Dispersed and deposited polyelectrolyte complexes and their interactions to chiral compounds and proteins Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von M. Sc. Wuye Ouyang geboren am 01.05.1979 in Zhenjiang, V.R. China Gutachter : Prof. Dr. Brigitte Voit Prof. Dr. Thomas Wolff Prof. Dr. Klaus D. Jandt Eingereicht am : 10.10.2008 Tag der Verteidigung: 14.01.2009 ABBREVIATION AFM Atomic force microscopy ASC Ascorbic acid ATR-FTIR Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy CD Circular dichroism COAC Coacervate phase CSA Camphorsulfonic acid CSP Chiral stationary phases D2O Heavy water / Deuterium oxide DLS Dynamic light scattering DMF Dimethylformamide EtOH Ethanol GA Glutardialdehyde GLU Glutamic acid H2SO4 Sulfuric acid H2O2 Hydrogen peroxide HCl Hydrochloric acid HSA Human serum albumin IEP Isoelectric point IR Infrared IRE Internal reflection element LB Langmuir-Blodgett film LBL Layer by layer LYZ Lysozyme MYO Myoglobin NaCl Sodium chloride NaClO4 Sodium perchlorate NaOH Sodium hydroxide i NC Nitrocellulose PANT Pantothenic acid PBS Phosphate buffer saline PCD Particle charge detector PDADMAC Poly(diallyldimethylammonium chloride) PDL Poly(D-lysine) PDI Polydispersity index PEC Polyelectrolyte complex nanoparticle PEC-0.66 Positively charged polyelectrolyte complex nanoparticle (n-/n+ = 0.66) PEC-1.50 Negatively charged polyelectrolyte complex nanoparticle (n-/n+ = 1.50) PEI Poly(ethyleneimine) PEI-m Poly(ethyleneimine-maltose) PEL Polyelectrolyte PEM Polyelectrolyte multilayer PLL Poly(L-lysine) PMAH-MS Poly(maleic anhydride-co-α-methylstyrene) PMA-MS Poly(maleic acid-co-α-methylstyrene) PREC Precipitate phase PSS Poly(styrenesulfonic acid) sodium salt PHE Phenylalanine PTFE Poly(tetrafluoroethylene) PVP Poly(4-vinylpridine) PVP-R* Poly(N-(S)alkylated-4-vinyl pyridinium iodide) PVS Potassium poly(vinylsulfate) R Dichroic ratio SF Scaling factor Si Silicon SE Enantiospecificity SEM Scanning electron microscope SUP Supernatant phase ii TYP Tryptophan TYR Tyrosine UV-Vis Ultraviolet/Visible (Spectroscopy) iii TABLE OF CONTENTS Motivation and Objective 1 1. Introduction and theory 3 1.1 Definition, properties and applications of polyelectrolytes 3 1.1.1 Physical properties of polyelectrolytes in solution 4 1.1.2 Polypeptides 5 1.1.3 Applications of polyelectrolytes 6 1.2 Polyelectrolyte multilayers 6 1.2.1 Layer by Layer self assembled polyelectrolyte multilayers 7 1.2.2 Formation of polyelectrolyte multilayers 9 1.2.3 Driving force and phase diagram of polyelectrolyte multilayers 11 1.2.4 Applications of polyelectrolyte multilayers 13 1.3 Polyelectrolyte complex nanoparticles 13 1.3.1 Formation of dispersed polyelectrolyte complexes 14 1.3.2 Applications of PECs in solution 17 1.4 Theory and methodology of chiral separation 17 1.4.1 Definition of chirality 17 1.4.2 Significance of chiral separation 18 1.4.3 Methodology of chiral separation 19 1.4.4 Principle of chiral recognition 22 1.5 Protein adsorption 25 1.5.1 Protein structure 25 1.5.2 Protein adsorption process 27 1.5.3 Factors for protein adsorption 27 2. Experimental 29 2.1 Chemical reagents 29 2.2 Preparation protocol 32 2.2.1 Polyelectrolytes synthesis 32 iv 2.2.2 Polyelectrolyte multilayer (PEM) deposition 32 2.2.3 Enantiospecific interaction to PEM 34 2.2.4 Membrane modification and enantiospecific permeation 35 2.2.6 Preparation of PEC dispersions 37 2.2.6 Preparation of PEC/protein dispersions 38 2.2.7 PEC particle films 39 2.2.8 Preparation of PEC/chiral probe conjugates 39 2.3 Instruments & Characterization 40 2.3.1 Attenuated total reflectance - fourier transform infrared spectroscopy 40 2.3.2 Colloid titration 43 2.3.3 Gravimetry 43 2.3.4 Dynamic light scattering 43 2.3.5 Circular dichroism 44 2.3.6 Atomic force microscopy 45 2.3.7 Ultraviolet/visible spectroscopy 47 2.3.8 Scanning electron microscope 48 3. Result and discussion 49 3.1 Polyelectrolyte multilayers 49 3.1.1 Polyelectrolyte multilayer deposition 49 3.1.1.1 PEM-PEI/PVS 49 3.1.1.2 PEM-PLL/PVS 51 3.1.1.3 PEM-PLL/PSS 54 3.1.1.4 PEM-PEI-maltose/PVS 63 3.1.1.5 PEM-PVP-R*/PVS (PSS) 65 3.1.1.6 Comparison of multilayer thickness 68 3.1.2 Enantiospecific interaction to polyelectrolyte multilayers 69 3.1.2.1 Properties of polyelectrolyte multilayer in contact with enantiomers 69 3.1.2.2 Influence of polyelectrolyte structure: chiral or non-chiral 75 3.1.2.3 Influence of aqueous medium 77 v 3.1.2.4 Influence of thickness 79 3.1.2.5 Influence of PEM types 81 3.1.2.6 Influence of orientation 84 3.1.2.7 Enantiospecficity of non-peptidic PEM 85 3.1.2.8 Enantiospecificty for other chiral probes 87 3.1.2.9 Chiral PEM modified membrane 88 3.1.3 Protein adsorption at chiral PEMs 91 3.1.4 Summary 92 3.2 Polyelectrolyte complex dispersion 94 3.2.1 Properties of PEC particles 94 3.2.1.1 Charge and mass of PEC dispersion 94 3.2.1.2 Size of PEC dispersion 95 3.2.1.3 Morphology of PEC dispersion 97 3.2.2 Properties of PEC/protein conjugates 99 3.2.2.1 Charge and mass of PEC/protein conjugates 100 3.2.2.2 Protein loading ability of PEC dispersion 101 3.2.2.3 Size of PEC/protein conjugates 103 3.2.2.4 Morphology of PEC/protein conjugates 105 3.2.2.5 Protein adsorption on PEC films 107 3.2.3 Enantiosepecific adsorption on PEC dispersion 109 3.2.4 Summary 110 Conclusion 112 Appendix 115 References 117 vi Motivation and Objective MOTIVATION AND OBJECTIVE Polyelectrolyte complexation is a rapidly growing field with applications in functional multilayer and nanoparticle generation. Polyelectrolyte complexes can be formed by consecutive polycation/polyanion adsorption (layer-by-layer self assembly) on the surface to get polyelectrolyte multilayer (PEM) films or the mixing of polycation/polyanion in solution to get polyelectrolyte complex (PEC) nanoparticles. PEM films and PEC particles are interesting and low cost products and their advantages (e.g. biocompatible) result in various applications in aqueous solution, especially in biological and biomedical fields. Therefore, the objectives in this study are to explore the specific interaction of PEM films and PEC particles with relevant probes. Low molecular chiral compounds and proteins are selected as probes to study their specific binding behaviour. Concerning chiral compounds, due to the rapidly growing demands for preparing optically pure compounds in the biomedical and pharmaceutical fields, scientists devote an increasing attention to find new enantioseparation techniques. The advantages of polyelectrolyte complexes (e.g. high stability, low producing cost, easy preparation) reveal that such ultrathin film and particles could be potential candidates for enantioseparation. [1] Hence, the first task of this work is to investigate the chiral discrimination using polyelectrolyte complexes (PEM and PEC) consisting of chiral polyelectrolytes. Homo-polypeptides (e.g. poly-L-lysine) consisting of a chiral amino acid and featuring high infrared sensitivity and some synthetic chiral polyelectrolytes are used as chiral selectors in the polyelectrolyte complexes to explore the chiral recognition. Low molecular weight compounds (e.g. amino acids, vitamins, drugs) are selected as chiral probes for the enantiospecific interaction. Further applications of such chiral recognition using PEM are explored. Concerning proteins, which are biomacromolecules made of numerous chiral amino acid units, huge interests are related to their binding under conservation of structure and function. Herein, they are chosen for the enantiospecific adsorption on the PEM containing different chirality. Because of the increasing interests in exploring new carriers for drugs, proteins and DNA, modal proteins are also used to evaluate the binding properties of dispersed 1 Motivation and Objective polyelectrolyte complex (PEC) nanoparticles. Different PEC systems and different proteins are tested and compared. The experimental routes to fulfil the objectives of this work are listed in the following (Scheme i): • Preparation of chiral and non-chiral PEMs using the Layer-by-Layer self assembly technique, which are recorded and analysed by ATR-FTIR and AFM. • Enantiospecific interaction of chiral probes, e.g. amino acid, vitamin, drug and protein on the PEMs. ATR-FTIR is the main analysis method for the evaluation of enantiospecificity. • Preparation of chiral and non-chiral polyelectrolyte complex (PEC) nanoparticles using mixing and centrifugation refinement technique. DLS, CD, titration etc. are applied for the characterization. • Enantiospecific interaction of low molecular weight chiral probes to PEC dispersions and protein binding to PEC dispersions. DLS and CD are the main methods for detecting the uptake of chiral probes and proteins. L L L L D L D D L L-Form probe L L L L + D-Form probe D L L L D L L L D Mixing and Polycation Layer-by-Layer centrifugation depostion + Polyanion P P P P P P P P P P + Protein P P P P P P P P Scheme i. Overview of the experimental routes in this work. 2 Introduction and Theroy CHAPTER 1 INTRODUCTION AND THEORY 1.1 Definition, properties and applications of polyelectrolytes Polyelectrolytes (PEL), by definition, are macromolecular species, which have repeating units bearing charged groups. These groups dissociate in the aqueous solution (water) forming a positively or negatively charged polymer chain and oppositely charged low molecule counterions. According to the types of electrolyte group, PEL can be classified into three parts, cationic PEL, anionic PEL and zwitterionic PEL. Poly(ethyleneimine), poly(L-lysine), etc. belong to cationic PEL; poly(acrylic acid), alginic acid etc belong to anionic PEL; proteins belong to zwitterionic PEL. PEL also can be further classified into: strong PEL, e.g. poly(vinylsulfate), whose charge is pH independent and weak PEL, e.g.