A NOVEL HYDROGEL ANCHORED LIPID BILAYER SYSTEM
by
Tuo Jin
A thesis submitted in conformity with the requirements for the degree of Ph.D. Graduate Department of Pharmacy University of Toronto
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ABSTRACT
The present shidy demonstrates the feasibility of a new self-assembled micro-particdate system which combines the complementary properties of liposomes and hydrogel microspheres. This system, termed "lipobeads", consists of a lipid bilayer shell anchored on a hydrated polyrner core (polyvinyl alcohol; PVA) resembling the structure of cytoskeleton. The experimental procedure starts with the preparation of PVA xerogel microspheres, followed by a surface-modification process, termed "lipocoating". The latter step consists of chemically grafting lipid molecules onto hydrogel bead surfaces followed by the spontaneous assembly of a phospholipid membrane shell after exposing these beads to a liposomal suspension. The chernical modification, which involves the reaction of surface hydroxyl groups of PVA, was charactenzed using attenuated total reflectance FTIR and angle-resolved XPS. It is shown that these analytical techniques can be used to indicate whether the surface modification is limited mainly to the top surface region as well as the proportion of hydroxyl sites in this region that have been involved in the reactions. Contact angle measurements and Langmuir-Blodget (LB) deposition were also carried out to characterize the process of self-assembly of the lipid membrane. Self assembly is driven by the hydrophobie interaction of liposomal components with the grafted surface anchors. The results obtained are consistent with the proposed structure of a unilamellar lipid bilayer on the hydrogel surface. The properties of the anchored lipid bilayer (lateral mobility and cross membrane permeability) were studied usïng fluorescence confocal rnicroscopy. With a photo-bleach protocol, the diffusion coefficient of fluorescent labeled lipids through the bilayer was measured to be 1.5 x 1 cm%ec, comparable to that observed in normal bilayers (10-8 cm2/sec). Results on the entrapment of calcium ions and a mode1 dnig (Lucifer yellow) confimi that this bilayer shell also functions as a permeability barrier to ions and lipophobic molecules. Also observed was that the entrapped calcium ions can be spontaneously released upon addition of an ionophore, consistent with the bilayer nature of the lipid coating. Based on these observed properties, the advantages and potential biomedical applications of are discussed. 1would like to thank Dr. Ping Lee, my thesis advisor, for his constant help, inspiration and enthusiasrn. His sustained support became the impetus behind this work.
1 would also like express my thanks to the members of my advisory cornmittee, Drs. M. V Sefion, Y.-L.Cheng, M. Thompson, U. J. Kull, and, especially P. S. Pemefather for many usefid suggestions. Dr. Pemefather provided the most fkequent discussion regarding biophysical aspects which complemented to the expertise of our group in phannaceutics, biomaterials and surface chernistry.
1 would like to extend my appreciation to Dn R N. S. Sodhi and X. Gu for their assistance and generous offers of the self-operating machine tirne for XPS and confocal microscopy which are located in Ontario Biomaterials Centre and Ontario Laser Centre, respectively.
Financial assistance fkom University of Toronto is gratefully acknowledged.
Finally, 1 appreciate the support and patience of my family who are my son, Brian, daughter, Frances, and particularly wife, Lee. This support is so valuable since it has been provided with the awareness that under today's situation getting a Ph.D. is no longer a factor which improves one's career option and family life. TABLE OF CONTENTS
Abstract Acknowledgement Table of Contents List of Figures List of Tables Glossary of tenns
Introduction Biomaterials and drug delivery Particdate drug delivery systems Investigation of surface properties of hgdelivery systems Objectives of the present research Outline of the thesis
Prepamtion and Characterization of PVA Xerogel Microspheres Supported lipid bilay ers and hydrogels Experimentai details Size distribution of PVA microspheres Surface smoothness and water swelling ratio of PVA microspheres Density and crystallinity of PVA rnicrospheres
Chernicd Modification of the PVA Surface Introduction Surface modification techniques Selection of surface reactions and surface analytical techniques Physicai principles of surface analpical techniques (ATR-FTIR & XPS) Experirnental details Results of surface modification reactions as detennined with ATR-FTIR and XPS Angle resolved XPS: depth distribution of and surface coverage by grafted molecules Discussion Characterization of Liposheets Supported planar Lipid bilayers Experimental setup Contact angle meanuement: interaction of modified surface with liposomes Langmuir-BIodget deposition on modifïed PVA sheets Discussion
Characterization of Lipobeads Prospective properties of supported lipid vesicles Materials and methods Preparation and stability of lipobeads Lateral mobility of supported membrane Penneability of supported membrane to ions Retention of mode1 dmg by supported membrane Summary of the features of lipobeads
Potential Applications of Lipobeads and future directions The nature of the technology Controlled release dmg delivery Delivery of synthetic polypeptide vaccines Red ce11 substitution Application in assessrnent of dmg absorption and distribution Reconstitution of trans-membrane protehs Future directions
Appendices 82 1 Preparation of glycerolphosphatidylethanolamine (GPE) 82 II Estimate of surface coverage of PVA by anchored phospholipids using angle resolved XPS 86 III Calculation of diffusion coefficient of lipid molecules along supported lipid bilayer 96
References 98 LIST OF FIGURES
Fime No, Figure title
1- 1 Scientific disciplines that are related to drug delivery 1-2 Structure and preparation strategy of lipobeads
2- 1 Size distribution of PVA xerogel beads as a fhction of surfactant concentration 2-2 Size distribution of PVA xerogel beads as a fimction of injection rate of polymer solution into oil 2-3 Confocai images of PVA beads suspended in ethanoi and water 2-4 Confocal image of PVA beads dned by acetone/ethanol treatment 2-5 Mode1 for estimating the average distance between polymer chahs
3-1 Reaction scheme for graffing fatty acid ch& on PVA surface 3 -2 Reaction scheme for grafted synthesis of phosphoiipids on PVA surface 3-3 Reflection and rehction of a light beam at the interface between a denser and a rarer media 3-4 Sampling arrangement for ATR-FTIR 3-5 Interaction of a X-ray photon with a core level orbital electron 3-6 Sampling arrangement for XPS 3-7 ATR-FTIR spectnim of PVA film after acylation with palmitoyl chionde 3-8 ATR-FTIR spectra of PVA film der each reaction step shown in Fig. 3-2 37 3-9 XPS spectra of PVA film before and after acylation with palmitoyl chlonde 40 3-10 XPS spectra of PVA film after each step of grafted synthesis of PE (Fig. 3-2) 41 3-1 1 XPS spectra of nitrogen containhg species deposited on PVA surface 45 3-12 Three possible morphologies of chemically grafted polymer surfaces as measured by XPS with 90' and 45' sampling angles 47
- vi- Expenmental arrangement for measuring contact angles Diagrammatic sketch of a Langmuir-Blodget trough Equilibriurn of surface tension of a solid-liquid system Observation during Langmuir-Blodget deposition of DOPC ont0 fatty acid modified PVA sheet
Protocol for photo-bleach experiment of a lipobead with a confocal microscope Liposomal coating of acylated PVA beads Lateral mobility of lipids in hydrogel anchored lipid bilayer labeled with fluorescent phospholipids Feasibility of using a slab mode1 for describing lateral mobility of lipid during recovery of photo-bleach Time course of recovery of fluorescence in the photo- bleached region Release of Ca2+ fkom lipobeads and bare PVA beads loaded with fluo-3 Release of Ca2+ from lipobeads made using PE anchors Confocal images of lipobeads and bare PVA beads loaded with Lucifer yellow Decay of fluorescent intensity of Lucifer yellow loaded lipobeads and bare PVA beads as a hction of time after 5 fold dilution
(Figures in Appendices)
1- 1 Reaction scheme for synthesis of glycerophosphatidyl ethanolamine (GPE) 1-2a 2D NMR spectm of GPE synthesized 1-2 b Phosphorus-decoupled NMR spectrum of GPE synthesized II- 1 Possible depth distribution of grafted molecules in the surface region of the substrate
- vii - LIST OF TABLES
Table No. TabIe title &
2- 1 Measured diarneter (p)and total weight (mg) of a set of PVA beads 20
3- 1 Surface modification tec biques for po lymeric materials 23 3 -2a XPS elemental fiactions of PVA sheets der each step of surface reaction 42 3-2b Sumrnary of cuve-fitthg results of high resolution XPS spectra 43 3-3 Detected elemental fiaction of N (1s) as a function of XPS sampling angle 47
1- 1 Chernical shift of GPE atoms in its NMR spectnim 83
- viii - GLOSSARY OF TERMS
(Poiymeers and matera) Crystallinity Weight portion of crystallized part with a polymer rnatrix. Hy drogel Three dimensional hydrophilic polymers whic h cm imbibe a large amount of water (20-90%) when swollen. IAM Immobilized artificial membrane: lipid monolayer anchoreci on Sioz. Liposome Fabricated vesicles made of lipid biiayers surroundhg an aqueous space Nimiber average mas Mn= xNiMi/xNi, where Ni is total number of polymer chains, i, of a given length. Mi is rnolar weight of these chahs. Weight average mas MW= wiMiEwi, where wi is total weight of poly mer chains, i, of a given length. Mi is molar weight these chains. Xerogel Dried hydrogel. (A TR-FTIR) Attenuated total reflection Multiple intemal reflection in which the light approaches in interface fiom the denser medium to the ruer medium and reflects at the interface. Vibration mode of molecules in which the lengths of chemical bonds are varying during vibration. Optical pnsm A crystal which is transparent to infiared light and so that is used for sampling surfaces to be measure to ATR-FTIR (xPS) Binding energy (Eb) Energy for an electron in a solid to reach Fermi level. In XPS Eb is characteristic of each elements and orbital. Chernical shift AEb caused by chemical environment of the electrons to be photo-excited. Escape depth Depth from the surface at which e-1 portion of photoelectron generated can Energy analyzer A component of XPS spectrometer which captures photoelectrons and measures the kinetic energy of the electrons. Fu11 width of half maximum of a XPS beak. penetrate the solid matrix without energy loss. Mg Ka x-ray source Ka line of the x-ray generated from a Magnesium target. Pass energy Energy of photoelectrons after retarded at the entrance of energy analyzer, with which the electrons pass the energy analyzer and reach the detector. Satellite peaks Binding energy peaks generated by the background Lues of a non- monochromatic x-ray source. Sensitivity factors Measure of relative sensitivity of each element and orbital, which are based on the unit transmission of the energy analyzer. (Others) Eady tirne approximation An approxirnated solution of Fick's equation valid when MM- 5 0.6. Late tirne approximation An approxhated solution of Fick's equation valid when MdMw 2 0.4. Surface pressure A lateral pressure required for lipids to form a well packed monolayer on water surface of a Langmuir-Blodget trou&. 1. INTRODUCTION
1.1 Biomaterials and drug delivery
The ultimate goal of dmg delivery is to deliver therapeutic agents to the target organ at a controlled rate for an effective duration in order to elicit desired pharmacological responses and to optimize therapy [l]. Significant advances have been made in the area of drug delivery technologies in the past two decades towards this goal and a nurnber of controlled-release products have already enjoyed commercial success [2]. Since the advent of biopharmaceutical products, controlled delivery technologies are facing new challenges as most of these compounds are susceptible to degradation and can not be administered as conventional chemical drugs [3].
Transformation of scientific discovery of rnany pharmacologically active peptides, proteins and nucleotides into clinically useful products generally requires an in-depth understanding of not only the mechanisms of drug action and its related pharmacokinetics but also appropriate delivery modalities and delivery system design, including relevant utilization of biomaterials [4]. Delivery systems which are biomimetic, biocompatible and responsive to physiological stimuli are the most desirable [SI.
The field of dmg delivery has evolved into a highiy interdisciplinary one. Figure 1.1 shows typical scientific disciplines utilized in drug delivery [6]. Often advances made in a related field can impact significantly in the hgdelivery area. Arnong these, advances in polyrnenc materials have played an important role as most dmg delivery systems employ polymeric materials of one kind or another [7]. Drug delivery technologies can be broadly classified into two categories: controlled release and dmg targeting [6]. Depending on the type of delivery system and properties of the polymer involved, the hgrelease rate may be controlled by mechanisms such as diffusion, polymer swelling and phase transformation, polymer degradation and erosion, and osmotic pressure [6].
Surface Science
Figure 1- 1 Scientific disciplines that are related to drug delivery [6].
More recently, delivery mechanisms which can respond to environment changes have attracted increasing attention. Arnong them, phase transformation of polymers as a hction of the extemal glucose level, or polymer degradation triggered by specific enzymes are a few examples [8,9]. In
the area of drug targeting, mechanisms based on antibody or receptor mediated pathways have
been utilized to achieve passive or active dnig targeting [IO]. Delivery systems for targeting
purpose are available as either biological or chernical carriers. Fcr biological carriers, replication
incompetent viruses have been employed as carriers for gene transfer due to their ability to infect a
specific ce11 line or tissue type [Il]. However, there are potential safety concems associated with
this approach due to concerns of possible recombination with endogenous viral gene sequences. In
the case of chernical carriers for dmg targeting, micro- (or nano-) particulates and macromolecular
approaches have been employed. With particdate carriers, a drug may be protected, transported and released at selected sites of action through a passive targeting process [6].Passively targeted systems oeen utilize existing reticular endothelid system (RES) as a transport pathway to reach destinations such as the spleen and liver [12,13]. On the other hand, macromolecular carriers may be potentially usefui as active targeting systems because they can provide a longer circulating time
[14] and may be conjugated with a targeting ligand [15]. These challenges have provided impetus for more research activities in the design of macromolecular carriers.
1.2 Particdate cimg delivery systems
Pharrnaceutical particulates encompass a wide variety of dispersed systems with charactenstic particle size ranging from nanometers to millimeters [16]. Particulatr delivery systems have been widely used in various routes of administration, ranging from oral, topical, subcutaneous, intramuscular to intravenous [I 51. For oral administration, particles of a variety of sites may be used depending on the release profile required for a given drug. For other routes of administration, especially for IV administration, particles with diameter less then a few microns should be used [16]. Perhaps the most actively studied particdates in drug delivery are liposomes and polymeric microspheres [16,17]. There are complementary advantages and limitations in these two delivery approaches.
Liposomes (fabricated vesicles made of lipid bilayers surroundhg an aqueous space) have a cell-like membrane surface which is flexible but highly impermeable to ions and other lipophobic molecules. Dmg release fiom these systems may be controlled by varying the composition, charge and phase transition temperature of the lipid component. With an appropriate selection of lipid molecules, stimuli-sensitive liposomes have been investigated for drug delivery applications [18].
One reason which has limired rapid cornmercialization of liposomes may be related to their mechanical instability and their limited drug loading capacity and efficiency [19]. To improve biological stability, liposomes with polyethylene glycol (PEG) modified surface (stealth liposomes) have been developed by which macrophage uptake of liposomes may be avoided and blood circulation time may be lengthened considerably [12]. To improve physical stability, polymerization of lipid bilayers have been employed to enhance the membrane strength of liposomes [20]. However, the polymerization process will alter bilayer properties such as membrane mobility and permeability.
Polymeric microspheres, including both reservoir or matrix system [21], on the other hand, are mechanically more stable and have a larger loading capacity, but are lacking many of the surface properties mentioned above. In reservoir system, drugs are entrapped by an impermeable membrane structure, whereas with a matrix system, hgsare dispersed in the entire matrix of the carrier as particles. Early approaches involve mostly hydrophobie polymers which require the use of organic solvents [22,23]. However, with increasing environmental concems and the susceptibility of biophannaceuticals (peptides, proteins and nucleotides) to organic solvents or reactive agents, hydrogels (hydrophilic polymer gels, for example polyvinyl alcohol (PVA) have become important for the delivery of these compounds, primarily because of the lmown tissue compatibility of hydrogel and their inertness towards biopharmaceuticals [24-271. Hydrogels, based on ionic or non-ionic polyrners, are three dimensional hydrophilic polymers which can absorb a large amount of water (20-90%) and thereby swell. In ionic hydrogels, a phase transition often occurs in response to physicochemical changes in the environment such as pH or temperature. The sensitivity of ionic hydrogels to pH and ionic strength may cause concems regarding the potential effect of other endogenous biomolecules on the charged hydrogel network and its dmg release characteristics [28]. Non-ionic hydrogels are chemically inert and therefore more attractive for this purpose. However, dmg release from highly hydrated network may be too fast to exert sufficient release rate control [2]. To overcome this challenge, a coating that regulates permeability rnay be applied to the surface of the hydrogel matrix to exert a certain degree of control over the rate of dmg release.
1.3 Investigation of surface properties of dnig delivery systems
The performance of polymeric drug delivery systems (e.g. drug release profile, blood circulation tirne, immunogenicity and dnig targeting) is often related to the physical and chernical properties of the surfaces of these devices. For example, Kwon et al. [29] found that the temperature sensitive "on-off' regulation of crosslinked poly(N-iso-acrylamide) was determined by its surface rather than bulk properties. Xu and Lee [30] reported that release of theophylline from cellulose acetate phthalate was controlled by the surface erosion of the polymer. In addition,
Kim and Lee [3 11 used a surface-limited crosslinking technique to encapsulate drugs in non-ionic hydrogel microspheres. Generally, the effect of surface properties become more significant for pdculate dmg delivery system with micro- and nano- size particles [16].
Surface properties which are important for delivery system performance generally include surface charge, sensitivity to environment, hydrophilicityhydrophobicity, penneability, biocompatibility and surface bio-functionality. These surface properties are govemed by the surface composition and surface morphology of the polymenc devices in question [32], thus they can be manipulated by the physical and chernical modification of the surface [33]. Some examples of using surface modification approach to irnprove hgdelivery systems can be found in the literature. Examples include: carboxylic groups which have been connected to the surface of polymer particles to improve bio-adhesion of the devices [34]. Levy et al. [35] have applied surface rnodi fied po 1ymer nano particles with organic functional groups for controlled and targeted drug release. Chernical grafting of long-chain molecules to polymer surfaces have been used for regulating hgrelease properties fiom the material [36,37]. Also polyethyleneglycol chahs have been immobilized ont0 liposome surfaces to lengthen blood circulation time[l2].
1.4 Objectives of the present research
The primary objective of the research presented in this thesis was to develop a hybrid PVA Solution
Dispersed in oil, followed by drying
PVA Xerogel Bead
Treated with fatty acid chloride
Acylated PVA Bead
Treated w ith liposomes
Lipobead
Figure 1-2 Structure and preparation strategy of lipobeads particulate system which retains the structural features and complernentary advantages of liposomes and non-ionic hydrogel rnicrospheres. The key steps of the preparation strategy is outiined in Figure 1.2. This involves a sequential surface modification which consists of chernical grafting of long-chah fatty acid anchors to the surface of polyviny 1alcohol (PVA) beads, fo llowed by the self-assembly of a lipid bilayer driven by the hydrophobicity of the surface fatty acid anchors. PVA is used to make the micro-spherical core structure by dispersing PVA solution in paraffin oil followed by appropriate cleaning and drying steps. Lipid molecules (fatty acids) are covalently grafted on the surface of dried PVA rnicrospheres through reactions with the surface hydroxyl groups. When treated with a liposomal suspension aftenvards, the surface modified polymer microspheres will allow the liposomal components to associate with the surface lipid anchors and distribute into a self-assembled overlayer on the beads through hydrophobic interaction. Dmg loading can be achieved by one of the following two steps. i) Relatively small and water soluble drugs can be loaded by impregnating the surface modified beads in a concentrated hgsolution pnor to liposomal coating so that the drug can be absorbed into the swollen polymer matrix. ii) Relatively large and insoluble dmgs cm be mixed directly into the polymer solution prior to dispersing it in parfin oil during the fabrication of unrnodified PVA beads.
To confirm the proposed structure (Fig. 1-2), the morphology and properties of the supported lipid membrane including unilamellar structure, lateral mobility of the assembled phospholipids, membrane permeability to hydrophilic dmgs and permeability changes to ionophores were characterized using several physicai techniques (Chapter 4 and 5). This research was aimed at establishing proof-of-principle that a hybrid system which
possesses some complimentar-y advantages of liposomes and hydrogel microspheres can be
achieved using a surface modification procedure involving molecular grafting of surface anchors for
lipids, followed by self-assembly of a surface lipid bilayers using lipids delivered to the surface in
the form of liposomes. Some aspects such as exploring practical applications and opthkation of preparation conditions, although technologically important, are considered as future improvements
of the present work. Aithough a nurnber of surface analytical techniques were employed, these
were only applied in a preliminary fashion, again to prove the feasibility of characterizing the
surface modification that is at the core of the lipobead technology developed here.
1.5 Outline of the thesis
The following chapters are presented in the order of experiments. Chapter 2 introduces the preparation and characterization of PVA xerogel beads. Chapter 3 focuses on chernical modification of the PVA xerogel surface. Chapter 4 describes attempts to obtain some mechanistic details regarding the interaction of the chemically modified PVA surface with phospholipids. The properties of the final product, lipobeads, are discussed in Chapter 5 with a series of characterization experiments. Finally, the potential application and fiiture improvements of the lipobeads technology are summarized in Chapter 6. 2. PREPARATION AND CHARACTERIZATION OF PVA XEROGEL MICROSPKERES
2.1 Supported lipid bilayers and hydrogels
Lipid bilayers have been formed on various solid surfaces such as electrodes, glasses and glassy hydrophobic polymers as mode1 membranes [38]. Formation of lipid bilayers on hydrophobically modified electrode surfaces through liposornal fusion ha recently been reported by Ringsdorf et al. [39]. Based on capacitance measurements and biosensor tests, Plant et al. [40] suggested that the lipid bilayers self-assembled on electrode sudàces are well packed and insulated to electrons. In addition, these supported bilayers have the ability to reconstitute membrane protein fimctions. However, the question addressed here is whether a well packed supported lipid bilayer cm be formed on a hydrated polymer surface.
Hydrogel surfaces, having different polymer chain lengths and chah foldings, are highly flexible in their fully hydrated state [41]. Contact angle and X-ray photoelectron spectroscopy
(XPS) measurements of a hydrogel surface containing small hydrophobic anchon, such as -CH3 group, suggest that the hydrogel surface morphology can change readily as a function of changes in the surrounding environment [42,43]. This flexibility is probably due to the fact that surface chahs are less entangled than those in the interior of the polymer matrix. On the other hand, the free energy change of hydrophobic interaction which is considered to be the driving force for lipid assembly is as large as several kcdmol [44]. It is therefore reasonable to assume that, upon formation of a bilayer between the liposomal component and the lipid anchors covalently linked to
PVA surface, such hydrophobic interactions will provides sufficient driving force to overcome the relatively small kinetic energy barrier of local movement of the hydrogel polymer chains thereby
facilitating the lipid alignment.
Fully hydrolyzed PVA was selected as the material to make the core structure for a number
of reasons. First, this type of PVA has a high content of hydroxyl groups to which lipid anchors can be linked. Second, PVA cm be physically crosslinked so that chemical crosslinking reagent can be avoided. For example, anneaihg of dehydrated PVA at an elevated temperature or treating
PVA gel in a fieeze-thaw cycle, dispersed crystalline domains will fom and serve as crosslinking junctions [45]. Moreover, PVA has been used as implant material and other devices for its proven biocompatibility [46, 471. Similarly, PVA has been used for artificial muscles [46], artificial vitreous body [47], and sofi contact lenses [48]. PVA has dso been studied extensively as carrien for small molecular as well as for protein drugs [49,50].
In order to form a well packed lipid bilayer on a polymer surface, the PVA microsphere substrate must also meet the following criteria: 1) the surface of the microsphere should be suficiently smooth (Qee of sharp edges and kinks) to avoid localized excessive curvature tension on the surface; 2) the microsphere should be dried (xerogel) for successive chemical modifications to take place selectively on the surface as many reagents involved are reactive to water; 3) hydrolysis reactions and polymerization should be avoided during the bead preparation as they may affect proteins and other high molecular weight dnigs which need to be incorporated during the preparation step.
The preparation of dehydrated hydrogel (xerogel) beads from hydrophilic polymers requires a drying step. However, aggregation and collapse of the surface structure can easily occur during
dryhg [51]. To solve this problem, a slow drying process has been used in the preparation of
PVA beads. By adjusting the flow rate of an air Stream bubbled through the bead suspension, the
drying process can be slowed sufficiently to prevent collapse of the bead surface. Further, the
viscosity of the suspending oil bath medium can be adjusted by temperature so that a sufficiently
high khetic banier to prevent aggregation can be achieved. To the best of our knowledge, the preparation of smooth surfaced PVA xerogel beads fiom fully hydrolyzed PVA polymer has not
been reported previously.
2.2 Experimental details
Materials Fully hydrolyzed PVA composed of a variety of polyrner chah length with a weight average molecular weight (MW)of 1 16,000 and a number average molecular weight (Mn) of 39,500
[52] (Elvanol 71-30) was purchased from Dupont. The solvents used in this study @araffn oil
[viscosity: 0.34 stokes], hexane, ethyl acetate, ethanol) were dl reagent grade, obtained from
Fisher Scientific. The surfactant, sorbitan monooleate, was supplied by Sigma.
Preparation Procedure PVA powder was dissolved in boiling water (25ml) to a concentration of
22 w/v% and subsequently injected at room temperature through a Teflon tube ( Cole-Palmer 4805-00 driver at its maximum rate (- 800 rpm). The particle size was controlled by selecting the appropriate injection rate and sorbitan monooleate concentration. AAer the PVA solution was successfully dispersed in paraffin oil, the oil suspension was cooled to 4'C under continuous stimng, and then frozen at -20'C ovemight to induce physical crosslinking. Then, the suspension was gradually warmed to room temperature, and the beads gradually dned in the oil bath by bubbling dry air through the suspension. During this procedure, the container containing the suspension was placed ont0 a Cole-Palmer "Roto-Torque" to maintain constant agitation for 1-2 weeks. After drying, the paraMin oil was removed by re-suspending the dried beads sequentially at 15 min intervals in hexane, ethyl acetate and ethanol. The beads appeared loosely aggregated after washing but were easily re-dispersed by stimng in ethanol. Determination of size distribution Dried PVA beads with a diarneter over 70 pm were hctionated using a Fisher Scientific Microsieve Set. On the other hand, PVA beads below 70 pm in diameter were fractionated according to their sedimentation rate in ethanol. Subsequently, PVA beads of different size ranges were weighed and a weight-distribution histogram was graded by counting the numbers of beads at a given weight (10 pl of beads/ethanol suspension of 10 mg/d in concentration which contains 16 to 240 beads depending on the size group) using a Spotlite Hemacytometer under an optical microscope (Car1 Zeiss Jeneval). Estimation of Density and Crystallinity Sphencal PVA beads of larger sizes (500 to 1000 pm in diameter) were prepared and weighed for the determination of the density of the material. The total volume of the selected beads was calculated according to their diarneter which was measured under an optical microscope. The density of the beads was calculated fiom their weight and volume denved from their diameten. Crystallinity is defined as weight eaction of crystalline part of the beads, and is estimated from the density on the basis of the following equation 1531: 1/p = w,/p, + (1 -W,')/p, where p, p,, and p, are the overall density, the density of 100% crystalline and density of 100% arnorphous domains, respectively. W, is the weight fraction of crystalline domain. This is a widely accepted method with px1.345 and p, =1.269 reported by Sakurada [53]. Microscopic image of beads Microscopic images of prepared PVA microspheres were recorded on a MRC-600 laser confocal microscope using a transmitted light mode. Confocai microscopie technique is widely used in imaging trace fluorescent probes [54]. However, here it was used because of its associated analysis software. 2.3 Size distribution of PVA microspheres Figure 2.1 descnbes the size distribution of the PVA beads as a function of the amount of surfactant at a fixed injection rate. The numbers given on the top of the solid bars are the average of two measurements. The standard deviation (S.D.) of the measurements are indicated on top of the solid bars. In the present experimental set up, the bead size ranges fiom 5 to 120 pm in diameter. For the particle size distribution, five size fractions of 5-20,20-45,45-70,70-90 and 90- 120 pm have been used. At an injection rate of O. 1 ml/min, the resulting bead size peaked at 5-20 pm in 8 wt/v% sorbitan mono-oleate (Fig. 2.1A). About 60% (or 27 wt%) of beads are in this fraction. Reducing the surfactant concentration caused the peak population to shift to a larger size. At a surfactant concentration of 2wtY0, most of the PVA beads (73%) were in the range of 20-45 pm in diameter (Fig. 2.1B). At 0.5 wt% surfactant (Fig. 2.1 C), the 45-70 pm fraction became the most prominent one (40%). Figure 2.2 shows the particle size distribution for different injection rates of PVA solution (O. t mVmin and 0.033 ml/min) at a given surfactant concentration (8 wt/vO/o). The beads with smallest diameter, 5-20 pn, showed the maximum size fraction (60% and 75%) for both injection rates. Reducing the injection rate fiom 0.1 to 0.033 dmin led to an increase in this size fraction fiom 60% to 75% and a decrease in the fraction of 20-45 group fiom 34% to 20%. Sue of beads / p m Figure 2.1 Size distribution of PVA xerogel beads as a function of surfactant concentration. A: 8 wt/v %; B: 2 wt/v %; C: 0.5 wt/v %. Size of beads I p m Figure 2.2 Size distribution of PVA xerogel beads as a function of injection rate at which the polymer solution was dispersed into padYn oil. A: 0.1 dmin; B: 0.033 mi/min. Figure 2-3. Confocal microscopic images of PVA beads suspended in ethanol (A) and water (B), respectively. The bar is IO mm in length. The surface of beads are smooth in that no local curvature of sub-micron radius are observed. The beads in water are swollen to twice the diameter of those in ethanol. Figure 2-4 Confocai images of PVA bead dried by an acetone/ethanol treatment (see Section 2.4) The remaining of size groups (45-70, 70-90, 90-120 pm) accounted for about 5% in total for both of the injection rates. The results shown in Figures 2.1 and 2.2 indicate that PVA beads with particular size distribution (to meet different applications - Chapter 1.2) can be prepared by using selected surfactant concentration and injection rates. For manufacturing purposes, the preparation condition will need to be refined to narrow the size distribution to within a designated range. 2.4 Surface smoothness and water swelling ratio of PVA microspheres Figure 2.3A shows an optical microscopic image of PVA beads suspended in ethanol (diameter around 10 p).In contrast, Figure 2.38 is the image of the PVA beads in the sarne size range but suspended in water. The surface of the these oil bath beads appears to be smooth under the optical microscope having subrnicron resolution. The dispersed sphencal beads were also dried using an alternative procedure, adding water-extracting solvent to the oil (acetone and alcohol). With this process, the beads can be dned instantly. However, surface roughness and irregular shapes resulted. The surface roughness was retained even after the beads were brought back to water swollen state (Figure 2-4). The PVA beads swelled when suspended in water but not in ethanol. To calculate "swelling ratio" , defined as the ratio of bead diameter at swelling equilibnum in water over that suspended in ethanol, diameters of five of the gradually dned PVA beads of shown size (1 0 pm) were measured before and afler water swelling. The measurements give an average swelling ratio of 2.0 + 0.1 (n = 5). 2.5 Density and crystallinity of PVA microspheres Crystallinity, density and population of crosslink junctions are parameters which determine the material flexibility and the water volume that the hydrogel matrix absorbs [53]. These factors can be important in determining swelling ratio, release rate and the "fiee volume" for proteins to maintain appropriate conformation. In the present study, crystailinity was estimated by direct measurement of density as mentioned above. Fourteen beads with diameter range of 500-1000 pm were weighed together, and the diameter of each bead was measured. The total volume of the beads was obtained by adding that of each bead calculated on the basis of the diameter of each bead. The diameter of each bead and total weight of the 14 beads are listed in Table 2-1. Based on the value in Table 2-1, the density of the PVA beads is calculated to be 1.271 gfcm3, slightly higher than the value of amorphous PVA (1.269 g/cm3), but considerably lower than that of crystalline PVA (1.345 g/cm3) [53]. Porosity of PVA is considered to zero [53]. This leads to a weight fraction of crystalline domain of 2.4% according to Sakurada's equation [53] (llp = WJp, + (1- WJp,; see Section 2.2). This value (equivalent to a crystalline volume fraction of 2.3%) suggests that the crosslink junctions formed by the crystalline particles only occupy a small fraction of the total volume. In the water-swollen state, these crosslink junctions will be pushed Merapart. A simple calculation can be performed based on the measured apparent density and swelling ratio to estimate the average distance between the polymer chahs. Table 2-1 Diarneter (pm)and total weight (mg) of PVA beads measured Since at a swelling ratio of 2 (the diameter of a bead is doubled), the volume increases 8 times, the density of the (amorphous part of) PVA bead decreases fiom 1.27 to 0.16 in the fully swollen state (if the mass of water is taken into account). Based on this density (0.16), each volume of 1000 A3 of swollen PVA bead contains 1.6 x 1002~g of PVA, which is equivalent to 2.2 of the repeating units, (-CHZHOH-). Since one PVA repeathg unit is about 2.8 A long, 2.2 units will be 6.2 A in length. Based on the assumption that each of these length are contained within a 1000 A3 hexagonal ce11 with a length of 6.2 A, then the average distance between polymer chah (center to center distance between two cells) will be 14 A (see fig. 2-5). Figure 2-5 Mode1 for estimating the average distance of polymer chahs. In summary, the experiments demonstrate that PVA beads prepared from fully hydrolyzed PVA polymer using a feeze-thaw treatment possesses a crystallinity of about 2.3 w/w??. The smoothness of the surface of PVA xerogel, which is required for Mersurface treatment, can be attained by using a slow drying process. The size distribution can be manipulated by modiQing surfactant concentration and the injection rate used when dispersing PVA solution to oil bath. 3. CHEMICAL MODIFICATION OF THE PVA SURFACE 3.1 Introduction Since the performance of polymeric drug delivery systems (drug release profile, blood circulation time, imrnunogenicity and hgtarg eting) is O ften adjusted by the phy sical/chernical properties of the surfaces rather than the bulk of these devices [29-311, surface modification is actively used as an effective approach in the development of novel drug delivery systems [34-31. In this chapter, chemical modification of PVA surface with long-chah lipid molecules is discussed and techniques for monitering the degree of surface modification are explored. 3.2 Surface modification techniques Functionalization (or defunctionalization) of the surfaces of polymeric materials cm be achieved by two means: creating specific surface sites by altering the existing surfaces or depositing a thin layer of chemical species on top of existing materials [55]. A number of physical and chemical methods have been used for these purposes. Table 3-1 lists some representative surface modification techniques. Some of these approaches have found commercial applications, while others are still at the early stages of development. The prirnary objective of this study was to establish the feasibility of a hydrogel-supported lipid vesicle systern as a novel drug carrier or ce11 model. Surface-supported bilayer membranes are finding increasing application in a wide range of biomedical disciplines including: limited due to steric hindrance [57]. In the present sîudy, two slrrface grafting procedures are used. One is to gr& fatty acid chains to the PVA smface by forming an ester bond with the surface hydroxyls. The other is to attempt a stepwise synthesis of phosphatidyl ethanolamine (PE) on the surface. Surfce grafting witlr fat@ acids DMAP, efher/ pyridine Figure 3 - 1 Reaction scheme for grafting fatty acid chains on PVA surface. DMAP stands for dirnethyl aminopyridine. This reaction is simple and easy to control. The formation of chernical linkage with the surface can be detected by the changes in carboxylic absorbance and oxygenkarbon ratio when the sample is subjected to appropriate surface analytical techniques. However, surface abundance of anchored fatty acids is difficult to detede quantitatively because the grafted molecules have the same elementary components (H, C and 0) as the substrate. In addition, the bilayer fomed by association with the fatty acid array will be asymmetric. For grafting of fatty acids, palmitoyl chloide or palmitic anhydride was allowed to react with the surface hydroxyls (Fig. 3- 1). With phospholipids, elements (N and P) which are not contained in the polymer are introduced, and these can serve as good indicators for estirnating surface coverage by the lipids. However, as indicated by XPS results (low nitrogen content and high nitrogen/phosphorous ratio (see Table 3-2a), earlier attempts to attach PE directly to the polymer surface resulted in low surface coverage and side-reactions. This may be due to cornpetition of small impurity molecules with the relatively larger PE for limited surface sites. Surface grafted synthesis of phospholipids An alternative strategy with multi-step reactions was employed to build PE stepwise on the PVA surface, in which a small "building block" was introduced at each step. For grafted synthesis of PE on the surface (Fig. 3-2), glutaric anhydride was first allowed to react with the surface hydroxyl groups. Subsequently, the carboxylic group formed by ring opening of glutaric anhydride was activated with pentafluorophenol (PFP) to form an active ester [69]. In step III, the head group of PE, glycerophosphoryl ethanolamine (GPE), was introduced to replace the PFP portion of the active ester by foming an amide bond. Finally, the two hyciroxyl groups of GPE were acylated by palmitoyl anhydride [70]. In the present study, two surface analysis techniques, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and x-ray photoelectron spectroscopy (XPS) were used for the characterization of biomedical polyrner surfaces. XPS is useful because it provides a total elemental andysis (except hydrogen and helium) of the top 10-100 A of solid surface with semi-quantitative capability 171,721. With angle-resolved sampling, the depth distribution of grafted materials may be estimated [72]. However, XPS is less informative for distinguishing transformation of chemical bonds due to limitations in resolution. ATR-FTIR complements XPS with the capability of directly monitoring transformations of chemical bonds, I DMAP 1 pyridine / hexane Q R OPVA -O-C w C-OH B NH2-CH2CH2-O-Pro-CH2CHCH2,Etfl 1 MeOH I I O OHOH I (RC=O)20 , DMAP ,ethyl ether / pyridine B 9 9 -O-C w C-NH2-CH2CHrO-5-O-CH2CH-CH2 O t, A t I Ç=OÇ=O Figure 3-2 Reaction scheme for grafted synthesis of phospholipids ont0 a PVA surface. especially for those with a carbonyl group, a strong oscillator in the infrared region. ATR-FTIR has been used as a sensitive surface analytical tool for protein adsorbates [73]. 3.4 Phy sical principles of surface analytical techniques (ATR-FTIR & XPS) A ften uated total refletance spectroscopy (A TR-FTIR) [74] FTIR is one of the most widely used analytical tools for chernical systems due to its ability to directly identie chemical bond transformation. However, this technique normally provide un- localized information regarding the sample. For the case when the chemical structure at the surface is being studied, a sampling technique (temed ATR-FTIR) which only sarnples the surface has been developed [74]. -Rarer media / 1 \ Rehcted Interface Incident light 1 Reflected light Figure 3-3. Reflection and rehction of a light beam at the interface of a denser and a rarer media Light incident upon an interface between two transparent media of different refiactive indices will be partially refiacted and partially reflected [74]. Figure 3-3 is a diagram showing that a light beam approaches the interface fiom the denser media to the rare media. The rehction angle follows Snell's law, n, sine = n2sincp (3-1) or n,/n, = n, , = sinWsinq (3-2) where n, and n2 are the refiactive indices of the involved media with different densities, respectively; nzl is the relative refiactive index; 0 and respectively. In the case that refractive angle, cp > 90°, total reflection occurs. The incident angle at which total reflection start to occur is called critical angle, &. From equation (3-2), OC = sin-in,, (3-3) In the present study, the refractive index of Ge, n, = 4; and that for organic polymers, n2 = 1.6, so that, n2,= n,/n, = 0.4, and 8c = 23.6'. The incident angle in this study is set as 45' (Figure 3-4), which will leads to total reflection. Upon total reflection, an electromagnetic disturbance exists in the rarer medium beyond the interface as described by Maxwell's equation [75]. This disturbance exhibits the fiequency of the incident wave but it is an evanescent electric field whose amplitude decays exponentially with the distance Fom the interface, E = ~~exp(-z/d~) (3 -4) where, Eo and E are the amplitudes of incoming wave and penetrating wave, respectively; z is the distance of penetration and dp is penetration depth defined as the distance required for the electric field amplitude to fdto e-1 of the value of the incident wave [76]. dp is given by where hi = hlis the wavelength in the denser medium. In the present experimental set up, dp/h = 0.27, thus the total sampling depth will be 3 x dp/h = 0.81 of the wavelength. Compared with transmission and extemal reflection sampling, ATR-FTIR offers solution compatibility and significantly supenor sensitivity to surface adsorbates [74,76]. IR bearn Polymer film To detector Figure 3-4. Sampling arrangement for ATR-FTIR [72]. X-ray pltotoelectron spectroscopy (XPS) [77] Irradiation of X-ray whose energy is greater than the binding energy of electrons in the atom wiil cause electrons to be ejected fkom the atom with a kinetic energy approximately equal to the difference between the photon energy and the electron binding energy, Ek = hv - Eb, or, Eb 2 hv - Ek (3-6) where Eb and Ek are binding energy and kinetic energy, respectively. With XPS the electrons ejected from core level orbitals are rneasured (Figure 3-5). The electrons excited by X-ray and ejected to vacuum, which are called photoelectron, can be captured by an energy analyzer by which the kinetic energy is measured and hence binding energy of the electrons can be obtained. Based on each key binding energy, elemental identification can be made. The binding energy of core level orbital can be shifted by the chemical environment that the atom fmds itself in [72,77]. This shift is called a chemical shift and provides the information regarding chemical bonding of the atom. For example, each carbon involved in a C-O single bond results in an approxirnately 1.5 eV higher binding energy from that just bonded to another carbon. Charging of insulators can also cause the apparent binding energy to shift significantly [77]. Thus it is normal to place the main aIkyl carbon at 285 eV [72] Core level obid / Nucleus Figure 3-5. Interaction of a X-ray photon with a core level orbital electron. Although X-ray photons penetrate many microns deep into a solid sample, the photoelectrons generated at that depth cannot necessarily travel through that distance to the vacuum and be detected. To be detected, the photoelectrons must emerge fiom the sample without any loss in energy and be comprised in the main photoelectron signal peaks. The population of this type of electrons decrease exponentially with the depth where the number of photoelectrons generated will be: N = N,exp(-xih) (3-7) where Nois the number of photoelectrons generated at the surface, N is the number of electrons at depth x A that emerged from the sample without energy loss, and h is a constant called mean fiee path or escape depth at which e-1of photoelectrons generated upon excitation emerge fiom the sample. The value of h is material dependent, ranging fkom few to several tens of angstroms. The sarnpling depth of XPS is generally accepted as three times of A, ranging fiom 10 A to 100 A [72,77]. Energy analyzer P' Vacuum chamber Figure 3-6. Sampling arrangement for XPS [77]. Figure 3-6 shows schematic diagram of a system equipped with an X-ray source and an energy analyser. The sample is held by a movable sample holder and can be presented to the energy analyser wiîh various angles. This allows the sample to be andyzed at difierent take-off angles so as to provide additional surface information. PVA powder (Elvanol 7 1-30, a fully hydrolyzed grade with MWaround 1 16,000 and Mn around 39,500 [52]) was obtained fiom DuPont. Al1 reagents, solvents and silica gel were reagent grade and purchased fiom Aldrich Chernical Company, Inc. For surface charactenzation, PVA films were subjected to the identical chernical treatrnent as beads. PVA films were prepared by casting an aqueous solution of PVA (10 w/v%) onto a glas plate using a film-casting knife, followed by drying in room temperature and annealing at 145'C for 5 min. The procedure preparing PVA beads was described in Chapter 2. Al1 reactions were carried out in round bottorn flasks at room temperature unless indicated otherwise. For fatty acid grafting, dry PVA beads or films were placed in a hexane solution of palmitoyl chloride (0.2M)in the presence of triethyl amine, or in a mixed solution (hexane:pyridine = 3:l) of palmitic anhydride (0.05M) in the presence of dimethyl aminopyridine as catalyst (0.08M). The reaction was allowed to proceed at room temperature for 2 days. For grafied synthesis of PE, PVA beads or films were first allowed to react with glutaric anhydride (0.2M) in a mixed solvent of hexane and pyridine (3: 1) containing DMAP (0.08M)as catalyst at 30'C for 16 hrs, followed by washing in sequence with hexane, acetone and ethanol. In step II (Fig.3-2), the acylated films were treated with pentafluorophenol (PFP, 0.M) and dicyclocarbodiimide (DCC, 0.3M) dissolved in dioxane at room temperature for 4 days. Mer washing with dioxane and ethanol, the films were Mertreated with a methanol solution of glycerophosphoryl ethanolamine (GPE, 0.2M) at room temperature for 20 hrs (step III of Fig. 3-2). As the final step (step IV of Fig. 3-2), palmitic anhydride (0.05M, reactant) and DMAP (0.08M, catalyst) dissolved in a mixture of diethyl ether and pyridine (33 were used to treat the PVA films at room temperature for 4 days. GPE used in the grafied synthesis of surface phospholipids (step III of Fig. 3-2) was prepared from equivalent solketal (2,l-dimethyl- l,3-dioxolane-4-methanol), phosphorus oxychlonde and ethanolamine according to a published method [78] as shown in Figure 1-1 of Appendix 1. The product was purified chromatographically using a silica gel column with methanol as mobile phase. The structure and purity of GPE was confmed using 2D NMR (Varian UNITY+SOOMHz [79], CD30D). Results of the NMR confirmation is presented in Appendix 1. ATR-FTIR spectra were recorded on a BIO-RAD-DIGILAB FTS-7 system with a thermocouple detector and a custom-designed sarnple holder. The polymer films were pressed against a Germanium pnsm (25 x 5 x 1 mm with 45" edge), through which the infiared beam passed and reflected repeatedly at the surfaces (See Figure 3-4 [74]). The XPS spectra were recorded on a Leybold MAX-200 system equipped with an unmonochromatic Mg Ka X-ray source (Ka line of x-ray emission fiom a magnesium target) operated at 12 kV and 25 mA (See Figure 3-6). Spectra were obtained in both a high resolution @as energy = 48 eV) and a low resolution @as energy = 192 eV) modes. Features arising from the X-ray satellite lines, inherent by use of a non-monochromatic source, were substrated fiom the spectnim by means of an algorithm [80] which was supplied with the instrument. The bindïng energy scale was established by placing the C (1s) peak of the main akyl group at 285 eV to account for sample charging. The pass energy was 192 and 48 eV for Low and high resolution measurements, respectively. Relative intensities of XPS peaks for each element were obtained fiom the low resolution spectra which were normalized to unit transmission of the electron analyser [8 11. The peak areas were obtained using the software provided with the instrument and applying the supplied sensitivity factors which are appropriate for the normalized spectra. The sensitivity factors used were 0.34, 0.78, 0.54, 0.61 and 1.O0 for C(ls), O(ls), N(ls), P(2p) and F(ls), respectively. Chernical shift information was obtained by anaiysing the spectra in high resolution mode. To obtain peak positions, areas and full width of half maximum (fwhm), curve-fitting routines (also supplied) were used. Prior to XPS measurernent, the sample was sonicated in sequence with hexane, dichloromethane-acetone mixture and ethanol . 3.6 Results of surface modification reactions as determined by ATR-FTIR and XPS ATR-FTIR Spectra Figures 3-7 and 3-8 summarize the ATR-FTIR results recorded after acylation of a PVA surface with fatty acids and afler each reaction step for synthesis of surface- anchored phosphatidyl ethanolamine, respectively. The ATR-FTIR spectra shown in the figures were obtained by subtracting the spectnim of bare PVA film fkom that after each surface reaction. For fatty acid grafiing (Fig. 3-7), the two selected surface reactions (with palmitoyl chloride and with palmitic anhydride) resulted in the same FTIR spectra (in both intensity and vibrational modes). The newly generated peak at 1730 cm-i is typicd for an ester carbonyl stretching vibration, while the peaks at 2889 and 2952 cm-' are typical for syrnmetric and asymmetric stretching of the -CHr of the hydrocarbon chains [82]. These absorption bands cannot be observed in transmission mode, suggesting that the acylation occurs only at surface hydroxyls. For the grafted synthesis of PE, when glutaric anhydride was reacted with the PVA film, two absorption peaks were detected at 1705 and 1730 cm-I, which can be attributed to the carbonyl stretching of the carboxylic acid and ester groups (Fig. 3-8a), respectively. The absorbante at 1705 cm-1 was reduced by 25-30% after reacîion with PFP in step II, while a new peak with intensity equivalent to the reduction of the peak at 1705 cm-' was generated at 1788 cm-1. This clearly indicates that some carboxylic acid g-roups have been esterified with PFP [77]. The peak at 1522 cm- l, which accompanied the one at 1788 cm-', is assigned to the stretching of the PFP ring [83]. No further conversion of the carboxylic groups to PFP esters (as evidenced by the spectroscopie transformation of the band at 1705 to 1788 cm-1) was observed by extending the reaction tirne beyond 4 days. Most likely, the PFP can only reach and esteri@ the carboxylic groups located at the surface region. Mer Merreaction with GPE in step III, both the peaks at 1788 and 1522 cm1disappeared while a new one with equivalent intensity was generated at 1 660 cm-1, suggesting the formation of an amide structure fiom the active ester with PFP. The absorption aîtributed to amide II at 1550 cm-1 appears too weak to be recognized [83]. Acylation of the two hydroxyl groups of GPE by palmitic anhydride in step IV is evidenced by the increase in peak intensity for the ester carbonyl stretch at 1730 cm- * and asymmeûic and syrnmetnc -CH2- stretch at 2952 and 2888 cm-'. respectively. The WAVE NUMBER i cmœ1 Figure 3-8. ATR-FTIR spectra of PVA film recorded d'ter each reaction step shown in Figure 3-2. a) Before treatment; b) Mer step 1; c) After step ri; d) Mer step III; e) Mer step IV. increased height of the peaks at 1730 cm- l is about twice that for amide (1660 cm-') and the PEI? active ester (1 788 cm-'). XPS Spectra Figures 3-9 and 3- 10 show the XPS spectra recorded before and after each reaction step, which are in good agreement with the ATR-FTIR results. The survey (wide scan) specûa are shown at the left side of both Fig. 9 and 10, in which the peaks with binding energies approximately at 285, 531, 400, 685 and 136 eV are assigned to carbon (ls), oxygen (1s) nitrogen (ls), fluorine (2p) and phosphoms (2p), respectively [84]. High resolution spectra of carbon (1s) are shown at right side of the two figures in which different components due to chernical bonding of carbon are denved by a curve fitting operation using software supplied with the instniment. Table 3-2a and 3-2b summarize the information obtained from low and high resolution spectra, respectively. For fatty acid grafting (Fig. 3-9), the two major components of in the high resolution spectnim of C(1s) of bare PVA are attributed to alkyl @.e.- 285; fwhm - 1.43) and alcoholic (b.e. = 286.3; fwhrn = 1.48) carbon [83]. The week and broad peak at 288.2 eV may result from impurities combined with un-hydrolyzed acetate groups. After acylation with palmitoyl chloride (Fig. 3-9, Table 3-2b), the alkyl component @.e. = 285) retained the same fwhm (1.441, but increased in relative intensity. The peak for alcoholic carbon (b.e. = 286.3) was, however, reduced in relative intensity and broadened (fwhm = 1.90). Since acylation cause the b.e. of alcoholic carbon to up-shift by 0.10 eV [83], this broadening may be due to the overlap of acylated and un-acylated alcoholic carbons. The new peak (fwhm = 1.46) that appeared at 288.9 eV is assigned to carboxylic carbon generated by acylation [84]. At the same the, the relative intensity of carbon peak versus that of oxygen in the wide scan spectra increased after the reaction (Fig. 3-9). This is attributed to the long-chah fatty acids deposited on the surface. This resuit is consistent with that of the ATR-FTIR measurements (Fig. 3-7). For grafted synthesis of phospholipids, the first reaction step in which hydroxyl acylation with glutaric anhydride was involved resdted in a reduction in relative intensity of the alcoholic carbon (b.e. = 286.5 eV) but an increase in fwhm (= 1.90 eV) (see Fig. 3-1 1b, wide scan spectrum). However, the oxygen/carbon ratio did not change significantly as compared with PVA (Table 3-2a). Introducing PFP in the second step (Fig. 3-2) caused generation of a new peak for F(2p) (b.e = 685) which was accompanied by an increase in relative intensity of the peak at 286.3 eV in the high resolution spectra (Fig. 3-10c). This intensity increase may be due to the aromatic ring of PFP. This peak rnay also overlap with those of acylated and un-acylated alcoholic carbons. Although fluo~ationof a carbon atom in a hydrocarbon chah will normally generate a binding energy peak at 287.9 eV [84], the chernical shifi resulting fiom halogenation of an aromatic ring can be substantially smaller (- leV) than that of a linear hydrocarbon [84]. This fluorine peak was rernoved after step III, while equivaient nitrogen (b.e = 400eV) and phosphorus (b.e. = 136eV) were detected instead (Fig. 3-l0d-wide scan and Table 3-2a). The same elemental abundance was observed for nitrogen and phosphorus, indicating that grafted GPE is the only source for the N(1s) and P(2p) signals. The peaks at 350 eV and 450 eV are assigned to the 2p and 2s electrons of calcium ion [77], which may be adsorbed by GPE during the purification process because no Ca was found when GPE was subjected to XPS prior to the separation. After step IV, the fractions of both total and akyl carbon increased in wide scan and high resolution spectra of Fig. 3-10e with accompany of a reduction of oxygedcarbon ratiofiom 0.5 to 0.3 (Table 3-2a), indicating the anchoring of palmitic chah Table 3-2a lists the survey After acylation Binding Energy 1 eV Figure 3-9. XPS Spectra of a PVA film recorded before and aller acylation with palmitoyl chloride. Left side: survey spectra (50-950 eV); right side: high resolution spectra (1 80-292 eV). Suwey Binding Energy 1 eV Figure 3-10. XPS spectra of PVA film after each step of grafted synthesis of PE shown in Fig. 3-2 a) before treatment; b) after step 1; c) after step II; d) after step III; e) der step IV. Left side: survey spectra (50-950). Right side: high resolution spectra (280-292) Table 3-2a. XPS elemental hctions of PVA sheets after each step of surface reaction # of Elemental fraction (% mean + S.D.1 Reaction step observations C(0.34) O(0.78) N(0.54) P(0.6 1) F(1.00)* Before reaction 2 68 4 1 322 1 Fatty acid graf%ng 2 79t 1 21 1 (Fig. 3-1) Grafted synthesis of PE (Fig. 3-2) AAer step I 2 70+2 3022 After step II 3 69t2 2722 3.9 + 0.2** After step III 4 6823 30+3 1.320.1 1.2k0.2 < 0.1 Af'ter step IV 3 7522 23 + 2 1.0+0.1 0.9+0.1 < 0.1 ------Direct PE grafhg*** 2 74 + 3 2443 1.O + 0.2 0.3 + 0.2 * The number in parentheses are sensitivity factors used with the system. ** XPS signals of halogen atoms gradually decay upon exposure to X-ray. * * * Reacted with phosphatidy 1 ethanolamine after step II. atomic fraction before and afier each reaction step of grafted synthesis of phospholipids which were determined by nonnalized XPS peak areas of wide scan spectra. There was no significant difference in oxygen/carbon ratio (OC) between un-treated PVA and those treated by the fvst three steps in Fig, 3-2. This is consistent with the stoichiometry in which the first three reactions do not involve a surface modification with significantly different OIC. However, the decrease in intensity and increase fwhm of the peak centered at 286.3 eV indicates the involvement of the surface hydroxyls in the reactions (Fig. 3-10 and Table 3-2b). Moreover, the presence of nitrogen and phosphorus after step III, although indicated by small peaks (Fig. 3-10), is significant (Table 3-2a). Table 3-2b Summary of curve-fitting of high resolution XPS specûa of C (1 s)+ untreated PVA 4623 1.4320.16 48 2 4 1.48 + 0.28 83-4 2.22 + 0.3 1 t Fatty acid grafting 60 + 3 1.44 2 0.07 32 2 2 1.88 +: 0.05 812 1.46 + 0.07 Step 1 45+3 1.47+0.17 48+4 1.90+0.14 7+1 1.56 20.11 Step II 3851 1.40+0.04 51 2 1 1.93 + 0.10 11 & 1 1.49+0,16 Step III 48'4 1.4420.15 45 2 5 2.22 + 0.35 711 1.59 0.3 1 Step IV 5453 1.41+0.06 38+3 2.09+0.18 811 1.48 f 0.02 * Al1 the values given are the average of two observations. Figure 3-1 1 shows the high resolution spectrum of the N (1s) @.e. = 400 eV, fwhm = 1.83) region after step III, as compared with amino (b.e. 399.1 eV) and ammonium (b.e. = 402.6 eV) groups deposited on the PVA surface. These values, which are identical to literature data [84], providing strong evidence that GPE was anchored on the surface through an amide bond. For the 1st reaction step, there is a possibility that the palmitic anhydride may also react with the hydroxyl groups of the PVA backbone left f?om previous steps. However, the nurnber of these hydroxyls is greatly reduced by the intermediate steps as evidenced by the XPS data. Moreover, palmitic anhydride is much larger and more hydrophobie than the other additives used in prior steps so that further penetration into the dry PVA matrix is unlikely. Among the synthetic steps reported here, coupling of GPE with the surface carboxylic groups through the pentafluorophenol (PFP) ester is unique. Direct coupling of amino groups with carboxylic groups using DCC cm lead to the formation of a by-product, N-acetyl urea [85], which is ineversible and cannot be removed from the surface. Although this process can be suppressed under acidic conditions [85], the amino group of GPE favors high pH in order to react as a nucleophile [86]. With the PFP ester, the coupling can be divided into two steps (Fig. 3-2). Moreover, the PFP ester selectively reacts with the arnino group of GPE and leaves the two hydroxy 1s intact. 3.7 Angle resolved XPS: depth distribution of and surface coverage by grafted molecules A major morphological difference between inorganic crystal and organic polymer surfaces is that the former are flat and dense while the latter are often porous, flexible and permeable. 406 402 398 394 Binding Energy / eV Figrne 3-1 1. XPS spectra of nitrogen-containing species deposited on a PVA surface. a) After PVA film was treated with 6-bromohexaoyl chlonde, followed by Mertreatment with ethanolamine; b)After step III shown in Figure 3-2; c) After treatment of a), followed by mertreatment with HCI (0.01N). Because of this structural complexity, it is difficult to modify and characterize polymer surfaces in a well defmed manner as compared with inorganic crystals. Unlike inorganic crystalline surfaces, during chernical modification of polymer surfaces, the reactions are not necessarily limited to the sites on the upper-most surface of the polymer substrate, but aiso possibly with those at the sub-surface or deeper regions. Therefore, the spectroscopically deterrnined composition of grafted molecules may include those fonned at both the surface and the buky structure of detectable depth. Consequently, the morphology of the modified surfaces (including surface coverage and composition) cannot be detemiined simply fiom the measured atornic fraction. In addition, the vertical distribution of the graf?ed species must be taken into account. On the other hand, the surface specificity of XPS is achieved by a mechanism such that the photoelectrons generated by the X-rays (See Chapter 3.4) must travel through the solid matrix and reach the energy analyzer (Fig 3-6) with sufficient kinetic energy so that they can be detected. The probability of occurrence of photoelectrons which can travel through the solid matrix and reach the energy anaiyzer without energy Ioss decays exponentially with increase in the distance traveled. By varying the angle between the simple surface and the axis of the energy analyzer (sampling angle), the through-solid travel distance of the photoelectrons generated at a given depth can be manipulated. Figure 3-1 2 shows some models approxirnating the relationship between sampling angle and fiaction of surface deposit to be sampled. Mode1 A in Fig. 3- 12 represents a layer of deposit on the top surface with thickness near zero A; in model B, layer has thickness comparable to photoelectron escape depth li (Equation 3-7); in model C, the deposited layer has thickness greater than the XPS sampling depth. For model C, changes in sampling angles will not make any difference (a constant ratio of 1) because the fiactional sampling depth To energy To energy To energy analyzer analyzer analyzer To energy To energy To energy analyzer anal yzer analyzer Figure 3-12. Three possible morphologies of chemicdy grafted polymer surfaces as measured by XPS with 90' and 45' sampling angles. A: with surface grafting of "near zero" thickness; B: Grafbg with thichess comparable to h; C: Grafting with thickness over XPS sampling depth. 1: at 90'; II: at 45'. x: XPS sampling depth, which reduces with sampling angles. The arrows represent the path of photoelectrons fiom their excitation site to the energy analyzer. for the deposit will be the same for any sampling angles. For model A, the ratio in fiaction of deposit between 0 sampling with 90' sampling will reach the theoretical maximum, sin 90°/sin 0. This ratio will drop exponentially with the depth of the deposit layer and approach a constant vdue of 1.O. Table 3-4 shows the elementai fiaction of nitrogen, N(ls), after reaction step III (see Fig.3- 2) based on XPS signal intensities recorded at sampling angles of 90' and 45'. The elemental fraction of N(1 s) increased fiom 1.3 to 1.8 when the sampling angle varies fiom 90' to 45', giving a ratio of 1.8/1.3 = 1.4. Since this ratio, obtained from reproduced meanirements (Table 3-3) is close to the theoretical maximum (1.414), model A (Fig. 3-12) is considered to describe the GPE, grafting, thus grafted GPE groups are mostly near the surface. A detailed mathematical denvation of thickness of and sdace coverage by the graf?ed layer is given in Appendix II. Table 3-3. Detected elernental fraction of N(1s) as a function of XPS sampling angle - - Experimental Runs* At 45" At 90" Ratio 1 1.7 1.2 1-42 2 1.8 1.3 1.38 3 1.9 1.3 1.40 ------*---- Average 1.8 1.3 1.40 * The results from each experimentai nin was obtained fiom a respect PVA sheet treated up to step III of Figure 3-2. By comparing Fig. 3-8a with 3-8b, about 25-30% of the peak height of carboxylic group (1 700 cm-') converted to PFP easter (1 780 cm-'), which in turn converted to an amide structure (1660 cm-') by replacing the PFP goup with GPE (Fig. 3-8c). The rest of glutaric spacers (70- 75 %) did not involve in successive reaction steps to link GPE. On the other hand, the fatty acid graftings showed the similar intensity of the absorbance for carbonyl stretching in ATR-FTIR spectra as that of glutaric groups (comparing Fig. 3-7 with Fig. 3-8), suggesting that the fatty acids chains gralted are more than those of phospholipids. 3.8 Discussion In chapter, we have demonstrated approaches of molecular grafiing of fatty acids and grafted synthesis of phospholipids ont0 PVA surface. By using two complementary surface analytical techniques (ATR-FTIR and XPS), each reaction step was confirmed. The ATR-FTIR measurements provided direct information of formation of new chernicai bonds between existing surface species and the molecules introduced to the surface. For example, grafthg fatty acids to the surface hydroxyls was evidenced by the infrared absorption of an ester stretching mode (1 730 cm-') which was accompanied by symmetric and asymmetric stretching of -CH2- chains at higher wave number (- 2900-3000 cm-', Fig. 3-7). Similarly, the ring opening reaction of glutaric anhydride associated with the surface hydroxyls resulted in the generation of two parallel IR absorptions at 1730 and 1700 cm-'attributed to an ester (attached to the surface hydroxyl) and an dangling carboxylic acid groups, respectively (Fig. 3-8a). The remaining reaction steps (II, III and IV) of grafted synthesis of phospholipids were also indicated by ATR-FTIR measurement with the IR absorption of PFP active ester, amide and easter (with -CH2- chah stretching), respectively (Fig. 3-8b, 3-8c and 3-8d). As a surface specific technique for elemental analysis, XPS measurement provided consistent information with those obtained fiom ATR-FTIR. For fatty acid grafting, the progress of the reaction was evidenced by ïncreased carbon content, decreased alcoholic carbon, and generation of carboxylic carbon when comparing the XPS spectm after the reaction with that before the reaction (Fig. 9, and Table 3-2a and 3-2b). For grafted synthesis, although no significant changes in C/O ratio was observed in the fmt three steps, progress of the reactions were indicated by decrease in the peak intensity of alcoholic carbon, generation of a fluorine peak, and generation of nitrogen and phosphorus peaks, respectively (Fig. 3-10, and Table 3-2a and 3- 2b). As a technique suitable for semi-quantitative analysis [72], the depth distribution of the grafled species were estimated using an angle resolved XPS measurement based on Ritrogen content (Table 3-3). The results suggest that phospholipid anchors only form at the surface of PVA. The experimental results presented in this chapter have demonstrated that with application of appropriate surface analysis techniques, a multi-step chemical modification of polymer surface can be monitored step by sep. 4. CHARACTERIZATION OF LIPOSHEETS 4.1 Supported planar lipid bilayers Supported bilayer membranes are finding several practical and fundamentai applications in a wide range of disciplines including biofunctionalization of inorganic solids, biosensors, artificial cells, dnig delivery, drug-membrane interactions, and ce11 surface phenomena [60-671. Lipid bilayers supported on planar surfaces cm be used as a convenient mode1 for a number of surface characterization techniques by which the physical-chemical properties of the bilayer and its interaction with the substrate surface can be studied. For example, a bilayer supported on an optical prism which is transparent to IR radiation has been studied by ATR-FTIR to detect its lipid orientation [87]. Surface plasmon resonance spectroscopy has also been used to characterize supported membranes [8 81. In addition, using an electrochemicd setup, the cross-membrane conductivity, irnpedance and capacitance has been measured [40]. Supported lipid bilayers are generally prepared using two deposition approaches: i) tmnsfer of pre-formed lipid monolayer fiom the watedair interface onto a substrate or ii) by self-assembly of fkee lipid molecules ont0 a surface [89]. With the former, which is referred to as "Langmuir- Blodget" (LB) deposition, a well defined bilayer with selected lipid composition can be immobilized on a surface, With the latter, a bilayer cm be formed on surfaces with a wide variety of surface shapes and morphology. Applications of both methods have been reported in the literature [89]. Among various supported bilayers, those supported on a polymenc gel network may offer certain structural similarities to that of biological membranes [60]. In the previously reported work [39,60], however, the bilayer is assembled ont0 a thin hydrogel layer which is supported on a metal electrode surface so that it can not be used as a ceil model or a diffusion barrier. Here, formation of a planar lipid bilayer on a fiee-standing surface-modified hydrogel sheet is described. We cal1 this assembly process which consists of chernical grafling and liposomal coating as "lipocoating", and the bilayer-coated hydrogel sheet as a "liposheet". UnIike other hydrogel-supported lipid bilayers in which the hydrogel itself is supported on another solid, liposheets can be used as model membranes based on which cross-membrane difiion and many other tram-membrane processes may be studied. In this chapter, two characterization techniques are used, namely contact angle measurement and Langmuir-Blodget deposition, to study the mechanisrn of the liposomal coating of modified PVA hydrogels. 4.2 Experimental setup In the present work, water contact angle was measured using a scope manufactured by Rame-Hart, Inc. (See Figure 4-1). The experirnent was canied out using following procedures: A PVA sheet with fatty acids anchored on the surface was placed in water. This sheet swelled and floated on the water surface. Then a droplet of water or of liposome suspension was placed on top of the the floating polymer sheet and allowed to stand an hour. The contact angle of the drop of water or the liposome suspension was measured with the equipment shown in Fig. 4- 1. Water drop Scope PVA sheet Water container i \ \ , Figure 4-1. Experimentai arrangement for measuring contact angle. Floating bar pressed against sensor Floating bar driven Pressure sensor \ Figure 4-2. A diagrammatical sketch of Langmuir-Blodget tmugh. Langmuir-Blodget deposition was perforrned using a LB trough (Mgw Lauda Filmwaage) show diagrammaticalIy in Figure 4-2. The lipid sample was diluted with chloroform to 0.5 mghi and spread over the surface of the water filled trough, followed by evaporation of the solvent. Afterwards, a well-packed monolayer was formed by compressing the monolayer with a floating bar driven by a steper motor until the membrane pressure reached an equilibriurn of approximately 30 dyne/cm. Subsequently, a pre-swollen surface-modified PVA film (2 x 3 cm? of which only 2 x 2 cm2 was dipped into the trough), attached onto a glass slide, was gradually dipped into and raised out from the water through the monolayer. Deposition of the lipid monolayer ont0 or stripping-off of the monolayer fiom the substrate film was indicated by a change in the monolayer surface area which was measured fiom the displacement of the floating bar. The membrane pressure was kept constant at 30 dydcm during the passage of the substrate through the lipid monolayer. Two lipid sarnples, dioleoyl phosphatidyl choline (DOPC)and 1,2-dioleoy, N-trimethyl propane (fiom Avanti Polar Lipids, diluted with chloroform to 5 mM) were used to form the monolayer on the water surface. DOPC was also used for the preparation of liposomes for contact angle measurements. 4.3 Contact angle measurement: interaction of the modified surface with liposomes Contact angle measurement are widely used to study the hydrophilicity/hydrophobicity of solid surfaces. Some physical chernical parameters, such as surface fiee energy, may be derived fiom the contact angle of the solid surface with the Iiquid [go]. Thus, contact angle measurement before and after the liposomal coating provides useful experimentd data for explaining the thermodynarnic forces responsible for fûnction during lipocoating. Figure 4-4 shows the forces on a solid-liquid system, where YL,YS and YSL are the surface tension of solid, liquid and interface, respectively. Solid Figure 4-3 Equilibrium of surface tensions of a solid-Iiquid system. At equilibrium, Here, we used contact angle as an indicator to monitor liposomal coating over PVA surfaces modified with fatty acid or phospholipid. For the water drop, the contact angle remained at 103' -+ 1' (n = 5) for days. This value is the same as measured with pamnin wax [gO,gl]. In the case of a liposome suspension, however, the droplet collapsed and fully wetted the surface. 4.4 Langmuir-Blodget deposition on modifïed PVA sheet Langmuir-Blodget deposition is a well-established way of preparing defmed lipid bilayers on planar solid surfaces [89]. With a Langmuir-Blodget trough, the formation of a well-packed lipid monolayer at water-air interface can be indicated by the surface pressure. Similady, the transfer of the monolayer ont0 the substrate surface can be observed by the change in area of the pre-formed monolayer. Here, we use Langmuir-Blodget technique to study the interaction of aligned phospholipids with surface modified PVA hydrogel sheets. Figure 4-3 diagramrnatically shows the observed changes during the L-B deposition of DOPC ont0 fatty-acid modified PVA sheets. When the PVA sheet was first dipped into the water phase through the DOPC monolayer, a monolayer was coated ont0 the surface as indicated by a the reduction of the DOPC monolayer surface area at constant surface pressure which is equivalent to the size of the sheet. Another layer of lipid was coated ont0 the surface when the sheet was gradually withdrawn through the water surface. However, this layer was easily stripped away when the sheet was re-immersed. This observation suggests that a lipid multilayer structure is unlikely to be formed on the polymer surface. Assembly of a well packed phospholipid mono- layer on alkalized electrode surfaces exposed to liposome suspensions have been demonstrated by several authors [38-40]. As will be discussed later, the dnving force in assembling the first layer is Surface modified A layer is deposited PVA sheet when imrnersing \ / -water surface Another layer is deposited when withdrawing / &yyUbl -water surface The 2nd layer is stripped out when imrnersing back into water / Figure 4-3 Observation during Langmuir-Blodget deposition of DOPC ont0 a fatty acid modified PVA sheet the hydrophobie interaction, which is much stronger than the interaction between the phospholipid heads groups that may drive successive coatings. This resuit also suggests that during contact angle measurement a single bilayer with the under leaflet formed by the anchored fatty acid and the upper leafiet formed by the added phosphoiipid is formed. To test the head group interaction, L-B deposition with 50 mol% positively charged lipids, 1,2-dioleoy, N,N,N-trimethyl propane, added was carried out. With monolayer formed by the charge lipids, the fmt layer was coated on the surface as depicted in Fig 4-3, but the second layer failed to form. Clearly electrostatic repulsion prohibited formation of successive coatings. 4.5 Discussion The hydration of dry phospholipids often leads to formation of multilamellar vesicles [93]. This may raise the question on whether a multilamellar structure is fomed on the modified PVA surface during contact with liposomal components. This question may be addressed by a thermodynamic analysis based on a cornparison of the surface free energy or enthalpy changes involved betweeri the different surface structures. Surface tension and contact angle (two closely related physical factors) for watedparaffin system are fiequently given in physical chemistry text books and handbooks as, YSL = 52 dynkm and 0 = 1O6', respectively [9O,911. Surface tension for water is, *Ir. = 73 dynkm [9 1,921. Since the fatty acid array at the PVA surface has similar chernical structure and contact angle (1 O3'), it is reasonable to assume that modified surface has a sirnilar surface tension to the waterkolid system, YSL = 52 dydcm (4-3) Since the dimension (forceAength) and numericd value of surface tension are the same as those of surface fiee energy (energylarea), this surface tension can be directly written as surface fiee energy change upon interaction with water, This high surface free energy is due to the hydrophobicity of the modified surface that resists wetting of the surface with water. When the surface is exposed to a liposome droplet, the spontaneous expansion of the liquid &op indicates that with the phospholipid coating, the free energy change upon interaction between water and the surface is negative. In another words, in this watedsurface system, the surface fiee energy change caused by the liposomal coating on the fatty acid array is at lest -52 erg/cm2. Because one phospholipid molecule in a well packed lipid layer occupies approximately 70A2 of surface area 1891, this surface free energy change can easily be converted to the unit of kcal/mol, This value falls in the range of hydrophobie interaction 1441. Here, the decrease in the surface fiee energy caused by forming an anchored bilayer should provide a sufficient driving force to overcorne the energy barriers of the local movement of hydrogel chains which facilitates the alignment of lipid molecules on the surface. Although the assembly of an organized lipid bilayer results in displacement of surface water and an entropy change against the assembly [91],these -60- factors are included in the Eee energy change which is estimated based on contact angle change. The change in cwature tension of liposomes rnay play an additional role in providing a themodynarnic driving force for drivhg fusion of the liposomes with the modified PVA surface. As evidenced by titration calorimetry, the enthalpy change between smail lipid bilayer vesicles (nano-size) and large vesicles (micro-size), bot. formed with uncharged phospholipids, fdls in the range of a few hundred caVmol [94]. Although fiee energy difference between lipid vesicles of different sizes cannot be denved since the entropy change of the process is dificult to obtain, it may be reasonable to assume that the entropy change due to liposome size is negligible. Since entropy is a measure of inegularity, unless the bilayer matrix is disrupted, the overall bending of the bilayer will not result in a significant entropy change. The difference in the enthalpy change as a function of size is due to the fact that the lipid bilayers of smaller vesicles have larger curvahue tension. However, this diflerence in enthalpy change between small and large vesicles (few hundred cal/mol) is at least one order of magnitude smaller than the expecting driving force associated with the hydrophobicity of the modified PVA surfaces (few Kcal/mol). This suggests that curvature tension (or elasticity) is unlikely to be an important contribution to the lipocoating process. In summary, hydrophobicity of the fatty acid anchors on PVA surface provide a suficient driving force (- 52 Kcal/mole) for spontaneous assembly of a lipid bilayer on the support surface when the sample is exposed to a liposome suspension. This energy preference, as suggested by LB-deposition, was not suffcient to drive multilamellar bilayer formation. 5. CHAliACTERIZATION OF LIPOBEADS 5.1 Prospective properties of supported lipid vesicles As discussed in Chapter 1, the prirnary objective of the present research is to develop a hybrid particdate system which retains the structural features and complementary advantages of liposomes and non-ionic hydrogel microspheres. This new hybrid system in the broadest sense also resembles biological cells in that it consists a iipid bilayer shell supported by a "cytoskeleton", in this case a swollen hydrogel core. Compared with liposomes, the present system is expected to have improved membrane stability, enhanced dmg loading capacity and stability for longer term storage. While at the same time, it is expected to possess a ceil-like surface which is flexible, ultra thin but highly impermeable to ions and lipophobic molecules. Flexibility of the supported bilayer is important for reconstitution of membrane proteins which often undergo conformation changes in a lipid bilayer environment [95]. The permeability bamer created by the supported bilayer ensures that loaded drug can be retained for a sufficient length of time before being released. The hydrogel core enclosed in a well packed, anchored lipid bilayer can also sustain an isolated aqueous interior, thus providing an ideal mode1 system to test the concept for a nurnber of potential applications. This aspect will be discussed Merin the next chapter. To veriQ these prospective properties, the lateral mobility and capability of the supported bilayer to entrap ions and lipophobic molecules was examined using the technique of fluorescent confocal microscopy (see Chapter 2). The self-repairing ability of the supported bilayer was also tested in a solution containing liposomes using a repeated disruption-reassembly protocol. 5.2 Materials and methods Dioleoyl phosphatidylcholine (DOPC),used for lipocoating the PVA bead surface, and 7- nitr0-2-l,3-benzoxadiazol-4-y1(NBA) labeled dioleoyl phosphatidylethanolamine (DOPE),a fluorescent labeled phospholipid, were purchased fiom Avanti Polar Lipids, the same source as described in Chapter 4. Fluo-3, a fluorescent Ca2+ indicator, and 4-bromo-A23187, a Ca2+ ionophore, were obtained fiom Molecular Probe. Lucifer yellow, which was used as a model lipophobic drug, was also supplied by Molecular Probe. For rneasuring lipid mobility and model àrug retention, a 100 mM KCl solution was used as the solvent system. For measuring membrane penneability to Ca2+ ions, a "calcium buf5errtcontaining 2mM CaC12, 1mM EGTA, 5mM HEPES and 140 mM KCl, and a "O calcium buffer" identical to the "calcium buffer" except without CaClz were used. Al1 rneasurements in which a fluorescent probe was involved were recorded on a MRC-600 laser confocal microscope (LSCM) using a filter with cut-off wave length of 480 nm. For measuring lipid lateral mobility in the supported bilayer, a photo-bleaching protocol was employed. First, the acylated PVA beads were treated with a liposome suspension containing 5% NBA labeled DOPE and 95% DOPC. Following the liposome suspension treatment and rinsing three times with the KCI solution, the resulting beads were placed under the LSCM for confocal imaging. The LSCM laser beam was parked over a portion of the bead for a few seconds to induce local photo-bleaching of the fluorescence probe. The recovery of the bleached part was monitored continually with LSCM. Figure 5-1 shows the instrumental set-up of the photo-bleaching experiment. The vertical dimension of the bleached area ittduced by parking a less focused laser beam on the surface is much larger than the thickness of the confocal plane which is imaged as a thin slice of the bead. For measuring the entraprnent of Ca2+ions, the acylated beads were first impregnated in a small amount of fluo-3 solution (200 FM), followed by adciing a liposome suspension prepared with the "calcium buffer". The concentration of fluo-3 after the liposome addition was 50 PM. Then a drop of this suspension of beads was examined under LSCM and diluted with the "O calcium buffer" by 5 folds. Confocal images were taken before and after the dilution as a function of time. As a control, unmodified PVA beads were subjected to identical treatment and measurement. For testing drug entrapment, a mode1 dmg, Lucifer yellow, was loaded by impregnating the acylated PVA beads in a Lucifer yellow solution (200 PM)in 100 mM KCl solution, followed by treatment with the liposome suspension prepared in the KC1 solution. The final concentration of Lucifer yellow after the liposomal treatment was 50 PM. Subsequently, the suspension of the PVA beads (now loaded with Lucifer yellow and coated with phospholipids) were examined by LSCM as for the calcium experiment mentioned above. Confocal images were taken before and after the suspension was diluted with 100 rnM KCl by 5 folds. Again, unmodified PVA beads were subjected to identical treatment and measurement. Lens Laser. Confocd plane Bleached area Bead coated with fluorescent labeled lipidî Photo-bleach protocol: 8 take the fist confocal image *park the laser beam on a portion of the coated surface take confocal images as a function of the after bleach. Figure 5-1. Protocol of photo-bleach experiment of a lipobead with a confocal microscope. 5.3 Preparation and stability of lipobeads Figure 5-2 illustrates one key observation during the liposomal treatment of the fatty acid acylated PVA beads. When acylated PVA beads were placed in a beaker of water, they aggregated and were floating at the surface due to their surface hydrophobicity and the water surface tension effect. However, when few drops of liposome suspension (prepared with DOPC) were added, followed by stirrhg, the PVA beads separated and sank to the bottom. This is a clear indication that the surface of the acylated beads had been successfûlly coated with the liposomal component (DOPC)and now has the hydrophilic head groups pointhg outward. + Liposomes Figure 5-2. Liposomd coating of fatty acid acylated PVA beads. The suspension of treated beads (lipobeads) cm be stored under nitrogen at room temperature for days without aggregation and floating back to the water surface. Removing the beads from water into air, however, caused darnage of the lipid coating as evidenced by the aggregation and floating of beads upon being placed in water again. However, these beads can be coated and made to sink by a few seconds of stimng in the liposomal suspension. This operation is quite reproducible. This result suggests a self-repairing capability of these lipobeads. 5.4 Lateral mobility of supported membrane Figure 5-3 shows the laser confocal images of the lipobeads containhg 5% NBA labeled phospholipids taken before and after the photo-bleach. The continuous ring of fluorescent emission before photo-bleach suggests that following lipocoating, liposomal phospholipids become evenly distributed over the lipobead surface. This observation is in keeping the considerable lateral mobility expected for lipid bilayers [96,97]. After the photo-bleach, an opening in the ring of fluorescent emission was observed (Fig. 5-3). This opening gradually closed over 15 minutes. Since photo-bleach of NBA is irreversible [97,98], the recovery is attributed to the diffision of labeled lipid molecuies through the bilayer into the bleached region. Figure 5-4B shows the time course of the fluorescence recovery in the bleached area. As shown in Fig. 5-1, the width of the bleached part in vertical dimension is rnuch larger than thickness of the confocal plane, thus the diffision process of labeled lipids into the bleached region can be approximated as an one dimensional (slab) mode1 (Figure 5-4A). By using a fiaction of release equation [99], MtMo = 4{Dt/icL2} (JI- 1) a diffusion coefficient @) of 1.54 x 10-9 cm2Isec can be calculated. The solid line is obtained when the value of D is incorporated into the equation. This value is within an order of magnitude as that observed in normal lipid bilayers 10-8 cm2/sec [96,97]. Details of the calculation will be described in Appendix II. Before bleach Righi after 2 min later 3 min Iater 5 min later 10 min later 1 5 mi 11 la ter Figure 5-3. Lateral mobility in hydrogel anchored lipid bilayer labeled with fluorescent phospholipid: Confocai images taken before and after a portion of lipobeqad surfec was photo-bleached. Time of capture is indicated below each image. B leac hed Confocd O 200 400 600 800 1000 Time / sec Figure 5-4 A: Feasibility of using slab model. B: Time course of recovery of fluorescence in the photo-bleached region. The solid line describes the expected recovery based on an early time estimate of the difiion coefficient [99]. 5.5 Permeability of supported membrane to ions Figure 5-5 illustrates the ability of lipobeads to trap ions. Figure 5-SA shows the fluorescence confocal image taken of lipobeads with Ca'+ and the fluorescent Ca2+ indicator, fluo- 3 [54], before and der a five-fold dilution of the lipobeads suspension with the "zero calcium" medium for a fmai fiee Ca2+ concentration of 60 nM. The fluorescent intensity of fluo-3 remained constant during the initiai 20 minutes of incubation but quickly decreased after adding a Ca2+ ionophore, 4-bromo-A23 187 [54], to bypass the pemeability bher created by the bilayer coating on the lipobeads. Adding the ionophore vehicle (methanol) alone to the same sample caused no change in fluorescent intensity, excluding the possibility that the bilayer membrane was simply disnipted by the solvent. We conclude that the decrease in fluorescence is due to facilitated Ca2+ diffusion across the bilayer membrane mediated by the ionophore. Figure 5-5B shows the confocal images of unmodified PVA beads loaded with Ca2+ and fluo-3. A five fold dilution with "zero calcium" medium caused the fluorescence intensity to decrease rapidly in the initial 2 minutes, followed by a more graduai decay until the fluorescent activity was totally lost (Fig. 5-5B). Since fluo-3 alone will cause some background fluorescent emission [54], the complete loss of fluorescent activity may be attributed to the loss of Ca2+ and fluo-3. These results indicate that the bilayer coating on the beads provides a permeability barrier to both ions and fluo-3. Lipobeads with surface modified by phospholipids were also examined for CaZf entrapment, and fond that loaded Ca*+ ions were released readily after dilution (Fig. 5-6). T'bis probably Ca iiiediuiii 20 min later 2 min later 20 inin later 100% v 45% 1% Dilution with "O Ca mediuin" Figure 5-5. Release of ca2+fiom lipobeads and bare PVA beads loaded with fluo-3. Numbers below the confocal images indicate relative fluorescent intensity. Tirne capture after dilution with non CI? buffer or addition of ionophore is indicated above l each image. Lipid layer on lipobeads prevent efflux until an ionophore is added, while bnre PVA bendç do not trap ca2+. 3 Dilution Before dilution 5 min later 20 min later Figure 5-6 Release of Ca2+ fiom phospholipid anchored üpobeads reflects the fact that the eEciency of adding phospholipid anchors to the xerogel surface was lower than that of adding fatty acid anchors (Fig. 3-7 & 3-8). In their swollen state, the surface area of the beads was enlarged by about 4 times, so that the coverage by the anchored phospholipids may be incornplete. For fatty acid modified lipobeads, however, the the lipid anchors are 3-4 times more than that of the phospholipids. In the water swollen state, these "extra" fatty acid anchors may move to the surface and align to an amy to fil1 the enlarged surface. This speculation, however, needs to be confimied. 5.6 Retention of model drug by supported membrane As discussed in chapter 1, non-ionic hydrogels possess a number of favorable properties such as biocompatible surfaces and chemically inert matrix. However, the release of small water soluble therapeutic compounds fiom this type of hydrophilic polymer matrix is often too fast [2]. The surface anchored lipid bilayer on the present lipobeads may provide a permeability barrier to render sufficient retention time for the loaded cornpound. The permeability of the supported bilayer to a model drug, Lucifer yellow, is demonstrated in Figure 5-7 and Figure 5-8. Lucifer yellow (MW = 458), a hydrophilic fluorescent dye, was loaded into both lipobeads and unmodified PVA beads, and examined by LSCM according to the procedures descnbed in Section 5.2. For the unmodified beads, the loaded Lucifer yellow was rapidly released and reached a plateau in a few minutes (Fig. 5-7 and 5-8). In the case of lipobeads, however, the fluorescent intensity decreased only slightly over 60 minutes. It is clear that the model drug was entrapped in the lipobeads by the lipid bilayer coating. As will be discussed later, the present lipobead system should be very useful for the delivery of many susceptible biopharmaceuticals such as polypeptides because the encapsulation process can be carried out without organic solvents and other contaminants. A PVA beads 20 40 60 Time (min) Figure 5-8 Decay of fluorescent intensity of Lucifer yellow loaded lipobeads and unmodified PVA beads as a function of the time after 5 fold dilution. 0:lipobeads with the surface pre- modified with fatty acids; A: unmodified PVA beads. 5.7 Summary of features of lipobeads We have demonstrated the structural features and a number of novel properties of lipobeads which are not found in other artificial micro-particdate systems. A surnmary of these properties are listed as follows. Lipobeads possess a structural similarïty to natural cells by having a lipid bilayer shell covalently anchored on a hydrated net work The anchored lipid bilayer has irnproved mechanical stability and self repairing capability, and can be self assembled with a wide variety of lipid compositions. Therapeutic reagents can be loaded into the hydrogel network by an absorptive process (impregnation) and encapsulated with a liposomal coating in an entirely aqueous environment. Therapeutic reagents cm also be loaded in the acylated hydrogel beads and stored in the dry form @fore liposornal coating) so that long term storage can be achieved. The hydrogel core is formed pnor to surface modification and liposomal coating, thus desired particle size and shape cm be better selected. The amhored lipid bilayer retains many properties of a fiee lipid bilayer, thus many technologies developed with liposomes can be applied to lipobeads. The lipocoat technology (defmed as hydrogel surface modification and liposomal coating) cm be applied to other hydrogels containing hydroxyls such as many natural and biodegradable polymers. 6. POTENTIAL APPLICATIONS OF LIPOBEADS AND FUTURE DIRECTION 6.1 The nature of the technology In the previous chapters, 1 have established a process termed "lipocoat" that modifies the surface of hydrogel beads in such a way that lipid bilayers will form spontaneously on their surface. This coating will protect the interior cornpartment of the bead and modiQ release of substances loaded into the bead. This process can be applied to the manufacture of products with applications for dmg and vaccine delivery, for hemoglobin carriers in artificial blood and for creating devices for predicting drug absorption and distribution. The process consists of a surface modification that covalently links amphipathic long-chah fatty acids or phospholipids via their polar ends to surface reactive groups of the hydrogel (e.g. ester linkages via surface hydroxyls). As a result, upon exposure to liposomes or other sources of lipids, the new surface hydrophobie groups will direct the spontaneous formation of a lipid biiayer coating. The assembly continues until the surface is completely coated and a continuous lipid vesicle now engulfs the bead creating a permeability barrier around the bead that has the same properties of a ce11 membrane. The hydrogel core acts as a scaffold, mimicking the role played by a cell's cytoskeleton. We have called this parîicular product "lipobeads". The naked lipo'oead precursor (acylated bead) cmbe stored in a stable xerogel or dried state, while the lipids can be stored in a lyophilized state. Addition of water is al1 that is required to drive the assembly of the product. This unique material system is expected to be potentially useful in several novel applications. 6.2. Controlled release drug delivery A number of particulate systerns have been explored by others for potentid drug delivery vehicles [16]. As a drug delivery system, non-ionic, water swelling, hydrogel microparticulates are superior to other particulate systems in that they are both biocompatible and have an inert interior, important properties for canying reactive therapeutic agents [2]. However, release of hydrophilic molecules (most biophannaceuticals fa11 in this category) from these carriers is often too fast to be usefid. Much effort has been invested in controllhg the release of substances from this material [3 11. The iipid bilayer anchored on the surface of Lipobeads is well packed (does not have defects or holes) and has al1 of the permeability properties of other lipid bilayers. It will entrap ions such as calcium despite a 1000 fold concentration gradient, but release that calcium on demand when the lipobead is exposed to a calcium carrier [61 1. Many other combinations of controlled release strategies can be used. Indeed the lipid coating of lipobeads will have the same permeability properties as liposomes and amenable to the same technology developed to control release fiom liposomes [17]. For example the release rate across the lipid bilayer permeability barriers can be controlled by selecting the types of Iipids used in generating the bilayer. These lipids cmVary according to their charge, chah length and phase transition temperature. Likewise al1 of the technology applied to engineering the interior of hydrogel dmg carriers can be incorporated into a lipobead device. Moreover, the naked lipobeads can be loaded just like any other hydrogel and then stored in a dried fom. Because chernical modification is restricted to the bead surface this will not interfere with the interior properties of the hydrogel particles. Also since water is the only reagent needed to drive the formation of lipobeads, contamination of the interior by organic solvents can be avoided. Lipobeads are as bio- compatible as liposomes and much more stable mechanically. They can thus serve as a depot delivery fodation. We expect that it will be possible to discover novel polymer cores that are biodegradable or at least recoverable making injectable formulations safer. 6.3 Delivery of synthetic polypeptide vaccines Synthetic vaccines are relatively small and hydrophilic polypeptides. They tend to be expensive and much effort is being applied to ensure their efficient delivery to the immune system. Optimum effects are found with slow delivery of small amounts. Much has been leamed about the rnovement of small peptides across lipid bilayers from bioavailability studies. This information can be applied directly to designing peptides and lipid coatings for the lipobeads with optimized release properties. 6.4 Red ce11 substitution Because of feus concerning the safety of the blood supply for operations, there is much interest in developing artificiai red ce11 substitutes or supplements. The problem is to develop an infusible preparation of an oxygen carier with properties similar to hemoglobin. As hemoglobin is readily available, a nurnber of polymer conjugates, polymer micro-capsules and liposomes are currently being studied as hemoglobin carriers [100]. Liposomes can encapsulate hemoglobin together with cofactors such as 2,3-DPG. They also have similar oxygen permeability properties to red cells. However, the amount of hemoglobin that cm be delivered by tolerated doses of liposomes is inadequate (low loading capacity). Also mechanical stability is poor and much hemoglobin is lost. Polymer microcapsdes are more stable but lack the ce11 like gas permeability properties of liposomes. Lipobeads will combine the advantages of both existing strategies in a synergistic manner. Hemoglobin cari be loaded at hi& capacities in the core and stored in the xerogel form until needed. The cell-like coating mimics both liposome and red-celi surfaces but is more stable than liposomes. In addition the shape and flexibility of the hydrogel core cmbe engineered to match that of red-cells. The larger size of lipobeads will reduce clearance by the reticulo-endothelid system, a major limitation of liposome formulations. 6.5 Application in assessrnent of dmg absorption and distribution For most therapeutic agents, their oral availability is limited by the rate of passive diffusion across the intestinal mucosa [101]. Drug discovery teams require rapid feedback about bioavailability of the lead compounds to direct their synthesis efforts and to plan animal testing. A typical animal test will require 25 mg of material and convincing evidence mut be generated to justify the expense of scaling up the synthesis procedure. An artificial system which offers a fast, simple, and reliable assay and at the sarne time only uses a small amount of material is, therefore highly advantageous. Two possible rate limit factors must be taken into account in estirnating drug absorption by passive diffusion through these mucosal walls: interaction of drug molecules with the polar head groups of the bilayer lipids (eg un-stirred water layer), and partition of the dmgs into the hydrophobic matrix of the bilayer [102]. The currently used systems, including octanowwater partitioning system [103] and octadecyl silica (ODS)chrornatography [104], can mode1 only the hydrophobic interaction with the membrane matrix, and therefore often lead to serious overestimation for hydrophobic dmg candidates. Liposomes [los] and immobilized artificial membranes (IAM) based chromatography [102] have been used to model both of the mechanisms. However, the liposomai method is experimentally tedious, tirne consumuig and with low repeatability for its lack of stability. The IAM method, on the other hand, overestimates significantly for very soluble molecules because what is being measured is related to the ease of entry into a hydrophobie environment and not tme tram-membrane transport. For this reason, the dnig retention the with IAM, which is the sole measurable parameter, reflects not only the membrane partition but also the hydrophilic adsorption ont0 the head groups. On the other hand, lipobeads potentially can be designed to measure tram-membrane flux. Moreover, the flux can be measured across a single bead thereby minimizing the amount of substance used. Furthemore, beads coated with membrane fragments ftom the tissue of interest will allow the incorporation of transporters into the assessment system. 6.6 Reconstitution of tram-membrane proteins Supported lipid bilayers on electrode and other solid substrate surfaces have been used as well defmed model bio-membranes to study membrane protein functions in bio-sensors and other bio-assays [38,40]. Applications of this type of supported membranes, however, are limited to partially associate membrane proteins. In the case of lipobeads and liposheets, the bilayers are anchored on a hydrated polymeric network, thus many tram-membrane proteins, such as ion channels and transporters, may be reconstituted. This may promote understanding of the mechanistic details of many ce11 surface phenomena by using fluorescent, electrochernical and spectroscopic techniques. 6.7 Future directions This work has established a proof-of-principle of the feasibility of a Iipid vesicle system supported on the surface of hydrogel beads as potential mode1 cells and drug carriers. As discussed in the previous chapters, this unique material system possesses a nurnber of advantages and potential applications as compared with other artificial particulate systems. Presently, following aspects of this technology required Merinvestigation and improvement. i) The supported bilayer structure needs to be Merexamined with better defined physical methods (say electron microscopie or X-ray ciiffiaction techniques). ii) Its ability to reconstitute trans-membrane proteins needs to be tested. iii) Development the supported vesicle system with bio-degradable hydrogel core is critical to exploring many of the potential applications discussed in the previous chapter. iv) The relationship between lipid composition and membrane properties (including stability, permeability, surface charge, biocompatibility, and sensitivity to chernical and biological stimuli) should be established with a systematic shidy. v) For red cell replacement, the effects of the hydrogel network on conformation and conformation change of entrapped proteins should be elucidated. For IV administration, in addition to the need to develop biodegradable core, rational surface modification in order to extend blood circulation time of lipobeads needs to be developed. vii) Refine the bead preparation procedure to achieve a narrow size distribution in designed range. APPENDICES 1 Preparation of glycerol phosphatidyl ethanolamine (GPE) GPE was a component in ou attempt at grafted synthesis of phosphatidyl ethanolamine (PE) on the surface of PVA xerogel beads. As the chernical is expensive, it was synthes~edin home. The synthetic scheme, based on a published method [78], is shown in Figure 1-1 Figure 1-1 Reaction scheme for the preparation of GPE [78]. Fkst, equivalent soketal (mixed with Et3N and THF) was added stepwise into C13P0 at OaC. Mer the reactant solution was warmed up to room temperature, equivalent ethanolamine (mixed with Et3N and THF) was added stepwise. Finally, the reactant solution was filtrated (rernoving the precipitate with Et3N) and added with solution of HCI. The product was purified chromatographically ushg a silica gel column with methano1 as mobile phase. 2D NMR spectroscopy was used to confirm the preparation of GPE. The spectrum is shown in Figure 1-la. Chernical shifts are listed in Table 1- 1. The protons at 6 3 17 and 409 are present in similar amount (2:2) and correlated. They are,therefore assigned to those bonded to the two adjacent carbons of the ethanolamine group. The chemical shifis at S 3 59 6 379 and 392 (with the ratio of peak area as 2: 1:2) are correlated, indicating that these protons are bonded to the three adjacent carbons of the glycerol group. The chemical shifts centered at 6 409 and 6 392 consist of very complex hyper-structures, that is simplified by phosphorus-decoupling (Figure 1-1 b), a routine operation used for measuring phosphate involved compounds, indicating that these protons are bonded to each carbon of the ethanolamine and glycerol groups next to the phosphate group, respectively. The two correlated shifts centered at 6 3.69 and 4.19 have a proton ratio of 4: 1 (with the high field one (6 4.19, 1H) possessing a hyper structure) are attributed to the isomer of GPE in which the secondary hydroxyl is esterified with the phosphate group (Fig. 1- 1b). The fraction of this isomer is about 10- 12%. The chemical shih at 6 3.30 and 3.34 are due to protonated impurity of the solvent (CH30H). Table 1-1 Chemical shifts of GPE in NMR spectrum Center of chem. shif? (6) Features Arnount (as proton #) Chemical species 3.17 triple 2H P-O-CH2CH2NH2 3.59 multiple 2H P-O-CH2CHOHCA20H 3.79 multiple 1H P-O-CH2CHOHCH20H 3.92 multiple 2H P-O-CH2CHOHCH20H 4.09 multiple 2H P-O-CH2CH2NH2 (r( rn JI*m Decoupled Figure 1-2b Phosphorus-decoupled NMR spectra of GPE synthesized II Estimate of surface covenige of PVA by anchored phospholipids based on angle resolved XPS. Depth distribution of gruied species As illustrated in Fig. 3-14, for molecular grafting of a polymer surface, the reaction rnay not necessarily Iirnited on the surface. The molecules to be grafied may penetrate into the polymer network and react with the sites in sub-surface or deeper region of the substrate. These penetrated species, if within XPS sampling depth (-3h), will also be sampled when subjected to XPS measurement, and their contribution to the detected XPS intensity depends on how deep these species are Iocated fiom the surface: (II- 1) where ibx is the contribution of species b at depth of x to the XPS intensity, iM, is that of species b at zero depth. (ibx and iboare proportional to N and No in equation 3- 1, respectively). The detected XPS intensity of a component does not oniy relate to the amount of detected elements, but also to their distribution over the sampling depth. To estimate the surface abundance of the grafied species by XPS, therefore, it is necessary to take the depth distribution of the species into account. This information cmbe obtained by an angle resolved XPS measurernent. In Section 3.5, the ratio between the nitrogen contents detected by XPS at sampling angle of 45' and that of 90' is 1.4. This value, which is close to the theoretical maximum (l/sin9 = 1.414, see Fig. 3-12A) for a layer of zero thickness on a surface, indicates that the majority of the nitrogen atoms (a component of grafted GPE at step III of Fig. 3-2) are dispersed within a narrow region near the surface (see the thicker cwein Figure 11-1). Therefore, an approximation that simplifies the depth distribution of GPE introduced in reaction step III (See Chapter 3.3) with a Distribution of added elernent Depth / A Figure 11-1 Possible depth distribution of grafted molecule in the surface region of the substrate. Thicker curve: the possible distribution; thinner line: approximation of the thicker curve; d: thickness of the layer where GPE is grafled. step function as shown in Figure 11-1 (the thin line) to represent the spread of grafted species over the surface region of the substrate within a certain thickness should be appropriate. In either case that the content of grafted GPE gradually decays versus the depth or the GPE disperses in a broad region fiom the surface (Fig.3-14B), the ratio between 45' and 90' sarnpling will be significantly less than 1.41 4. Even if an error may be introduced by the approximation, it will generally over estimate on thickness and lower the estimated surface coverage. Therefore, if the calculated thickness is aiready smdl and coverage is already high (approaches 100%) bbased on the approximation, the error will be smdl. Mathemaricc2 derivation Before presenting the mathematical denvation, some tenns need to be defiend. Noxmalized XPS intensity by the substrate elements at the dace im Normahd XPS intensity by the element of added species at the surface la Relative amount of substrate elements detected by XPS. Ib Relative amount of grafted elements detected by XPS. d Thiclaiess of the added species fkom the dace(in A) A Escape depth (in A) 0 Sampling angle Fb(8) Elementai hction of grafted species as measured by XPS at sampling angle 8 From equation II- 1 and Fig. 11-1, the relative amount of &ed element (b) and substrate element (a) detected by XPS can be described by following equations. X dI a- I. = LOJ~e adedr+ (to + iso)j e d d d P = iaoAsin8(1-e Me)+(Lo+ibo)Asin& In the case that 8 = 90°, equations (11-2) and (II-3) can be simplified as, d Zb(90°) = id(1- eT) d d L(90') = Loil(l- eT) + (bo + &)hi -d If we set y = e A, then equations (II-4), (II-5), (II-6) and (11-7) will be, The detected XPS fiaction of grafted element for 90' and 45' sampling will be, Then, the ratio of the fiactions of elements between the two sampling engles can be given by cornparison of equation (II-1 3) over (II-12), followed by simplification: d Recail that y = e-l and (L+ ibo) = 1, then Since Fb(45') and Fb(90') are measured value and h is a constant, the sole variable, 4 should be able to resolved from equation (II-15). Equation (II- 15) also indicates that the ratio Fb(4S0)/Fb(90') is determined ody by the thickness of the layer where the grafted species dispersed in, and is independent of the surface coverage. It should be noted that in above discussion we have introduced an approaximation regarciing the escape depth h To simpli& the denvation, we used an average h for C(ls), O(1s). This approximation is vaiid since later calculation shows that the values of escape depth for the Is orbital electrons of nitrogen, carbon and oxygen are suficiently close. It is aiso valid to use normalized iao and ibo in the above calculation because the measured values, Fb(45') and Fb(90) are fiactions of each element. Surface coverage is defined as ibo/(ibo + iao). Since the thickness of the layer of grafted species, d, can be obtained from Fb(6')/Fb(90'), a measured value,the two variable ibo can be calculated fiom equation (II- 16) or (II- 17). Stoichiometrical analysis In chemical modification of polymer surfaces, both substrates and introduced species are not simple substances but molecules or repeated hgments with multiple elements. On the other hand, however, XPS is an anaiytical tool providing elemental information. To detennine the surface morphology with XPS, chemical structure of grafted species and substrates must be taken into account. After reaction step III (Chapter 3), three types of repeating units as shown in the following scherne may exist in the region accessible to the reac tion: Ç-OH CH2 1 Where A is the unreached nydroxyl; B is the hydroxyl linked with a glutaric acid after reaction step 1; and C is the surface hydroxyl linked with a glutaric acid and a GPE after reaction step III. (Reaction step II is for activation the carboxylic group formed in step 1). The elemental content of the species above are listed under each chemicd formula of the species. For surface analysis with XPS, nitrogen is used as an indicator. The elemental fractions obtained fiom XPS should be re-grouped according to the chemicd formulas and the elemental contents of each species. For each of species C which is indicated by one nitrogen, there are 13 carbons, 9 oxygens and one phosphorus as well. Based on the ATR-FTIR result show in Fig. 3-1 0, species B is approximately three times as much as species C, thus for each nitrogen, there are three species B which contain 3 x 8 = 24 carbons and 3 x 4 = 12 oxygens. Species A contains two carbons and one oxygen, and its quantity relative to species C is to be determined fiom XPS measurements and the calculations discussed above. The population of phospholipids, which is related to GPE (Fig 3-2), is defined as C/(A + B + C), while the surface coverage of hydroxyls is defined as (A + B)/(A + B + C). Later calculation will show that species B are al1 located below sub-surface region. Calculation of escape depth A According to equation (II-2), escape depth A is the distance of a material through which 63% of photoelectrons can penetrate without decay of kinetic energy. This is a measure of the easiness of a matenai that photoelectron cm pass through. For organic polymers, the value of h is dependent on the molecular weight of repeat unit (M), number of valence electrons (n), density of the material (p) and kinetic energy of the photoelectron (Ek).h can be calculated with an equation developed by Ashley [106]: )C = (lVIEk/pn)/[l 3 .6h(Ek)- 17.6 - 1400/Ek] (II- 1 8) For repeating unit A (PVA), the parameters in equation (1-18) have following values, respectively , M= 44 n = 18 P = 1.27 (see Chapter 2) Then, Mm)= 1.93 For repeating unit C, M= 367 n = 140 P = Assume same to PVA. M/(pn) = 2.02 For approximation, we chose M/(pn) = 2.00. The beam energy of X-ray Eh = 1253 eV (See Chapter 3) Since the binding energy of C(ls), N(1s) and O(1s) are 285, 400 and 53 1 eV, respectively, the kinetic energy for the elements are Ek(C, 1s) = 1253 - 285 = 968 eV Ek(N,l s) = 1253 - 400 = 853 eV Ek(0,I s) = 1253 - 53 1 = 722 eV By incorporating the values of the related parameters into equation (11-1 8), escape depth for the related elements can be obtained as following k(N-1s) = 24 A h(C-Is)= 26A k(O-ls)= 21A Because for each of the three species, the ratio of carbon over oxygen is stoichiometrical, we use carbon signal as the indicator for the substrate and nitrogen for species C, and use the average value h = 25 A to approximate h(N-1 s) and h(C- 1s). For the same reason, Fb(45') and Fb(90°) here represent the fraction of measured nitrogen of the surn of nitrogen and carbon, respectively. By incorporating the data in Table 3-2 into equation (1- 17), the fiaction of nitrogen derstep III is Fb(90a)= 1.3%/(1.3% + 66.2%) = 1.9% (II- 19) Here we set nitrogen intensity as Ib and carbon intensity as Ia. Incorporating the h value and Fb(45')/Fb(9o0) ratio (See Table 3-3) into equation 01-15), we have Fb(4S0)/Fb(90")= (i-e-1.414a25)/(~-ed25) = 1.40 01-20) The limit of the ratio (1 -e-r.414d/25)/(l-e-d25) as d approaches O is 1.4 14. This agrees with the physical mode1 that if al1 the grafted elements are distributed at the surface with depth of zero, the ratio of detected content of the element between two sampling angles (8,and e2) equds the reverse ratio of the sine of the two angles (sinWsin0 1). With a numerical solution based on equation (LIS), d can be detennined as 3 A, eg. the nitrogen introduced with GPE is distributed within a layer of 3 A thick nom the surface. XPS measurement is known to have an accuracy withh 10% [68]. This resdts in Fb(45')/Fb(90e) varying fiom 1.27 to 1.414 and d from 2 to 12 A. For later calculation, we take the average value of d, 7 A. To estimate the surface coverage by GPE, the values for d, 5 and Fb(0) (see equation II- 18) have been incorporated into equation (11-17) to obtain the surface fraction of nitrogen, ibo, (The height of the cwein Fig, 11-1). Based on equation 01-2) and (II-19), the measured nitrogen fraction, N/(N + C), is, Fb(90°) = ib~(l-e-~)= 1.9% (II-2 1) in which the nitrogen content refers to those distributed within the 7A surface layer, and carbon is the suof those in both of the surface region (74and the buiky structure (See Fig. 3-12). Based on equation (I-21), the nitrogen fiaction within the surface region, bo, is therefore, ibo = ~b(90')/[i-e-dk) = 1.9%/(1-e-7/25) = 7.7% (11-22) Since for each nitrogen refers to species C which contains 12 carbon atoms, there are 92.4% of carbon content assigned to.species C corresponding to 7.7% of nitrogen. Since 7.7% + 92.4% = 100%, species C dominates the population in the region of 7A thick fiom the surface. Although the species B (which is the glutaric spacer) present 3-4 times as abundant as species C in total according to ATR-MR (Fig. 3-7), it may be located below the surface region. Although the nurnbers calcuiated above may not be quantitative, the repeatable high ratio of Fb(45')/Fb(90e) strongly suggests that the grafting of GPE only occurred at the surface region. On the other hand, the observed nitrogen content (Table 3-2), if distributed at surface ody, leads to a conclusion that al1 the surface hydroxyls were used for grafting GPE. III Calculation of ciifhision coefficient of lipid molecules along supported lipid bilayer. According to the illustration in Chapter 5.4 and Fig. 5-4 and , the diffusion process of Iabeled lipids into the bleached region can be approximated as an one dimensional (slab) model. For a monolithic solution system [93], the mathematical solution of the diffusion process based on Fick's first law reduces to two approximations which are valid for different parts of the diffusion curve: For early tirne (O < MdMo 0.6), MdMo = 4{DthL*) Il* (III- 1) For late time (0.4 MMo5 1.O) MtlMo = 1 - 8/n**exp(-n*Dt/L2) 011-2) where Mt/Mo is the portion which has difised into or out of a slab with thickness of L at the t; D stands for diffusion coeficient and t is time in second. The thickness L is measured from confocal message (Fig. 5-3) to be 25 p.Equation (111-1) and (111-2) can be re-written as: (MuM0)2(~L2)/16= Dt (1113) L*/rln(x2/8.(1 -Mt/Mo)) = Dt (nI-4) Here, diffusion coefficient D became the slope of equation (III -3) and (III-4). By using equation (111-3) for early time approximation, Da,,, can easily be calcdated based on 5 early date points listed in following. With a linear least square regression, the slope Dearlywas optimized to be 0.150 pd/sec. 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