameters. We have demonstrated different force interactions of the weak polyacid, PAA, that depend on pH and ionic strength. The PAA-grafted chains were uncharged at pH of 5.6 as shown by the similarity of interacting force which is independent on different salt concentrations (Figure 10B). No alteration in surface roughness was observed (Table 4) showing that the chain conformation was not affected by salt concentration. When the pH of the solution was increased (Figure 10C), the system varied with the different salt concentration. At 0.1 mM salt solution, steric repulsion extended to a longer range of separation distance and decreased when the salt concentration was increased. This may indicate a decrease in the extending steric force. In contrast, a purely electrostatic repulsion dominated the system at the highest salt concentration, 10 mM, due to a decrease in the Debye screening length which may induce conformational changes in the grafted polymer chains. All hydrophilic/hydrophilic systems designed in this work could be applicable to particle-based drug delivery systems because they demonstrate long-range repulsive interactions whether steric or electro-steric which overcome particle agglomeration.

————— Conclusions —————

33

5 CONCLUSIONS

Sterically-stabilized particles prepared by a covalent ‘grafting-from’ technique were successfully designed. In addition, the ‘grafting-from’ technique has been shown to be a nondestructive method as confirmed by SEC. Long-range repulsive interactions were mainly observed when hydrophilic polymers were surface grafted onto the PLA substrates. Some symmetrical systems, e.g. PLA-g-PAAm/PLA-g-PAAm and PLA-g-PAA/PLA-g-PAA and the asymmetrical PLA-g-PAAm system resulted in purely steric interactions at pH 5.6, while PLA-g-PAA/PLA-g-PAA also demonstrated electro-steric interactions at pH 7.4. These hydrophilic/hydrophilic systems and one hydrophobic/hydrophilic PLA/PLA-g-PAAm system could be used to overcome aggregation due to attractive interactions. They dominated symmetrical system of hydrophobic substrate, i.e. neat PLA/neat PLA system, although a short-range repulsion was observed before the PLA colloidal probe experienced a jump into contact at a separation distance of about 10 nm at the lowest salt concentration. Alterations in the surface chemistry of the PLA particles of two different geometries, characterized by FTIR and XPS, confirmed the success of the surface grafting technique. The surface morphology and topography of PLA particles of two different geometries also indicated the success of the ‘grafting-from’ technique. Hydrophilic polymers covalently grafted onto the PLA surfaces exhibited the long-range repulsion necessary to inhibit particle agglomeration in drug delivery systems for example.

————— Future work —————

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6 FUTURE WORK

The present study has presented sterically-stabilized systems between hydrophilic polymers grafted onto a PLA substrate. It was observed that long-range repulsive interactions mainly operated in hydrophilic/hydrophilic systems and in one hydrophobic/hydrophilic system, PLA/PLA-g-PAAm. The hydrophobic/hydrophilic PLA/PLA-g-PAA system showed an attractive interaction which is in contrast to the interaction between neat PLA and PLA-g-PAAm. In a future study, it would therefore be of interest to determine the characteristics of the end-grafted polymers prepared by the ‘grafting-form’ technique, i.e. molecular weight and chain contour length. Since this work was carried out in monovalent salt solution of different concentrations, it would also be interesting to investigate steric stabilization in salt solutions with higher cation valency and alcohol-based solvents with which the end-grafted polymers are miscible. Another challenging work is to couple either synthetic or natural occurring polymers such as collagen to the end-grafted PLA substrates and to evaluate the force interactions between different substrates for other purposes in the medical field.

————— Acknowledgements —————

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7 ACKNOWLEDGEMENTS

First I would like to express my gratitude to my main supervisor, Professor Ann-Christine Albertsson, and my co-supervisors, Dr. Karin Odelius and Dr. Anders Höglund for giving me the opportunity to be a part of Polymer Technology group and to work on this topic. I would also like to convey my gratitude for your excellent scientific guidance, support and encouragement throughout this work. The ERC Advanced Grant PARADIGM (Grant Agreement No: 246776) and the Swedish Research Council (ID: 621-2010-3478) are thanked for their financial support for this work. Professor Mikael Hedenqvist is thanked for taking the time to proof-read this thesis. Co-authors Dr. Prasun Roy Kumar and Dr. Torbjörn Pettersson are gratefully thanked for great discussions, comments and motivation. For Roy, I will visit New Delhi one of these days. All senior scientists, PhD students, and the administrative staff at the Polymer Technology group are thanked for their assistance, good discussions and advice and more importantly the nice working atmosphere. Special thanks I would convey to professors, researchers, and PhD students of other groups, I could mention one by one for advice, motivation, discussions, funny jokes, private fika, lunch and dinner invitations, and nice company. Thanks also to all my friends from different nations wherever they are now, for their assistance, motivation as well as the good time we have had. Ngaturaken sewu agunging panuwun ingkang tanpa winates, kawula aturaken kunjuk ibu punapa bapak ingkang sampun kondur ing kasedan jati sumrambahipun adhi-adhi sutresna punapa dene kulawarga mbah putri ing Cawas mboten sanes kulawarga

————— Acknowledgements —————

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pakdhe Wal sumrambah sadaya tiyang ingkang nyengkuyung lampah kula ingkang mboten saged kawula aturaken satunggal-satunggalipun. Mugi jumbuh ingkang ginayuh sumbada ingkang kajangka saking swargi bapak ingkang tansah kaparingaken dhateng kawula, sarana pandungo dados kasunyatan ing mangke inggih karana Gusti rena paring kamulyan dhumateng kulawarga kita. Wonten ing Gusti kawula estu badhe saged ngunduh sadaya ingkang ginayuh.

————— References —————

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