Bio- and Multifunctional Polymer Architectures E-Book

Brigitte Voit

• Herausgeber: John Wiley & Sons
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 491

Veröffentlichungsjahr: 2016

Beschreibung

This reference/text addresses concepts and synthetic techniques for the preparation of polymers for state-of-the-art use in biomedicine, synthetic biology, and bionanotechnology.

Leseprobe

Table of Contents

COVER

TITLE PAGE

PREFACE

ACKNOWLEDGMENTS

1 INTRODUCTION

1.1 WHAT MAKES POLYMERS SO INTERESTING?

1.2 MACROMOLECULAR ENGINEERING AND NANOSTRUCTURE FORMATION

1.3 SPECIFIC NEEDS IN BIONANOTECHNOLOGY AND BIOMEDICINE

REFERENCE

2 TERMINOLOGY

2.1 POLYMER ARCHITECTURES

2.2 MULTIFUNCTIONALITY

2.3 BIOCONJUGATES

2.4 BIOCOMPATIBILITY

2.5 BIODEGRADATION

2.6 BIOACTIVITY

2.7 MULTIVALENCY

2.8 BIONANOTECHNOLOGY

REFERENCES

3 PREPARATION METHODS AND TOOLS

3.1 GENERAL ASPECTS OF POLYMER SYNTHESIS

3.2 CONTROLLED POLYMER SYNTHESIS

3.3 EFFECTIVE POLYMER ANALOGOUS REACTIONS

3.4 PEGYLATION

3.5 BIOCONJUGATION

3.6 ENZYMATIC POLYMER SYNTHESIS

3.7 SOLID PHASE SYNTHESIS AND BIOTECHNOLOGICAL APPROACHES

3.8 HYDROGELS AND HYDROGEL SCAFFOLDS

3.9 SURFACE MODIFICATION AND FILM PREPARATION

3.10 MICROENGINEERING OF POLYMERS AND POLYMERIC SURFACES

REFERENCES

4 ANALYTICAL METHODS

4.1 MOLECULAR STRUCTURE AND MOLAR MASS DETERMINATION OF POLYMERS AND BIOHYBRIDS

4.2 CHARACTERIZATION OF AGGREGATES AND ASSEMBLIES

4.3 CHARACTERIZATION OF HYDROGEL NETWORKS

4.4 SURFACE CHARACTERIZATION

4.5 BIOPHYSICAL CHARACTERIZATION AND BIOCOMPATIBILITY

REFERENCES

5 MULTIFUNCTIONAL POLYMER ARCHITECTURES

5.1 MULTIFUNCTIONAL (BLOCK) COPOLYMERS

5.2 DENDRITIC POLYMERS

5.3 GLYCOPOLYMERS

5.4 PEPTIDE-BASED STRUCTURES

5.5 BIOHYBRID HYDROGELS

REFERENCES

6 FUNCTIONAL MATERIALS AND APPLIED SYSTEMS

6.1 ORGANIC NANOPARTICLES AND AGGREGATES FOR DRUG AND GENE DELIVERY

6.2 POLYMER THERAPEUTICS AND TARGETING APPROACHES

6.3 MULTI- AND POLYVALENT POLYMERIC ARCHITECTURES

6.4. BIORESPONSIVE NETWORKS

6.5 BIOFUNCTIONAL SURFACES

REFERENCES

ABBREVIATIONS

INDEX

END USER LICENSE AGREEMENT

List of Tables

Chapter 03

Table 3.1 Differences and Common Features of Living Anionic and Controlled Radical Polymerization Processes (Kinetic Constants According to Fig. 3.4)

Table 3.2 PEGylated Proteins Currently on the Market

Table 3.3 Classification and Examples of Enzymes as Well as Typical Polymers Synthesized by

In Vitro

Enzymatic Catalysis [49]

Table 3.4 Summary of Surface Modification and Film Preparation Techniques

Table 3.5 Methods to Structure Polymers or Polymer Surfaces by Microengineering Techniques

Chapter 04

Table 4.1 Molecular Structure and Molar Mass Determination: Important Polymer Characteristics and Important Analytical Methods for Their Determination

Table 4.2 Assignment of Diagnostic CD [5] and IR Spectral Data [6, 7] on Protein Solutions (H

2

O) to Typical Secondary Structure Fractions

Table 4.3 Surface Characterization Methods and Information Gained

Table 4.4 Characterization of Adsorbed Proteins

Table 4.5 Structure of ISO 10993

Table 4.6

In Vitro

Hemocompatibility Assays

Chapter 06

Table 6.1 Examples of Polymer-Based Nanoconjugates Used Clinically

Table 6.2 Bioactive Molecules

List of Illustrations

Chapter 01

Figure 1.1 Structure and form of polymer chains in solution and melt (top) and possible ordered bulk structures (bottom).

Figure 1.2 Supramolecular aggregate formation and drug encapsulation of dendritic core–multishell architectures (left) and cryo-TEM structure elucidation of the formed drug complexes (right).

Figure 1.3 Scheme of biohybrid starPEG–glycosaminoglycan networks for recapitulating and modulating cell-instructive ECM signals.

Chapter 02

Figure 2.1 Schematic representation of linear homopolymer formation from one type of monomer unit and the possibly resulting coil structure; supplemented by the examples of polystyrene and poly(ε-caprolactam) (or nylon 6).

Figure 2.2 Monomer arrangements in linear homopolymer chains.

Figure 2.3 Schematic representation of linear polymers, short- and long-chain branching (brush) polymers, star-type polymers, and cross-linked polymer chains. The dendrimer structure on the right is an extreme form of highly short-chain branched macromolecules.

Figure 2.4 Examples of homo- and copolymer architectures.

Figure 2.5 The mixture of macromolecules of chains with different degrees of polymerization results in a distribution curve. These can be monomodal (broad or narrow), multimodal, or bimodal.

Figure 2.6 Schematic representation of possible bioconjugate structures; top: protein—linear synthetic polymer chain conjugate, bottom: synthetic polymer chain conjugated in the side chain with dye or drug molecules, for example, oligonucleotides.

Figure 2.7 Schematic representation of the concept of biomolecular recognition exemplified for enzyme–substrate interactions. Note that the interaction involves a combination of geometrically matching intermolecular forces that are additionally modulated by thermodynamic and kinetic effects.

Figure 2.8 Comparison between monovalent and multivalent interactions of multifunctional polymers with biological receptors, for example, proteins.

Figure 2.9 Schematic representation of a magnetic nanoparticle with the protective polymer layer and attached targeting, therapeutic, or imaging moieties.

Figure 2.10 (a) The concept of DNA origami for the fabrication of 2D and 3D nanostructures; (b) Examples of realized structures through DNA assembly.

Chapter 03

Figure 3.1 Classification of reaction mechanisms used to build up polymers.

Figure 3.2 Schematic representation of the development of molar mass, for example,

M

n

, with monomer conversion for chain growth (free radical), chain growth (living) and step growth mechanisms for the polymer buildup reaction.

Figure 3.3 Examples of commercially important polymers prepared by chain growth processes (Note: PVA is formed from poly(vinyl acetate) after an additional polymer analogous reaction).

Figure 3.4 Polymerization steps and kinetic equations relevant in the chain growth polymerization exemplified for the free radical polymerization of styrene initiated with azobisisobutyronitrile (AIBN) (

k

index

denote the kinetic constants for decomposition (d), initiation (i), propagation (p), termination (t), and transfer (tr)).

Figure 3.5 Examples of commercially important polymers prepared by step growth polymerization.

Figure 3.6 Cationic ring-opening polymerization (CROP) of

meso

-lactic acid dimer toward poly(

D

,

L

-lactide).

Figure 3.7 Synthesis of poly(vinyl alcohol) by hydrolysis of poly(vinyl acetate).

Figure 3.8 Living anionic polymerization of styrene initiated by

n

-butyllithium (

n

BuLi).

Figure 3.9 Important monomers for anionic polymerizations.

Figure 3.10 Formation of the distyryl dianion as bifunctional initiator for the anionic polymerization.

Figure 3.11 Poly(ethylene glycol) synthesis having active functional end groups (Nu

−

, nucleophile or OH

−

; E

+

, electrophile or proton).

Figure 3.12 Anionic

N

-carboxyanhydride (NCA) polymerization toward polypeptides.

Figure 3.13 Typical monomers for the cationic polymerizations.

Figure 3.14 Typical initiation reaction of isobutylene via protonation and the two main types of products with unsaturated end groups formed.

Figure 3.15 Cationic polymerization of aziridine toward highly branched poly- (ethylene imine).

Figure 3.16 Monomer synthesis and cationic polymerization of oxazolines (Nu

−

 = nucleophile, e.g., amine) followed by acid hydrolysis toward linear poly- (ethylene imine) for R = Me, Et.

Figure 3.17 General principles of controlled radical polymerization reducing the concentration of active radicals.

Figure 3.18 Simplified mechanism of ATRP.

Figure 3.19 General concept of NMRP highlighted using TEMPO as stable radical and some examples for advanced stable nitroxide radicals and Hawker adducts.

Figure 3.20 RAFT polymerization process based on a dithioester RAFT agent and some examples of RAFT agents (Z = modifies addition and fragmentation rates; R is free radical leaving group and defines initiating ability).

Figure 3.21 Synthesis of polyarylenes via CC coupling by A

2

 + B

2

Stille and Suzuki polycondensation (M

1

, M

2

 = arene like phenyl).

Figure 3.22 Synthesis of functional and branched polyethylene core by chain walking polymerization (CWP) (a: NaBAr

4

; b: deprotection of alcohol).

Figure 3.23 Selection of Grubbs-type initiators of first to third generation.

Figure 3.24 ROMP of norborn-5-ene-2-methanol with a Grubbs-type initiator in solution.

Figure 3.25 Mechanism of the controlled polycondensation in a biphasic system.

Figure 3.26 Concept of the Kumada catalyst transfer polycondensation. dppp, propane-1,3-diylbis(diphenylphosphane); TM, transmetallation; RE, reductive elimination; OA, oxidative addition.

Figure 3.27 Examples of active esters used for efficient polymer analogous reaction toward functional esters or amides; (a) 4-nitrophenylester, (b)

N

-hydroxysuccinimide (NHS) ester, and (c) pentafluorophenylester.

Figure 3.28 Some examples of efficient reactions used for polymer modification that allow also orthogonal coupling (REO chemistry).

Figure 3.29 Commercial strained cyclooctynes (dibenzylcyclooctyne (DBCO)-NHS, difluorinated cyclooctyne (DIFO) acid, bicyclenonyne (BCN) carbonate) for introduction into (bio)macromolecules.

Figure 3.30 Blood level of native versus PEGylated protein.

Figure 3.31 Molecular structure of pure interferon α-2a (left) and a schematic representation of its PEGylated analog [35, 36].

Figure 3.32 Structures of polymers that have been used for PASylation (top), HESylation (middle), and PGylation (bottom).

Figure 3.33 Conjugation strategies.

Figure 3.34 Examples of reactions of click chemistry used in bioconjugation (cf. also Section 3.3).

Figure 3.35 Schematic representation of DNA or RNA nanoparticles (polyplexes) by cationic polymers through electrostatic interactions and noncovalent conjugate formation.

Figure 3.36 Bioconjugation strategies for cysteine (left) and lysine (right) residues.

Figure 3.37 Polycondensation reaction via (a) esterification and (b) transesterification reactions.

Figure 3.38 Mechanism of enzyme-catalyzed ring-opening polymerization (ROP).

Figure 3.39 Examples of cyclic monomers for enzyme-catalyzed ring-opening polymerization reactions.

Figure 3.40 Examples of polyesters.

Figure 3.41 Examples of enzymatically derived types of polymers.

Figure 3.42 Structure and the three-letter code of the main mammalian amino acids. The amino acids are aligned toward the increasing relative hydrophobicity under physiological conditions.

Figure 3.43 Synthetic scheme for solid phase peptide synthesis.

Figure 3.44 Deprotection mechanism of alpha amino groups in Fmoc and Boc protection strategies.

Figure 3.45 Orthogonal side chain protection strategies for cysteine and their application for site-directed disulfide bond formation.

Figure 3.46 Bottom: chemical structure of a polynucleotides (DNA, RNA). Top: chemical structure of nucleoside bases.

Figure 3.47 Left: schematic view of the solid phase polynucleotide synthesis. Right: the structure of DNA building block with the protection (red) and activation groups (blue).

Figure 3.48 The chemical structure of monosaccharides currently applied in the solid phase synthesis.

Figure 3.49 Schematic view of the solid phase polysaccharide synthesis.

Figure 3.50 Schematic view of PCR cycle (left) and propagation (right).

Figure 3.51 Schematic view of DNA replication.

Figure 3.52 Schematic view of recombinant protein expression.

Figure 3.53 Schematic view of recombinant hyaluronic acid biosynthesis.

Figure 3.54 Schematic diagram of (a) a chemical hydrogel with covalent point cross-links (e.g., poly(ethylene glycol) (PEG) hydrogel), (b) a physical hydrogel cross-linked by ion–polymer complexation (e.g., calcium alginate hydrogel), and (c) a physical hydrogel with crystalline regions (e.g., poly(vinyl alcohol) (PVA) hydrogel).

Figure 3.55 Formation of macroporous starPEG–heparin cryogels by combined cryotreatment of the aqueous gel-forming reaction mixture and lyophilization of the incompletely frozen gel. Yellow rods, heparin; grey crosses, starPEG.

Figure 3.56 Representative confocal microscopy image of human umbilical vein endothelial cell colonization on RGD-modified cryogels after seven days in culture in

xy

direction (3D projection) indicating three-dimensional cell growth. Green: cryogel dyed by Alexafluor488. Red: actin of endothelial cells dyed by Alexafluor633-labeled phalloidin.

Figure 3.57 Schematic diagram of a micromasonry assembly process. Microgels of desired shapes were produced by photolithography and mixed with a solution containing the prepolymer (a). The solution was poured on the surface of a high-affinity PDMS mold (b) where it spread on the PDMS surface (c). The removal of the excess prepolymer solution induced a further packing of the microgels (d). The system was exposed to light to cross-link the prepolymer remaining by the units, and the structure was subsequently separated from the PDMS template (e).

Figure 3.58 Schematic representation of the

in situ

gelling of poly(ethylene glycol)–peptide and glycosaminoglycan–peptide conjugates by Michael-type addition.

Figure 3.59 Schematic representations of alkane SAMs immobilized: (a) on Si-wafers by trialkoxysilane groups and (b) gold wafer by thiol groups, where X—functional (terminal) group like amino or carboxylic acid groups.

Figure 3.60 Langmuir–Blodgett monolayer formation on the (a) hydrophilic substrate by withdrawing from the trough and (b) hydrophobic substrate by immersion in the trough.

Figure 3.61 Layer-by-layer deposition of polyions.

Figure 3.62 Schematic representation of the immobilized molecules by (a) (3-glycidyloxypropyl) trimethoxysilane, (b) 3-(trimethoxysilyl)propyl methacrylate incorporated in the poly(methyl methacrylate) (PMMA), (c) poly(glycidyl methacrylate), (d) glycidyl methacrylate incorporated in the PMMA, and (e) PEMA on 3-aminopropyl-dimethylethoxy-silane. Where Rx—pendant molecules, R—polymer.

Figure 3.63 Schematic representation of multistep surface modification procedures for polymeric materials that comprise a low-pressure plasma treatment either for activation/functionalization (a, b, and d) or cross-linking (c).

Figure 3.64 Reactions on hydrocarbon polymer chains induced by e-beam radiation.

Figure 3.65 Schematic illustration of the major steps involved in soft lithography and three major soft lithographic techniques: (a) replica molding, (b) micromolding, (c) microtransfer printing, and (d) microcontact printing.

Figure 3.66 AFM and SEM images of a 100-nm gold film, patterned with polymer multilayers and etched. (a) AFM image, one PEI/POMA bilayer (the line scan shows a 100-nm-wide hole); (b) SEM image, one PEI/POMA bilayer.

Figure 3.67 Schematic representation of the PRINT process.

Figure 3.68 Schematic representation of an electrospinning setup.

Figure 3.69 Schematic representation of microgel formation by microfluidic droplet gelation. At the first cross-junction, these three fluids formed a laminar coflowing stream in the microchannel. This stream is broken to form monodisperse premicrogel droplets at the second cross-junction by flow focusing with immiscible paraffin oil. The droplet formation induces a rapid mixing of all the components inside the droplets, which leads to a subsequent cross-linking of the macromonomers.

Chapter 04

Figure 4.1 (a) Synthetic scheme of complex biohybrid structures composed of hyperbranched poly(ethylene imine) (PEI) and maltose units (R). The abbreviations T, L, and D represent terminal (T = NH

2

), linear (L = NHR), and dendritic (D = NR

2

) units. (b)

13

C NMR spectrum of pure PEI in D

2

O. The abbreviations T, L, and D represent neighboring terminal (T = NH

2

), linear (L = NHR), and dendritic (D = NR

2

) units.

Figure 4.2

13

C NMR of structures B and C (Fig. 4.1) of maltose-modified hyperbranched PEI showing the influence of different degrees of maltose attachment on hyperbranched PEI scaffold (Fig. 4.1).

Figure 4.3 Typical CD spectra of LYZ (0.1 mg/ml, solid line) and CONA (0.2 mg/ml, broken line).

Figure 4.4 Typical FTIR spectra of LYZ (10 mg/ml) and CONA (10 mg/ml).

Figure 4.5 Jablonski diagram.

Figure 4.6 Schematic outline of ionization mechanism in MALDI-MS.

Figure 4.7 Simplified TOF process of mass analysis. Ion source (matrix with analyte molecules) and sample slide (opening to drift region) possess a potential difference for accelerating different charged analyte ions.

Figure 4.8 LILBID mass spectra of fifth-generation glycodendrimer (a) and its complexes with Re clusters (b) for determining complexation ratio between Re cluster and fifth-generation glycodendrimers (as 2 in b) using LILBID-MS technique. Molecules with differing number of negative charges appear as separate peaks in (a and b). In (b), M

−

presents the mass distribution where glycodendrimer can complex x-fold Re clusters from twofold up to twelvefold.

Figure 4.9 Dependence of the hydrodynamic size on the molecular weight for lysine dendrimers. Mono = first-, second-, and third-generation monodendron, Thia1 = thiacalixarene core decorated with two monodendrons, Thia2 = thiacalixarene core decorated four monodendrons. The dashed lines indicate the theoretical slopes for the scaling exponents 1, 2, and 3.

Figure 4.10 2D electrophoresis NMR spectrum of PAMAM G2 at pH 3.4. Protonated part structures of dendrimer show the same mobility in average and also confirming the monodisperse property of G2 dendrimer in electric field.

Figure 4.11 (a) Scheme of channel profile with perpendicular forces during the separation of a sample mixture and (b) AF4 separation channel setup with specific components.

Figure 4.12 Principle of AF4 method for analyte filtration and analyte@polymer complex fractionation.

Figure 4.13 TEM of polymersomes at pH 3 (a) and 10 (b). Membrane consists of protonable poly(diethylaminoethyl methacrylate) (PDEAEM), which is responsible for the swelling/deswelling properties of polymersome.

Figure 4.14 Swelling of a cross-linked bulk polymer and network structure of the swollen hydrogel characterized by the average mesh size

ξ

. Network defects: 1. dangling end, 2. entanglement, 3. ring.

Figure 4.15 The degree of cross-linking is an important parameter that controls the equilibrium swelling: The image shows a series of biohybrid hydrogels synthesized by covalent cross-linking of four-arm poly(ethylene glycol) and heparin using carbodiimide chemistry. With decreasing molar ratio of poly(ethylene glycol) to heparin, the cross-linking degree decreases, which results in increased swelling of the networks.

Figure 4.16 Uniaxial compression stress–strain curves obtained for star-(polyethylene glycol)-heparin hydrogels with three different cross-linking degrees. Black: high. Dark gray: medium. Light gray: low cross-linking degree.

Figure 4.17 Oscillatory shear experiments can be performed on a rotational rheometer, where the polymer network is placed between two parallel plates.

Figure 4.18 Frequency dependence of storage und loss modulus for star-(polyethylene glycol)-heparin hydrogel obtained by means of oscillatory rheometry.

Figure 4.19 XPS C1

s

(left) and O1

s

spectra (right) of poly(3-hydroxybutyrate) before (top) and after NH

3

plasma treatment (bottom).

Figure 4.20 Scheme of the experimental setup for sessile drop experiments (left) and for captive air bubble measurements (right) using ADSA.

Figure 4.21 Poly(

N

-isopropylacrylamide-co-

N

-(1-phenylethyl) acrylamide) film thickness versus temperature during first and second heating/cooling cycles in distilled water (heating/cooling rate 1 K/min).

Figure 4.22 QCM-D data of a PNiPAAm-based copolymer film at temperatures around the phase transition. Solid lines correspond to data obtained ramping up temperature, while dashed lines correspond to ramping down temperature.

Figure 4.23 Setup and principle of surface plasmon resonance (Kretschmann configuration).

Figure 4.24 (a) High-resolution AFM topograph of a nanopatterned matrix composed of aligned collagen type I fibrils. (b) Cells seeded onto the collagen matrices strongly polarize along the direction of the fibrils (white arrow) and deform matrix perpendicular to the fibril direction. Collagen fibrils are bundled at the front and back of the cell without rupturing.

Figure 4.25 Force versus displacement curves on PNiPAAm gel in pure water at 10 and 35°C and on mica in pure water at room temperature. The same

z

-piezo displacement results in a smaller cantilever deflection on the soft gel surface in comparison with the hard mica sample because of elastic indentation.

Figure 4.26 SEM/ESEM images of mouse fibroblast cell morphology. (a) SEM images of 3 T3 mouse fibroblasts (20 kV). (b–d) ESEM images of 3 T3 mouse fibroblasts adhesion and proliferation on biomaterials (4.60 Torr, 5°C, 7 kV).

Figure 4.27 Cell adhesion on a primary protein layer and the formation of secondary protein layers by cell-secreted proteins.

Figure 4.28 Principle of streaming potential/current measurements combined with reflectometric interference spectroscopy. The charge of the polymer film is determined by streaming potential and streaming current measurements via nonpolarizable electrodes positioned at the inlet and outlet of the channel. Structural variations of the polymer layer and adsorption or desorption processes are followed simultaneously through the evaluation of the interference pattern resulting from the interference of the partial beams

I

1

and

I

2

. The relative dimensions in the scheme are not in scale.

Figure 4.29 Schematic illustration of an SCFS experiment and of the adhesion events detected. (a) A single cell is attached to an AFM cantilever (1) and approached to a substrate (1 and 2). Once in contact, cell adhesion molecules diffuse into the contact zone (2). The adhesive strength between cell and substrate increases. After a predefined contact time, the cell is retracted and the cantilever bends because of the adhesive strength between the cell and the substrate (3). Once the restoring force of the cantilever exceeds the strength of the interactions between cell and substrate, the cell starts to detach (3 and 4). The force detected at this point corresponds to the maximum detachment force (

F

D

). During further retraction of the cantilever, the contact area between the cell and the substrate shrinks (4) and the cell sequentially detaches from the substrate (5) until the cell and the substrate are completely separated (1). (b) Force–distance (

F–D

) curve showing steps (1–5) corresponding to those outlined in A. During approach (gray line) and retraction (black line), the force exerted on the cantilever, which is proportional to cantilever deflection, is recorded in an

F–D

curve. The retraction

F–D

curve is characterized by the maximum detachment force (

F

D

). This force is generally followed by steplike events that correspond to the unbinding single cell adhesion molecules from the substrate (

s

and

t

events).

Figure 4.30 Optical principle of reflection interference contrast microscopy (RICM). The optical path (a) and the formation of constructive and destructive interference with the resulting reference pattern (b) are depicted on Ref. 61.

Figure 4.31 Adhesion characteristic of HPC after 24 h of cultivation on matrix coatings is shown by RICM images and an overlay of RICM and DIC images taken at the same objective position. RICM images show adhesion areas; DIC images visualize the cell above. Surfaces: (a) fibronectin, (b) tropocollagen I. Scale bar, 5 µm.

Figure 4.32 Screening chamber: Two stainless steel cover plates (left and middle) that fix the test surfaces are pressed together by a screw (right). The PTFE spacer forms a cavity. Blood is filled into the mounted chamber through holes in the PTFE spacer, which are sealed with a closure.

Figure 4.33 The number of adherent thrombocytes on glass surface (left) after 3 h incubation with blood in a screening chamber is higher than on PTFE surface (right).

Chapter 05

Figure 5.1 Multifunctional random copolymer of four monomers: methyl methacrylate (MMA), semifluorinated methacrylate (sfMA-H2F8), acetylacetonato methacrylate (AAMA), and benzophenone methacrylate (BPMA) [2].

Figure 5.2 Ringsdorf model of polymer therapeutics [3], showing a polymer chain with a given biocompatible backbone where the solubility is fine-tuned by the solubilizing groups incorporated by copolymerization. Targeting groups and cleavable prodrugs (here: doxorubicin) can be introduced by polymer analogous reactions.

Figure 5.3 Schematic representation of a dendritic multifunctional carrier molecule.

Figure 5.4 Schematic representation of multifunctionality in various polymer architectures.

Figure 5.5 (a) Self-organization structures of block copolymers and surfactants: spherical micelles, cylindrical micelles, vesicles, foc- and boc-packed spheres (FOC, BOC), hexagonally packed cylinders (HEX), various minimal surfaces (gyroid, F surface, P surface), simple lamellae (LAM), as well as modulated and perforated lamellae (MLAM, PLAM). (b) AFM pictures (insert: SAXS analysis) of phase-separated poly(styrene-

b

-4-vinylpyridine) diblock copolymer films containing a low molar mass additive 2-(4-hydroxyphenylazo)benzoic acid (HABA) and showing in vapor-annealed films standing up (A) (dioxane) and laying down (B) (chloroform) cylinders, suitable for templating.

Figure 5.6 Nanomorphologies (TEM pictures, OsO

4

stained) in styrene–butadiene–

tert

-butyl methacrylate triblock SBT copolymers of different compositions (numbers indicate repeating units of the blocks). (a) S

27

B

29

T

44

: triphasic lamellae. (b) S

19

B

57

T

24

: two phases from cylinders; one phase forms the matrix.

Figure 5.7 Principle of block copolymer lithography for spatially defined placing of gold nanoparticles on surfaces [16]. (a) Block copolymer structure, (b) formation of micelles with a metal ion core, and (c) formation of thin films by dip coating and plasma treatment to remove organic layer.

Figure 5.8 Scheme for the control of cell’s integrin clustering at nanostructured and biofunctionalized substrates (based on spatially defined deposition of gold nanodots through block copolymer lithography).

Figure 5.9 Amphiphilic block copolymers form different structures with increasing length of the hydrophobic segment. The resulting curvature forces the formation of micelles, polymersomes, or wormlike structures.

Figure 5.10 TEM pictures showing examples of spherical and linear multicompartment micelles formed by poly(styrene-block-2-butadiene-block-methylmethacrylate) (SBM) triblock copolymers with various core volume ratios (

V

PS/

V

PB) resulting in the structures shown in (a–e). Staining was achieved with OsO

4

(B black, S gray, M corona not visible) [18]. Scale bars correspond all to 100 nm.

Figure 5.11 Schematic representation of a multifunctional polymersome able to interact with a cell.

Figure 5.12 Examples of dendritic polymer structures.

Figure 5.13 Divergent and convergent synthetic routes toward dendrimers. G: generation.

Figure 5.14 Synthetic steps toward PAMAM dendrimers by the divergent method.

Figure 5.15 General scheme of a unimolecular amphiphilic core–shell dendritic architecture as drug carrier.

Figure 5.16 Hyperbranched polyglycerol (hPG) and poly(ethylene imine) (PEI).

Figure 5.17 Common carbohydrate-based materials.

Figure 5.18 Schematic representation of a cell membrane decorated with glycopolymers indicating their specific biointeractions.

Figure 5.19 Examples of glycomonomers used in RAFT polymerization reactions.

Figure 5.20 Amphiphilic glycoblock copolymers that self-assemble into vesicles.

Figure 5.21 Enzymatic sugar elongation of a synthetic glycopolymer (only one reaction step is shown [46]; TcTs = 

Trypanosoma cruzi trans-sialidase

; PNPNeu5Ac = 

p

-nitrophenyl-5-acetamido-3,5-dideoxy-α-

D

-glycero-

D

-galacto-2-nonulopyranosidonic acid; BSA = bovine serum albumin; HEPES = 

N

-2-hydroxyethylpiperazine-

N

′-2-ethanesulfonic acid).

Figure 5.22 Main functional groups for the introduction of oligo-, di-, and monosaccharide units on monodendron and dendrimer surfaces.

Figure 5.23 General synthetic approaches toward hyperbranched glycopolymers and related structures.

Figure 5.24 Sialic acid conjugated dendritic polyglycerol nanoparticles with diameters in the range of 60 nm efficiently block viral infections by polyvalent inhibition of the hemagglutinin in contrast to their smaller multivalent analog.

Figure 5.25 Simplified schematic illustrations of the hierarchical self-assembly processes involved in the formation of hydrogels from peptide molecules.

Figure 5.26 Schematic illustration of different secondary structures formed by polypeptides: (a) β-sheet, (b) β-hairpin, (c) α-helix, and (d) the supercoiled multistranded protein motif coiled coil.

Figure 5.27 Possible applications of spider silk materials in biomedicine. Recombinant spider silk proteins can be processed into morphologies other than fibers, broadening the spectrum of possible applications.

Figure 5.28 Target ssDNA-induced volume change of hairpin DNA (top) and DNA without secondary structure cross-linked polymer hydrogels.

Figure 5.29 Screening of peptide motifs coupled to starPEGs that can form hydrogels with 14 kDa heparin.

Chapter 06

Figure 6.1 Formation and architecture of block copolymer micelles, which spontaneously form by self-assembly in water. The characteristic features are a pronounced core–shell architecture, which can be controlled by the individual polymer blocks. Typical examples for block copolymers are PEO-

b

-PPO, PEO-

b

-PCL, and PEO-

b

-PAsp.

Figure 6.2 Preparation methods for polymeric capsules that can be used for multicompartmentalization: in type I, the polymer in the shell is arranged vertically along the core surface. These systems can be obtained by self-assembly of amphiphilic copolymers into polymersomes and subsequent cross-linking or by surface-initiated polymerization (SIP) from the surface of nanoparticle templates; in type II, the polymer in the shell arranges horizontally along the core surface, mainly synthesized by layer-by-layer (LbL) assembly of polyelectrolytes onto a particle; in type III, the polymer in the shell arranges disorderly along the droplet surface, commonly synthesized by an emulsion-based method where a polymer is deposited at an aqueous/organic interface, yielding a polymer wall around a stabilized droplet.

Figure 6.3 Polymeric units used in block copolymers for polymersome preparation.

Figure 6.4 Mechanisms of polymersome formation from block copolymers. For solvent inversion, film rehydration, and electroformation, the polymer is first dissolved in an organic solvent and polymersomes are initiated after water is added to the system. In contrast, pH-sensitive polymers are dissolved in acidic water and polymersomes are formed by switching to basic conditions.

Figure 6.5 Schematic representation of polymersomes, with non-cross-linked and cross-linked membranes. Only in cross-linked polymersome membranes, transport can be reversibly activated upon polarity switch.

Figure 6.6 Cross-linking units that have been integrated in block copolymers used for polymersome formation.

Figure 6.7 Schematic illustration of the constructed pH-sensitive polymeric capsule with a dendritic glycopolymer inside the core and the formation of a porous wall by switching the pH to 6 or lower, which can lead to the release of the encapsulated dendritic glycopolymer tuned by the shear rate [9] (for TEM images of collapsed and swollen membrane, cf. Fig. 4.13).

Figure 6.8 (a) Schematic illustration of the procedure for the synthesis of polymeric capsules based on surface-initiated RAFT polymerization using silica nanoparticles as templates. (b) TEM image of polymer grafted silica nanoparticles. (c) SEM and (d) TEM images of the synthesized polymeric capsules.

Figure 6.9 Schematic illustration of the morphology of liposomes embedded in a multicompartment polymeric capsule (left) and cryo-TEM image of a (PAH/PSS)

4

/PAH/liposomes

NBD

/PSS/PAH/PSS multicompartment polymeric capsule embedded in ice (inset) and a close-up of the polyelectrolyte shell, which contains intact liposomes (right).

Figure 6.10 (a) Synthetic scheme for “thermal click” reaction to form PG megamers. (b) Synthetic pathways toward pure PG-nanogel and surface-functionalized PG-nanogel particles: (i) cyclohexane/DMSO/block copolymer surfactant, sonic tip miniemulsification 4 × 1 min; (ii)

p

-TSA (cat.), 115°C, 16 h; (iii)

p

-TSA (cat.), 115°C, varied time; (iv) NaN

3

, DMF, 60°C, 24 h; (v) propargyl derivative, CuSO

4

 · 5H

2

O, sodium ascorbate, H

2

O, 24 h.

Figure 6.11 (a) Droplet microfluidic templating of micrometer-sized droplets using a glass microcapillary device. (b) Pre-microgel emulsion obtained from the experiment in Panel a. (c) Optical micrograph of water-swollen microgel particles formed by gelation of the droplets in Panel b.

Figure 6.12 Fluorescence microscopy shows clear evidence for cellular uptake of fluorescently labeled PG nanogels via an endocytotic pathway.

Figure 6.13 Synthetic route to biodegradable polyglycerol nanogels, showing a generalized depiction of a nanogel and degradation fragments [31, 32].

Figure 6.14 Thermo-responsive polyglycerol-based nanogels synthesized via precipitation polymerization. The nanogels showed a tendency to shrink with increasing solution temperature as shown by DLS measurements.

Figure 6.15 Dendritic core–multishell nanocarriers as novel amphiphilic architectures for drug delivery having a hydrophobic inner shell and a hydrophilic outer shell: with linear PEG (left;) or dendritic polyglycerol outer shell (right;).

Figure 6.16 Rhodamine B penetration into pig skin: staining of pig skin following the application of 0.004% rhodamine B-loaded cream (a), SLN (b), and CMS nanotransporters (c) for 6 h. The representative pictures taken from the identical donor animal are obtained by superposing normal light and fluorescence images of the same area. (d) The arbitrary pixel brightness values (ABU) were obtained by fluorescence picture analysis (cream, black columns; SLN, gray columns; CMS nanotransporters, white columns,

n

 = 3). The inserted numbers give the respective enhancement of penetration over cream, *differences (

p

 ≤ 0.05).

Figure 6.17 TEM (A) and AFM (B) pictures of dendrimersome structures derived from (3,4)-12G1-PE-(3,5)-12EOG1-(OCH3)4 (see chemical structure shown).

Figure 6.18 Proposed mechanism of gene transfection having the following elements: formation of the DNA/siRNA polymer complex (polyplex), endocytosis of the polyplex, fusion of endosome and lysosome, release of the polyplex into the cytosol, incorporation of the polyplex into the nucleus, and transcription of the DNA into mRNA followed by release of the polyamine backbone into the cytosol. Alternatively, direct binding of siRNA to mRNA via RISC complex and knock down is possible.

Figure 6.19 Idealized fragment of poly(glycerol amine) (PG–NH

2

) (top) (reproduced with permission from Mehrabadi et al. [47b]) and

in vivo

silencing of the luciferase gene by siRNA–PG–NH

2

(bottom). 3D bioluminescence image of mice treated with 16 mg kg

–1

43 kDa PG50: light emission of tumors before (day 0) and after (day 3) treatment with 16 mg kg–1 43 kDa PG50 complexed with non-targeting (nt) siRNA and luciferase specific (a-Luc) siRNA, respectively on three consecutive days.

Figure 6.20 Schematic representation of (a) the EPR effect and (b) further cellular uptake mechanisms.

Figure 6.21 Bivalent binding of a ligand to the tetravalent cGMP receptor. (a) If the polymeric spacer is too short, only one binding site may be occupied in the multivalent protein receptor. (b) The highest bond strength is achieved with an adequate spacer length and optimal operating range for the second bond. (c) Too long a spacer increases the number of unproductive degrees of freedom and reduces the binding strength again.

Figure 6.22 The selectin–ligand interaction recruits leukocytes to the vascular endothelium, which allows them to adhere. Following the inflammatory mediators, leukocytes migrate from the blood vessels toward the focus of inflammation.

Figure 6.23 Structure of a sialyl Lewis

X

ligand (sLe

X

) und its selectin specific interaction.

Figure 6.24 Schematic structure of a dendritic galactose conjugate with high L-selectin binding; IC

50

 = 2.45 mm (RH) and 35 nm (RSO

3

Na).

Figure 6.25 (a) A multivalent binding of a virus to a cell surface is compared to (b) a noncompetitive binding with monovalent ligands. (c) Multi- and polyvalent ligands are considerably more effective in binding and shielding a virus surface than monovalent ligands, thus preventing viral adhesion.

Figure 6.26 A polyvalent interaction of sialic acid-functionalized polyglycerol nanogels with hemagglutinin receptors on the virus surface. The viral binding and thus the cellular infection of the influenza virus can be reduced by up to 80% through efficient competition between the nanogel and glycan structures, such as sLe

X

, presented on the cell surface.

Figure 6.27 (a) Structure of dPGS, (b) therapeutic study of contact dermatitis in a mouse model involving ear swelling after stimulation by trimellic acid anhydride (TMA) and dPGS (blue bar) compared to commercial prednisolone (dose: 30 mgkg

−1

, yellow bar), and (c) an inflammation selective fluorescence diagnosis with a dPGS–dye conjugate.

Figure 6.28 Different types of bioresponsive hydrogels that change properties in response to (I) small molecules via receptor/ligand interactions; (II) (cell-secreted) enzymes via cleavable linkers; and (III) small molecules that are converted by immobilized enzymes. The macroscopic response (swelling/collapse of the hydrogel) is shown.

Figure 6.29 Autoregulation of heparin release from a thrombin-sensitive bioresponsive hydrogel. (a) Thrombin formation. (b) Responsive heparin release. (c) Heparin-catalyzed thrombin inhibition. (d) No further heparin release.

Figure 6.30 Release profile of insulin-loaded microparticles in response to a glucose stimulus.

Figure 6.31 Top: Representative surface and cross-sectional images indicating three-dimensional growth of HUVECs within MMP-cleavable gels after 7 days. Bottom: Representative cross-sectional images illustrating enhanced three-dimensional cell migration in VEGF-loaded MMP-cleavable hydrogels after 1 day of culture, scale bars = 50 µm.

Figure 6.32 Binding modes for biofunctionalization.

Figure 6.33 Schematic illustration of the maleic anhydride copolymer thin film system used to covalently immobilize biofilm-degrading enzymes. (a) Initial state–PEMA film covalently attached to amine-functionalized glass. (b) Immobilization of biofilm-degrading enzymes through reaction of the primary amino groups of lysine side chains to the anhydride groups of the polymer.

Figure 6.34 Springtail skin morphology and process scheme for manufacturing polymer membranes with similar structural features. (a) Habitus image of

Folsomia candida

. Insets show scanning electron micrographs (SEMs) of the characteristically contained bristles, granules, and ridges. The nanoscopic granules and interconnecting ridges form cavities, are arranged in a comb-like pattern, and provide a template for the developed polymer membranes. (b) Process scheme for membrane fabrication: Firstly, a two-tier silicon master structure is fabricated by optical lithography. Secondly, the master structure serves as template for reverse imprint lithography. (c) SEM image of the two-tier silicon master structure (inset: detailed view of a small pillar centered on a larger pillar, scale bar: 1 µm). (d) SEM image (inset: cross-section after focused ion beam preparation, scale bar: 1 µm) and (e) photograph of the springtail-skin-inspired polymer membrane. (f) Water droplet (colored with red dye) deposited on the membrane, which was transferred to a 4 mm diameter glass rod.

Figure 6.35 Structure of selected benzamidine-type inhibitors [137].

Figure 6.36 Scheme of the inhibitor modified polymer film where R represents one of the benzamidine-based inhibitors (Fig. 6.35) [137].

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BIO- AND MULTIFUNCTIONAL POLYMER ARCHITECTURES

Preparation, Analytical Methods, and Applications

BRIGITTE VOITRAINER HAAGDIETMAR APPELHANSPETRA B. WELZEL

Copyright © 2016 by John Wiley & Sons, Inc. All rights reserved

Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, [phone], fax [phone], or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, [phone], fax [phone], or online at http://www.wiley.com/go/permissions.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

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Library of Congress Cataloging-in-Publication Data:

Names: Voit, Brigitte, author. | Haag, Rainer, author. | Appelhans, Dietmar, author. |  Welzel, Petra B., author.Title: Bio- and multifunctional polymer architectures : preparation,  analytical methods, and applications / by Brigitte Voit, Rainer Haag,  Dietmar Appelhans, Petra B. Welzel.Description: Hoboken, New Jersey : John Wiley & Sons, 2016. | Includes  bibliographical references and index.Identifiers: LCCN 2015041264 | ISBN 9781118158913 (cloth)Subjects: LCSH: Polymers–Biotechnology. | Polymers in medicine. | Biomedical materials.Classification: LCC TP248.65.P62 A67 2016 | DDC 668.9–dc23LC record available at http://lccn.loc.gov/2015041264

PREFACE

Synthetic polymers have revolutionized modern life over the last century and provide highly versatile materials for everybody’s daily comfort. In contrast, bio- and multifunctional polymers are about to change our lives with innovative system solutions, especially in the biomedical area. This research field is currently subject of intense efforts and bears a large innovation potential for the future, especially at the interphase between materials and biomedical sciences. Also in the context of emerging research areas such as chemical biology, nanobiotechnology, and synthetic biology, the control of material-biointerphases is of outmost relevance.

In this textbook we highlight recent developments in designing and realizing synthetic multifunctional polymer architectures, which are of special importance for application in bionanotechnology and biomedicine. In the last 20 years, a significant progress has been made in controlling the structure and molar mass of macromolecules, implementing functional units, inducing specific self-assembly behavior, and combining synthetic structures with biological function. In addition, a much deeper understanding of the biological and physical interactions between a biosystem and a synthetic material as well as strong interdisciplinary cooperations between polymer chemists, cell biologists, and medical scientists evolved, which allow today a much more precise and rational approach toward custom-made biocompatible, bioactive synthetic macromolecular architectures and biointerfaces as well as bioconjugates for biomedical applications.

Due to this progress that has been made in the recent years and the high impact on biotechnology and biomedicine, this textbook will provide the basic synthetic tools available to tailor-make multifunctional polymers and to control their biointeractions and self-assembly, and on the other hand, it will highlight functional materials and system applications that are based on the availability of such complex and multifunctional macromolecules.

This textbook will allow an easy access into the field especially for advanced and graduate students as well as experienced researchers in natural sciences and biomedical specialists entering the field or being interested in and working at the interface between polymeric materials and biomedicine. Target study areas are bioengineering, biomaterials science, biomedical science, chemistry biophysics, polymer science, and regenerative medicine.

Brigitte VoitRainer HaagDietmar AppelhansPetra B. Welzel

ACKNOWLEDGMENTS

The book would not have been possible without the help of numerous people for which we are deeply thankful. Firstly, we would like to acknowledge the major contributions of Prof. Carsten Werner for the concept and details of the biomaterial and biomedical application parts of the book. Various coworkers contributed to subchapters; this includes Dr. Mikhail Tsurkan, Dr. Mirko Nitschke, Dr. Ulrich Scheler, Dr. Albena Lederer, Dr. Susanne Boye, Dr. Martin Müller, and Dr. Ulrich Oertel who contributed to Chapters 3 and 4. Dr. Juliane Keilitz and Dr. Wiebke Fischer took over major parts in Chapters 3, 5, and 6 and helped significantly in editing and finalizing the full book. A number of students and postdocs have to be named who greatly helped with making schemes and figures: Tim Erdmann, David Gräfe, Jörg Kluge, Banu Iyisan, and Dr. Emanuel Fleige. Marlen Groß and Maximilian Keitel are highly acknowledged for their technical support.

1INTRODUCTION

Materials that can be applied in bionanotechnology and biomedicine are a subject of current research. Bio- or multifunctional polymeric materials might help solving many of today’s medical problems and allow, for example, a safer use of medicinal products and implants, a more targeted and specific drug administration, and finally even in vivo tissue engineering for effective regenerative medicine. Furthermore, specially designed functional materials provide new perspectives in diagnosis and fundamental studies of biological processes as well as significantly increase the number of controllable targets in medical treatments.

The aim of this book is to outline why and how synthetic bio- and/or multifunctional polymers are particularly promising in this context. Therefore, chemical and physical tools that are available to custom-make polymers and to control specific biointeractions will be introduced. Combining up-to-date polymer synthesis knowledge with a fundamental understanding of the biosystem and ways to control specific biological interaction has led to highly promising advances in the design of specific polymers for biomedical applications, which has been recently successfully demonstrated.

1.1 WHAT MAKES POLYMERS SO INTERESTING?

Various types of materials like metals and alloys, ceramics, different inorganic scaffolds, and low and high molar mass organic molecules are proven instrumental for the broad variety and the specific needs of bionanotechnology as well as biomedicine applications. Synthetic polymers play a very special role in this context because they are organic in nature and can be tailor-made in many forms to mimic the complexity of the natural biomacromolecules that define and control life. Thus, polymer scientists have taken up the challenge of identifying important design rules that come from nature and at least partially implemented them, essentially reduced, into synthetic polymer structures. Biomacromolecules in the form of polynucleotides and polypeptides contain a large complexity of information in a single molecule that is the base for tertiary structure formation, recognition, bioactions, and biointeractions. This is achieved in biology by a full sequence and molar mass control during the synthesis of the biomacromolecules as well as by an amazing control of the interplay of noncovalent interactions such as found in hydrophobic or electrostatic interactions and hydrogen bonding.

Synthetic macromolecules have similar basic structural features as biomacromolecules, which has given rise to many different kinds of polymers that can seamlessly interface with biosystems and provide particular advantages for new biomedical applications. The first of these features are that they are formed by a large number of repeating units (monomers). Secondly, they can be prepared in different molar masses. Constitution (composition) and connectivity (linear, branched) of the repeating units are already two important parameters that can be varied in synthetic macromolecules. In addition, the characteristics of polymers can be significantly broadened by combining several comonomers in one polymer chain. These can be randomly distributed within a linear polymer chain or added in a special sequence and in a specific topology (see polymer architectures), which results in block, star, and graft copolymers, for example.

Since the variety of monomer structures is nearly unlimited synthetic polymers offer many more variation possibilities with regard to introduction of specific chemical units and functions than, for example, proteins where a limited number of amino acids is found in nature. Similarly, there is theoretically also no limit to the number of different monomer units that can be combined in one polymer chain. However, so far, the exact sequence of the monomers has not been controllable by common synthetic approaches since polymerization is usually a statistical process.

A specific feature of polymers and the major difference to naturally occurring proteins and polynucleic acids is their dispersity. This can account on the one hand for the chemical composition in copolymers, whereby each individual chain may have a different sequence of the comonomers (= isomers). However, it is especially prominent when one looks at the molar mass. The statistic nature of the polymerization process always results in a mixture of macromolecules of different lengths with a specific distribution in molar mass.

In analogy to proteins, however, one can further define a “primary structure” in synthetic polymers, which describes not only the constitution but also the configuration of the monomer units within the polymer chain. Although monomer units are usually introduced head-to-tail, sometimes, head-to-head or tail-to-tail connections are observed that reduce the potential order in the chain. Similarly, cis- and trans-configuration within individual monomer units may have to be considered that can significantly change the material’s properties as can be seen in the comparison of poly(cis-1,4-isoprene) (natural rubber) to poly(trans-1,4-isoprene) (a brittle material without commercial use). A specific feature in polymers is tacticity, which describes the arrangement of the substituent in a repeating unit and can be isotactic (always in the same direction), syndiotactic (controlled alternating), or atactic (random) (Fig. 1.1). Isotactic polypropylene is a million-ton-scale technical thermoplastic material that is used widely in packaging, whereas atactic polypropylene is a viscous oil with no practical use (see Chapter 2).

Figure 1.1 Structure and form of polymer chains in solution and melt (top) and possible ordered bulk structures (bottom).

In further analogy to proteins, macromolecules can also have a

2TERMINOLOGY

3PREPARATION METHODS AND TOOLS

In this chapter, some basic fundamentals and the most common methods and techniques for the synthesis and modification of functional and well-defined macromolecules and bioconjugates are described. Please note that it is not the aim to give all details and aspects of polymerization methods used widely today. For that, the reader is referred to textbooks in polymer chemistry [1, 2].

3.1 GENERAL ASPECTS OF POLYMER SYNTHESIS

The formation of synthetic polymers is a process that occurs via chemical connection of many hundreds up to many thousands of monomer molecules. As a result, macromolecular chains are formed. They are, in general, linear but can be branched, hyperbranched, or cross-linked as well. However, depending on the number of different monomers and how they are connected, homopolymers or one of the various kinds of copolymers can result. The chemical process of chain formation may be subdivided roughly into two classes, depending on whether it proceeds as a chain growth or as a step growth reaction (Fig. 3.1).

Figure 3.1 Classification of reaction mechanisms used to build up polymers.

The buildup of the polymer chain with monomer conversion differs significantly depending on the class of polyreaction used. In a chain growth process, the molar mass increases rapidly and reaches a plateau value already at low monomer conversion; if chain growth is well controlled or “living,” then a linear dependency of degree of polymerization versus monomer conversion can be achieved. In contrast, in a step growth process only at very high functional group conversion (>90%) high molar mass products can be achieved (Fig. 3.2).

Figure 3.2 Schematic representation of the development of molar mass, for example, Mn, with monomer conversion for chain growth (free radical), chain growth (living) and step growth mechanisms for the polymer buildup reaction.

3.1.1 Chain Growth Polymerizations

Chain growth polymerizations (also called addition polymerizations in the English terminology, which should not be mistaken as polyaddition that belongs to the step growth processes) are characterized by the occurrence of activated species, so-called initiators and active centers. The initiator adds one monomer molecule after the other in a way that at the terminus of each new species formed by a monomer addition step, an activated center is created, which again is able to add the next monomer molecule. Active initiators are formed from compounds that create radicals via homolytic bond scission, from metal complexes, or from ionic (or at least highly polarized) molecules in the initiating steps. From there the chain growth can start as a cascade reaction upon manifold repetition of the monomer addition and reestablishment of the active center at the end of the respective new product. Finally, growth of an individual macromolecule is arrested in either a termination or a transfer step. While termination leads to the irreversible disappearance of an active center, chain transfer results in the growth of a second chain while the first one is terminated. Here, the active center is transferred to another molecule (solvent, initiator, monomer, etc.) where it is able to initiate further chain growth. The resulting “dead” polymer, on the other hand, can continue its growth only when activated in a subsequent transfer step. Because in general this reactivation does not occur at the terminal monomer unit but somewhere in the chain, branched or cross-linked products will result. In conclusion, chain growth polymerizations are typical chain reactions involving a start-up step (initiation) followed by many identical chain reaction steps (propagation)—stimulated by the product of the first start-up reactions. Transfer processes may continue until, finally, the active center disappears in a termination step.

Monomers appropriate for chain growth polymerizations either contain double or triple bonds or are cyclic, having a sufficiently high ring strain. Depending on the nature of the active center, chain growth reactions are subdivided into radical, ionic (anionic, cationic), or transition metal-mediated (coordinative, insertion) polymerizations. Accordingly, they can be induced by different initiators or catalysts. Whether a monomer polymerizes via any of these chain growth reactions—radical, ionic, and coordinative—depends on its constitution and substitution pattern. Also, external parameters like solvent, temperature, and pressure may also have an effect. The two main processes for the synthesis of ton-scale commercial polymers (Fig. 3.3) are free radical polymerization (done in bulk or heterogeneous systems like in emulsion and dispersion polymerization) and insertion polymerization for polyolefins.

Figure 3.3 Examples of commercially important polymers prepared by chain growth processes (Note: PVA is formed from poly(vinyl acetate) after an additional polymer analogous reaction).

4ANALYTICAL METHODS

In general all characterization methods applied for characterizing large organic molecules and polymers are also relevant in the field of bio- and multifunctional polymer architectures. Details on that can be found in common polymer text books [1, 2], and the readers are referred to those since it is impossible to cover all characterization aspects in this book. Especially the field of thermal analysis that allows determining of important features like glass transition temperature, melting temperature, and degradation behavior will not be addressed here, even though these determine significantly the application range of the material. In addition, bulk material property characterization (mechanical properties as well as bulk morphology) will not be addressed with the important exception of gel characterization. However, special aspects like a full structural analysis, verification of meaningful molar masses and dispersities, understanding the solution and aggregation behavior, and, finally, determining surface and biophysical interactions are very essential for any application of synthetic polymers and biohybrids in biomedical application, and hence, the most important characterization techniques will be briefly described from the basic features and their potentials and limitations will be outlined providing some relevant examples.

4.1 MOLECULAR STRUCTURE AND MOLAR MASS DETERMINATION OF POLYMERS AND BIOHYBRIDS

In Table 4.1, the most important characteristics that have to be elucidated for complex polymer structures and suitable analytical methods are listed.

Table 4.1Molecular Structure and Molar Mass Determination: Important Polymer Characteristics and Important Analytical Methods for Their Determination

Characteristics

Analytical Methods

Molecular structure

Chemical composition

NMR, elemental analysis, UV–Vis, IR, pyrolysis–GC–mass spectrometry, MALDI-TOF, LILBID-MS

End groups

Spectroscopy (NMR, UV–Vis, fluorescence, Raman), titration

Branching and cross-linking

NMR, solution viscosity, melt viscosity, light scattering, solubility tests

Stereoregularity, head-to-tail,

cis–trans

Spectroscopy

Optical isomerism, optical activity

Polarimetry, IR spectroscopy

Refractive index

Refractometry

Molar masses and sizes

Molar masses

Absolute methods:

end group analysis, membrane osmometry, vapor pressure osmometry, static light scattering, mass spectrometry (MALDI-TOF, ESI) sedimentation measurements

Different average values:

M

n

,

M

w

Relative methods:

solution viscosity, melt viscosity, size-exclusion chromatography exclusion, field-flow fractionation

Dispersity (

M

w

/

M

n

)

Fractionation, size-exclusion chromatography

Structure,

M

w

, and shape

Static light scattering, dynamic light scattering, sedimentation measurements, small-angle X-ray, solution viscosity, imaging methods ((

in situ

) AFM, cryo (HR-)TEM, electron tomography)

Aggregation (in solution)

Dynamic light scattering, field-flow fractionation, small-angle X-ray, fluorescence spectroscopy, UV–Vis spectroscopy, LILBID-MS, imaging ((

in situ

) AFM, (cryo) (HR-)TEM, electron tomography)

4.1.1 Structural Characterization

4.1.1.1 High-Resolution NMR Spectroscopy

Chemical constitution, steric configuration, and, in some cases, details about chain conformation, aggregation, association, and supramolecular self-organization behavior of macromolecular substances can be determined using high-resolution nuclear magnetic resonance (NMR) spectroscopy. This spectroscopic technique is sensitive towards nuclei with a nuclear spin different from zero. Identical nuclei (e.g., protons) incorporated at different places of a molecule, or bond to different molecules, have different shielding constants s and thus – at constant external field H0 – different resonance frequencies n1. This effect is called “chemical shift” d and is usually given relative to that of a standard compound like tetramethylsilane (TMS). Because of the smallness of this shielding constant, the value of the chemical shift of a nucleus i is given in parts per million (ppm). For protons, the chemical shifts d are between 0 and approximately 12 ppm and for 13C between 0 and approximately 220 ppm. Just by analyzing the chemical shifts of the signals found in an NMR spectrum, a first rough analysis of the polymer constitution is possible. Moreover, the intensity of the absorptions of each nucleus is independent of the chemical environment but proportional to their relative concentration. This feature – together with the characteristic chemical shifts – is of special importance for qualitative and quantitative structural elucidation via NMR spectroscopy: Position (d/ppm) and intensity of absorption give clear and direct information about constitution, configuration, and other features of the material to be analyzed. And there is one more dominant effect that consolidates and deepens the structural information obtained from NMR investigations. This is the indirect spin–spin coupling of neighboring, nonequivalent nuclei of a molecule via the bond electrons. It leads to a fine structure (multiplet structure) of the absorption signals, which is caused by the generation of additional small magnetic fields at the locus of the observed nucleus and, thus, provides important information on neighboring molecule groups.

The NMR spectra of dissolved polymers can be interpreted in the same way as those of low molecular weight compounds. Hence, it is a powerful tool for constitutional analyses: The chemical constitution of repeating units and end groups, the content of comonomers, or the steric configuration (tacticity) of macromolecules can be determined in dilute solution using high-resolution NMR spectroscopy. Also, NMR spectra of linear polymers of low molar mass often show unique absorptions due to their end groups. By referencing these absorptions to those of the nuclei in the repeating units, it is possible to obtain the ratio of the number of end groups to the number of repeating units. Thereby it is possible to evaluate the Mn