University of Groningen
Polymeric surfactants based on the chemical modification of alternating aliphatic polyketones Araya Hermosilla, Esteban Alejand
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Polymeric surfactants based on the chemical modification of
alternating aliphatic polyketones
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus prof. E. Sterken
and in accordance with the decision by the College of Deans.
This thesis will be defended in public on Monday 6 May 2019 at 11.00 hours
by
Esteban Alejandro Araya Hermosilla born on 10 February 1984
in Providencia, Chili
Prof. F. Picchioni
Assessment Committee
Prof. M. Kobrak Prof. G.J.W. Euverink Prof. R. Rossi
Polymeric Surfactants Based on the Chemical Modification of Alternating
Aliphatic Polyketones
Esteban Alejandro Araya Hermosilla
Dedicated to my beloved family.
Contents
***
1 Introduction 1
1.1 Supramolecular systems . . . 1
1.1.1 Supramolecular systems . . . 1
1.1.2 Self-Assembly . . . 2
1.1.3 Active molecules . . . 3
1.1.4 Antibiotics . . . 4
1.1.5 Dyes . . . 4
1.1.6 Redox-active molecules . . . 6
1.2 Polymeric surfactants . . . 7
1.3 Polyketones . . . 8
1.3.1 The Paal-Knorr reaction . . . 10
1.4 Aim of the thesis . . . 10
2 Novel polyketone with pendant imidazolium groups as nanodispersants of hydrophobic antibiotics 13 2.1 Introduction . . . 14
2.2 Experimental . . . 16
2.3 Results and discussion . . . 18
2.4 Final remarks . . . 24
2.5 Conclusions . . . 24
3 Amphiphilic modified polyketones as stabilizers of the tetra-anionic form of 5,10,15,20-Tetrakis-(4-sulfonatophenyl)porphyrin in aqueous acid con- ditions 27 3.1 Introduction . . . 28
3.2 Experimental . . . 30
3.3 Results and discussion . . . 32
3.4 Conclusions . . . 41 v
4 Supramolecular structures based on pH-sensitive surfactant polyketones and 5,10,15,20-tetrakis-(sulfonatophenyl)porphyrin 45
4.1 Introduction . . . 46
4.2 Experimental . . . 48
4.3 Results and discussion . . . 51
4.4 Conclusions . . . 65
5 Totally organic redox-active pH-sensitive nanoparticles stabilized by am- phiphilic aromatic polyketones 77 5.1 Introduction . . . 78
5.2 Experimental . . . 80
5.3 Results and discussion . . . 82
5.4 Final remarks . . . 88
5.5 Conclusions . . . 90
6 Functionalization of polyketones: beyond the synthesis of polymeric sur- factants 91 6.1 Introduction . . . 91
6.2 Polymeric emulsifiers based on polyketones . . . 93
6.2.1 Introduction . . . 93
6.2.2 Experimental . . . 94
6.2.3 Results and discussion . . . 95
6.2.4 Conclusions . . . 99
6.3 Catalysts Based on Polyketones . . . 99
6.3.1 Introduction . . . 99
6.3.2 Experimental . . . 103
6.3.3 Results and discussion . . . 106
6.3.4 Conclusions . . . 112
A Appendix 115
Bibliography 123
List of Figures
***
1.1 Schematic representation of the components of an organic dye molecule,
4-Hydroxyazobenzene. . . 5
1.2 Scheme of aliphatic polyketone. . . 9
1.3 Schematic representation of polyketones chemical modification via the Paal-Knorr reaction. . . 11
1.4 Schematic representation of the Paal-Knorr reaction . . . 11
2.1 Schematic representation of the chemical modification of polyke- tones (PK50) via Paal-Knorr reaction with histamine. . . 15
2.2 Structure of oxolinic acid and flumequine. . . 16
2.3 OA precipitation pH at 1 ⋅ 10−3M and 2 ⋅ 10−4M. . . 19
2.4 FLU precipitation pH at 1 ⋅ 10−3M and 2 ⋅ 10−4M. . . 19
2.5 Correlograms obtained by DLS of PK-Im at concentration of 1 ⋅ 10−3, 1 ⋅ 10−4, and 1 ⋅ 10−5M at different values of pH. . . 21
2.6 Size and zeta potential of particles composed of OA, PK-Im, and FLU and PK-Im as a function of their relative concentration. . . 22
2.7 Size and zeta potential of OA/PK-Im nanoparticles as a function of time for OA/PK-Im at different molar ratios. . . 22
2.8 Size and zeta potential of FLU/PK-Im nanoparticles as a function of time for FLU/PK-Im a different molar ratios. . . 23
3.1 Acid and basic forms of TPPS. . . 29
3.2 Schematic representation of the chemical modification of polyketone 50 via Paal-Knorr reaction with N-(2-hydroxyethyl)ethylenediamine (HEDA). . . 30
3.3 1H-NMR spectra of pyrrole-HEDA model compound. . . 33
3.4 1H-NMR spectra of PK50 modified with HEDA at different x. . . . 34
3.5 FT-IR spectra of PK50 modified with HEDA at different x. . . 35
3.6 Surface tension graph of PK50-HEDA80 as a functions of pH and concentration. . . 36
3.7 Transition pH of TPPS at a concentration of 1 ⋅ 10−6M. . . 37 vii
3.8 Normalized UV-vis spectra of 1 ⋅ 10−6M TTPS at different pHs. . . 37 3.9 Schematic structure of J-aggregates and H-aggregates formed by TPPS. 38 3.10 Normalized UV-vis spectra of 1 ⋅ 10−6 M TTPS in the presence of
PK50-HEDA80 1 ⋅ 10−4M at different pHs. . . 39 3.11 Fluorescence spectra of 1 ⋅ 10−6 TTPS in the presence of PK50-
HEDA80 1 ⋅ 10−4at different pHs. . . 40 3.12 Transition pH shift of TPPS at 1 ⋅ 10−6in presence of the polymer at
1 ⋅ 10−4: PK50-HEDA80, PK50-HEDA60, and PK50-HEDA40. . . 41 3.13 Normalized visible spectra of 1 ⋅ 10−6M TTPS in presence of PK50-
HEDA60 and PK50-HEDA40 at different pHs. . . 42 3.14 Fluorescence spectra of 1⋅10−6M TTPS in presence of PK50-HEDA60. 43 3.15 Fluorescence spectra of 1⋅10−6M TPPS in presence of PK50-HEDA40. 44 4.1 Acid and base form of TPPS. . . 47 4.2 Schematic structure of J-aggregates and H-aggregates formed by TPPS. 47 4.3 Schematic representation of Paal-Knorr reaction on polyketones with
the amines used in this study. . . 48 4.4 1H-NMR spectra of pyrrole-IM, pyrrole-PI, and pyrrole-HEDA model
compounds. . . 52 4.5 1H-NMR and FT-IR spectra of polyketones before and after modifi-
cation. . . 54 4.6 Surface tension of PK50-IM, PK50-PI and PK50-HEDA as a func-
tion of the concentration at various pH values. . . 56 4.7 Correlograms, size and zeta potential of the polymer solutions at a
concentration of 1 ⋅ 10−4M of functional groups and at different val- ues of pH. . . 58 4.8 Size and zeta potential of PK50-IM interacting with TPPS at differ-
ent concentrations. . . 60 4.9 Size and zeta potential PK50PI interacting with TPPS at different
concentrations. . . 60 4.10 Size and zeta potential PK50-HEDA interacting with TPPS at differ-
ent concentrations. . . 61 4.11 Normalized UV-vis spectra of TTPS at different concentrations in
the presence of PK50- IM80 1 ⋅ 10−4M at different pH. . . 63 4.12 Normalized UV-vis spectra of TPPS at a different concentrations in
the presence of PK50-PI 1 ⋅ 10−4M at different pH. . . 64 4.13 Normalized UV-vis spectra of TTPS at different concentrations in
the presence of PK50-HEDA 1 ⋅ 10−4M at different pH. . . 65 4.14 Fluorescence spectra of TTPS at 1 ⋅ 10−6M in presence of PK50-IM
at 1 ⋅ 10−4M at different pHs. . . 67 4.15 Fluorescence spectra of TTPS at 5 ⋅ 10−6M in presence of PK50-IM
at 1 ⋅ 10−4M at different pHs. . . 68
LIST OF FIGURES ix 4.16 Fluorescence spectra of TTPS at 1 ⋅ 10−5M in presence of PK50-IM
at 1 ⋅ 10−4M at different pHs. . . 69 4.17 Fluorescence spectra of TTPS at 1 ⋅ 10−6M in presence of PK50-PI
at 1 ⋅ 10−4M at different pHs. . . 70 4.18 Fluorescence spectra of TTPS at 5 ⋅ 10−6M in presence of PK50-PI
at 1 ⋅ 10−4M at different pHs. . . 71 4.19 Fluorescence spectra of TTPS at 1 ⋅ 10−5M in presence of PK50-PI
at 1 ⋅ 10−4M at different pHs. . . 72 4.20 Fluorescence spectra of TTPS at 1 ⋅ 10−6 M in presence of PK50-
HEDA at 1 ⋅ 10−4M at different pHs. . . 73 4.21 Fluorescence spectra of TTPS at 5⋅10−6M in presence of PK50HEDA80
at 1 ⋅ 10−4M at different pHs. . . 74 4.22 Fluorescence spectra of TTPS at 1⋅10−5M in presence of PK50HEDA80
at 1 ⋅ 10−4M at different pHs. . . 75 5.1 Functionalization of aliphatic polyketones with ABA via the Paal-
Knorr reaction. . . 79 5.2 1H-NMR and ATR FT-IR spectra of PK50ABAx at different values
of x. . . 83 5.3 Optical images of 10−3M of PK50ABAx at basic and acid pH, and
correlograms obtained upon titration of the corresponding basic so- lutions with HCl, for different values of x. . . 83 5.4 1H-NMR spectra in D2O of TTC 10−3M, PK50ABAx 10−2M, and
their corresponding mixtures at different x values . . . 85 5.5 600 MHz NOESY spectra in D2O of 10−3M of TTC in the presence
PK50ABA37, PK50ABA53, and PK50ABA69. . . 86 5.6 Optical images of samples containing 10−2M of PK50ABA37, PK50-
ABA53, and PK50ABA69. . . 87 5.7 Apparent size (left) and zeta potential (right) of TF nanoparticle sam-
ples containing PK50ABA37, PK50ABA53, and PK50ABA69 after reduction of variable amounts of TTC with ASC. . . 88 5.8 Optical images after reduction of 5 ⋅ 10−4M of TTC with ASC in the
presence of PK50ABA37, PK50ABA53, and PK50ABA69 at acid and basic pH. . . 89 6.1 Polyketones modified with HEDA and ABA. . . 94 6.2 Surface tension graph of PK50-HEDA and of PK50-ABA as a func-
tions of pH and concentration. . . 96 6.3 Emulsion stability in a function of the concentration of PK50-HEDA
and pH. . . 97 6.4 Stability of the emulsions produced with PK50ABA at a concentra-
tion of 3.0 mg/mL. . . 97
6.5 Phase inversion point of emulsions assisted by PK50HEDA at differ- ent concentrations. . . 98 6.6 Possible products of the reaction CO2with epoxides. . . 100 6.7 Scheme of a catalytic cycle via the cooperative activation of epoxide
with a Lewis acid (Mg) and a nucleophile (Br−ion). . . 101 6.8 Schematic representation of a polyketones about to undergo a Paal-
Knorr reaction with IM and the alkylation of imidazole group. . . . 102 6.9 Schematic representation of a polyketones about to undergo a Paal-
Knorr reaction with IM, FU and the alkylation of imidazole group. . 103 6.10 1H-NMR spectrum of pyrrole-imidazole model compound and after
the alkylation of the imidazole group. . . 107 6.11 1H-NMR spectrum of PK50, PK30 modified with IM and PK50
modified with IM and FU. . . 109 6.12 Mechanism proposed for synthesis of cyclic carbonates through the
cyclic intermediate using inonic liquids immobilized onto polyketones.110 6.13 1H-NMR spectrum of a reaction aliquot from the reaction of CO2
with styrene oxide, CO2with 1,2-epoxyhexane. . . 113 6.14 FT-IR spectrum of a reaction aliquot from the reaction of CO2with
styrene oxide, CO2with 1,2-epoxyhexane. . . 113
List of Tables
***
2.1 Size, PDI values, and zeta potential of PK-Im aggregates at different
pHs at a PK-Im concentration of 1 ⋅ 10−3M. . . 25
2.2 Size, PDI values, and zeta potential of PK-Im aggregates at different pHs at a PK-Im concentration of 1 ⋅ 10−4M. . . 25
2.3 Size, PDI values, and zeta potential of PK-Im aggregates at different pHs at a PK-Im concentration of 1 ⋅ 10−5M. . . 26
2.4 Final pH and component concentration of the formulation containing OA and PK-Im. . . 26
2.5 Final pH and component concentration of the formulation containing OA and PK-Im. . . 26
3.1 Amounts in the feed and carbonyl conversion (x) in the Paal-Knorr reaction between PK50 and HEDA. . . 32
4.1 Functionalization of PK50 with HEDA, IM, and PI . . . 50
5.1 PK50 and ABA feed ratios and the corresponding x. . . 81
6.1 Functionalization of PK50 and PK30 with IM. . . 105
6.2 Alkylation of PK50IM and PK30IM. . . 105
6.3 Cyclic carbonates conversion by PK50IM and PK30IM. . . 111
xi
Chapter 1
Introduction
***
1.1 Supramolecular systems . . . 1
1.1.1 Supramolecular systems . . . 1
1.1.2 Self-Assembly . . . 2
1.1.3 Active molecules . . . 3
1.1.4 Antibiotics . . . 4
1.1.5 Dyes . . . 4
1.1.6 Redox-active molecules . . . 6
1.2 Polymeric surfactants . . . 7
1.3 Polyketones . . . 8
1.3.1 The Paal-Knorr reaction . . . 10
1.4 Aim of the thesis . . . 10
1.1 Supramolecular systems, self-assembly and active molecules
1.1.1 Supramolecular systems
Supramolecular systems are complex architectures formed by molecular building blocks held together by noncovalent intermolecular forces such as hydrogen bond- ing, aromatic-aromatic interactions, hydrophobic interactions, metal-ligand coordi- nation, electrostatic interaction, and dispersion forces, among others. [1–7] There are two types of supramolecular systems: the so-called “supramolecular arrays”, which arise from the spontaneous organization of a large number of components, and the
“supermolecules” formed by the intermolecular association of few components. [8]
Supramolecular chemistry has been inspired and stimulated by living systems. [9,10]
1
Although biomolecules comprise essentially covalent bonds, they exhibit novel at- tributes and structures owing to the non-covalent interactions. The formation of the quaternary structure of proteins and the double helix of DNA are paradigmatic exam- ples of the noncovalent interactions value in biological systems. [11] Non-covalent interactions are considered to be labile and dynamic. Therefore, the linking of molecules via noncovalent intermolecular forces may result in self-assembled supra- molecular systems with adaptive response to diverse stimuli. [12–14] Indeed, these forces are necessary for the fabrication of stimuli-responsive materials with the po- tential of self-repair and rectification of defect as in living systems. For instance, supramolecular polymers are endowed with stimuli-responsive attributes. [1, 15] In addition, the reversibility in the supramolecular system is crucial for some function displayed such as chemical sensing [16, 17] and catalysis. [18, 19]
1.1.2 Self-Assembly
The creation of nanostructured materials with dynamic and refined functions is vital for nanoscience and nanotechnology. Nowadays the production of nanostructures can be performed by two strategies, i.e. “top-down” and “bottom-up” nanofabri- cation. [20] In reality, the significant progress in the fabrication of nanomaterials mainly stems from the improvement of top-down microfabrication techniques, since they allow designing extremely complex functional geometries and patterns with a high precision. [21, 22] Nevertheless, this approach encounter significant manu- facturing limitations due to the difficultness to reach geometries and patterns lower than 100 nm (for instance photolithography), it is a intrinsically two-dimension tech- nology, and their expensive fabrication process. [23, 24] Consequently, bottom-up process have become prominent and relevant alternative approaches. [25, 26] They rely strongly on self-assembly of functional molecules based on supramolecular chemistry principles. [27, 28] Supramolecular self-assembly, also called molecular self-assembly, consists in the spontaneous organization of a group of pre-designed molecules without human intervention, leading to the formation of complex supra- molecular entities and functional structures. [24, 26, 28–30] The self-assembly ap- proach has been used for the fabrication of polymeric micelles, [31] nanoreactors, [32] and self-assembled monolayers. [33] It is also important in life; many organelles, folded proteins, lipid membranes, nucleic acid are formed by self-assembly. This synthetic approach can be classified as static and dynamic. [24, 26, 34, 35] Static self-assembly occurs in absence of external influences and the spontaneous organi- sation of the building block in ordered systems is driven by the energy minimization, therefore, the system is at equilibrium and does not dissipate energy. [20, 24] For this type of self-assembly, it is very important to control the noncovalent interac- tion to form steady and well-defined structures. In other words, the molecules in a self-assembled aggregate must be connected by as many noncovalent interactions as possible in order to drive the system towards the most energetically favourable state, thus overcoming the competing interaction with the solvent. Moreover, they have to
1.1. SUPRAMOLECULAR SYSTEMS 3 overcome the favourable entropy that occurs when the aggregate is fragmented into a dissociate state. [28] Dynamic self-assembly (out-of-equilibrium systems) [36–39]
is the less studied of the two self-assembly structures and is found mainly in living systems. It depends strongly on the dissipation of energy and on the presence of an external stimulus; the system is organized by reaching an energy minimum due to the influx of energy in the system, which is subsequently dissipated via an entropic pro- cess directly correlated with the interaction of the system building blocks. [40] Once the influx of energy disappears, the system may dissemble. [20] The best example of dynamic self-assembly can be found in a cell; it decreases the entropy by consuming energy from the environment (for instance in the form of food). However, stopping the source of energy (food) to the cell, it might dies. [24] An examples of dynamic self-assembled structures are magnetohydrodynamic self-assembly. [37] These dy- namic structures depend on a continuous energy supply and its dissipation to the environment, and therefore, when the flow of energy conclude they breakdown. [40]
Static and dynamic self-assembly can be subdivided into hierarchical self-assembly, co-assembly and direct self-assembly. Hierarchical self-assembly systems, mainly found in nature, are constructed by the self-organization of molecular components in a first order assembly that becomes the foundation structure for a bigger assembly (second order), and so on, “growing up” in complex functional structures. [41–43]
Co-assembly refers to the association process of different building blocks for the fabrication of a supramolecular system that it is not possible to produce by the self- assembly of an isolated molecule. In addition, this system may possess the prop- erties and/or functionalities of their constituents. [44–46] Direct self-assembly is a self-assembly process influenced by an external stimuli or a template. [47] For in- stance, a pre-patterned surface can be used to control the orientation and position of the building blocks in the fabrication process of a new nano-object. [48] Another self-assembly strategy, which is directly related to the noncovalent interactions, and used through this thesis, is the ionic self-assembly [49, 50] also called electrostatic self-assembly. [51–53] This is an interesting approach to produce supramolecular structures due to simplicity, cheapness, and flexibility. [54] This uses the electro- static interactions between charged molecules and it is accompanied by a coopera- tive binding mechanism. This means that the first bond stimulates the formation of the next one and this process is propagated towards the formation of a supramolecu- lar structure. [54, 55] In addition, other noncovalent interaction can be present such as aromatic-aromatic interaction, hydrogen bonding, and hydrophobic interaction, among others.
1.1.3 Active molecules
The main goal of supramolecular chemistry and self-assembly techniques is the design of systems with a desired structure and specific functionalities via the self- assembly of molecular building blocks in certain conditions. [56] To produce such systems, one often relies on the noncovalent interactions between molecules and in
their chemical properties. [20, 57, 58] Therefore, the first step of self-assembly is the synthesis of molecules (building blocks) with specific dimensions, forms, and chem- ical characteristics such as hydrophobicity, hydrophilicity, polarizability, charge and functionality. [11, 28] These “functional molecules” may possess photoactive, elec- troactive, ion-active or switching features. In this thesis, we made use mainly of bi- ological (antibiotic), redox, and photo (dye) organic active-molecules for the forma- tion of self-assembly structures with pH-sensitive polymer surfactants via the elec- trostatic self-assembly approach. These active molecules will be briefly discussed below.
1.1.4 Antibiotics
Antibiotics are biologically active molecules of natural origin or synthetics that are capable of inhibiting the growth (bacteriostatic) or kill bacteria (bactericidal). [59]
They are still used to treat bacterial infections in human and veterinary medicine. [60]
The action mechanisms of antibiotics are generally classified in inhibition of the cell wall synthesis, [61] DNA replication and repair, [62] and protein synthesis of bac- teria. [63] The over-utilization and misuse of antibiotics have created an environ- mental pressure for bacteria to evolve resistance, which is currently a global health problem. [64] Indeed, the discovery of new antibiotics for the resistant pathogens is of paramount interest. [65] The encapsulation of antibiotics may provide a solu- tion to this problem as it protects the drug from the environmental condition while, at the same time, resulting in a controlled release and a targeted delivery. [66, 67]
Therefore, this is an option to decrease the antibiotic dosage and, as a consequence, it would help to control bacterial resistance. Antibiotics have been encapsulated in liposomes, polymeric nanoparticles, solid lipid nanoparticles, dendrimers, and poly- mersomes. [68–71] The encapsulation of antibiotics can be accomplished by the electrostatic self-assembly method due to some antibiotics (for instance quinolones, antibiotics used in the second chapter of this thesis) are composed of ionic and aro- matic groups. In addition, the aromatic planar architecture, the ionic character, and the hydrophobicity, which can be controlled externally by adjusting the solution pH due to the presence of weak acid groups, are essential characteristic that can be used for the encapsulation in polymeric nanostructures.
1.1.5 Dyes
Dyes are among the most import photoactive molecules for the humankind. They have been applied during our history mainly for imparting colour to fabrics. [72–74]
In fact, they are described as “coloured substances, which are soluble or go into solution during the application process and impart colour by selective absorption of light”. [75] Nevertheless, with the recent technological advancements, they are being used in different areas either at the scientific or industrial level. Depending on the
1.1. SUPRAMOLECULAR SYSTEMS 5
N N HO
Auxochrome Chromophore Chromogen
Figure 1.1: Schematic representation of the components of an organic dye molecule, 4-Hydroxyazobenzene. [75]
type of dye, namely its the molecular structure, aggregation attributes, solvent sol- ubility, absorption and fluorescence characteristics, application possibilities might involve photodynamic therapy, [76] solar cells, [77] biologic probes, [78, 79] ink-jet printing, [80] dermatology, [81] and photography, [82, 83] among others. Organic dyes molecules are constituted by a chromophore, a chromogen, and an auxochrome (Figure 1.1). [84] They also must possess a structure with alternating double and single bonds (conjugate system) and exhibit resonance of electrons, which is a sta- bilizing force since it lower the potential energy of the molecule. Overall, all the components and chemical characteristics provide to the dye the capacity of absorb- ing light in the visible range (400-700 nm); if one of them is missing the molecule loses the property of absorbing visible light (the colour is lost). [75] The components of the dye may play others important roles besides being responsible for the ability to absorb visible light. For instance, the auxochrome (so-called the colour helper since they can shift the colour of the chromogen) can be ionic groups such as carboxylic acid, hydroxyl, amino, and sulfonic acid groups, provide to the dye the solubility in water and the ability of interact with other molecules by means of electrostatic interactions.
Dyes have been used to form supramolecular structures. In fact, depending on the dye concentration and environmental conditions, dyes might self-assemble to- wards aggregates with different absorption and fluorescent characteristics in com- parison to their monomeric form. They can be aggregate either as J-aggregates or H-aggregates. The former is characterized by a new sharp absorption band, which is red shifted with respect to the long-wavelength absorption band of the monomer (bathochromically shifted) and with a high absorption coefficient, and resonance flu- orescence. [85–87] Conversely, aggregates with absorption bands blue shifted (hyp- sochromically shifted) in comparison to the monomer band and with small or no fluorescence are called H-aggregates. [88] Supramolecular structures have also been produced by the association of water-soluble polymers and dyes through the electro-
static self-assembly approach. [89] It has been found that polyelectrolytes enhance the aggregation of the dye since they induce a high local dye concentration by means of long-range electrostatic interactions. [90,91] In contrast, aromatic polyelectrolytes avoid the dye aggregation due to the specific short-range aromatic-aromatic interac- tions. [92, 93] The dye dispersion provided by these polymers rely on the linear aro- matic density and flexibility of the polymer, and the relative concentration between the polymeric charge aromatic group and the dye. [90, 94] Furthermore, the for- mation of polymer/dye supramolecular structures may improve the qualities of the dyes. For instance, the polyelectrolyte-porphyrin nano-assemblies display higher catalytic activity in comparison to the porphyrin not interacting with the polymer template. [53] Moreover, the improvement of phototoxic reactive oxygen species (ROS) production via the type I mechanism upon irradiation of the complex com- posed by an aromatic polyelectrolyte and methylene blue has also been reported.
This is explained by the ability of the polymer to prevent the dye aggregation and enhance the dye-to-dye contact. [76]
1.1.6 Redox-active molecules
Oxidation-reduction reactions (redox) involve the transfer of electrons between atoms, ions, or molecules. These redox species can act as reducing or oxidizing agents; the former is a species that donates electrons and the latter accepts them. In this sense, active-redox molecules, also mentioned in the literature as electron donor-acceptor (D-A) molecules, can be described as molecules that are able to donate or receive electrons. Furthermore, these molecules possess the ability to adjust their molecu- lar conformation and physicochemical characteristics reversibly when they undergo redox reaction. [95–97] Redox-active molecules can be synthetic or present as such in nature. For instance, redox proteins, which are constituted of connected catalytic sites (defined as multi-electron redox centres or single-electron redox centres that in- teract with substrates and serve as reservoirs or sinks of electrons), perform essential functions in biological processes that request electrons transfer such as photosyn- thesis, respiration, and metabolism. [98] Other cases of natural D-A molecules are antioxidants or antiradicals (for instance carotenoids and flavonoids). [99, 100] Their function is to reduce free radicals by transferring electrons (becoming oxidized), and therefore, inhibits that other molecules endure an oxidation reaction with these free radicals. [101] In addition, antioxidants may also accept an unpaired electron from the free radical, becoming reduced and the free radical oxidized. [102] Synthetic D- A molecules have been used to produce redox-fluorescence switch molecules, [103]
for the fabrication of batteries [104, 105] and memory devices. [106] In addition, there are synthetic redox-active molecules that can change their luminescent prop- erties when they experience a redox reaction. Due to the change from a colourless to coloured molecule after reduction, they have been used for the detection of bac- teria, [107] to quantify reducing carbonyl groups in cellulose, [108] to determine mammalian cell growth, [109] and for sensing metal ions. [110]
1.2. POLYMERIC SURFACTANTS 7 The synthesis of sub-micron or/and nanoparticles using redox-active molecules that can switch their solubility and luminescence properties in aqueous solution (i.e become hydrophobic and coloured reversibly) through their reduction/oxidation, and in addition, stabilized by ionic or anionic surfactants or polymeric surfactant, is a promising approach for the production of smart stimuli-responsive materials.
1.2 Polymeric surfactants
Polymers are macromolecules consisting of covalently bonded segments, referred to as monomers. They can be grouped in a general form as natural polymers (for in- stance polysaccharides, proteins and nucleic acids) and synthetic polymers, which include common plastics and adhesives. Other types of polymers are amphiphilic polymers [111, 112] also known as polymeric surfactants [113] (surface active poly- mers) or micellar polymers [114], which can be found in nature or synthesized through diverse polymerization reactions. Natural surfactants or biosurfactants (for example lipopolysaccharides) are abundant in nature and can be obtained either from the plant and animal kingdoms. These types of surfactant are utilized by the biolog- ical systems to overcome solubility problems, as emulsifiers and dispersants, and to modify surfaces. [115] They are biodegradable, available from renewable sources, and show low toxicity. [116, 117] Nevertheless, they exhibit separation problems from the natural source, as a result the separation and isolation cost exceeds the cost production of synthetic surfactant. [118] In view of this, the majority of scien- tific investigations and industrial applications have been focused on synthetic surfac- tants. Indeed, the latter have been used for the preparation of emulsions and suspen- sions, [119, 120] drug delivery systems, [121, 122], in photodynamic therapy, [123]
and oil recovery. [124–126]
Polymeric surfactants are made of lyophilic and lyophobic segments (also named as hydrophilic and hydrophobic segments when the solvent is water) linked together by covalent bonds. Due to their amphiphilic properties, they are capable of:
- decreasing the surface tension of the solvent and the interfacial tension with another phase; [127]
- being absorbed by surfaces and interfaces such as dispersed solid or liquid phases; [128, 129]
- self-assembly as micelles when the polymer concentration reaches a certain value, called the “critical micelle concentration” (CMC).
In a micelle, the lipophilic segments are in the interior, constituting the hydropho- bic core, and the polar segments are directed to the solvent, making the hydrophilic corona. In the case that the solubility of the polymer in aqueous medium depends
on external stimuli such as pH and temperature, the polymer might show a stimuli- responsive behaviour. [130] For instance, pH-responsive polymers contain acidic or basic groups, they can accept or release protons by adjusting the pH, resulting in an alteration of the hydrophilic-hydrophobic balance. [131] Thermosensitive polymers assume a water-soluble extent coil conformation at low temperatures. However, as the temperature increase, the water molecules are released in bulk, following the associative contact between the hydrophobic segments of the polymer. [132, 133]
The macromolecular nature of polymeric surfactants permits a diverse and com- plex distribution of hydrophilic and hydrophobic segments. [114] Indeed, the hy- drophobic pendant groups may be grafted onto a hydrophilic polymer backbone or, conversely, the hydrophilic pendant groups grafted onto a hydrophobic backbone. A different approach is the alternating arrangement of the hydrophobic and hydrophilic monomers. Besides, these methods to synthesize surface active polymers can be combined. [134] The diverse polymeric surfactant architectures have been classi- fied in different groups such as block copolymers, stars and graft copolymers, and amphiphilic dendrimers. All those polymers are identified by possessing large and well separated hydrophilic and hydrophobic monomers. [114] On the other hand, other polymeric surfactants exhibit an architecture where hydrophilic and hydropho- bic segments are scattered all over the macromolecule; these polymers are referred as polysoaps. They are defined as polymers that contain surfactant-like segments in the polymer repeating unit. [118]
A fundamental requirement for several applications is the precise control of the polymeric surfactant in terms of composition and polymer structure, molecular weight, and balance between the hydrophilic and hydrophobic segments. The best candidates to meet those criteria are the living radical polymerization approaches such as atom transfer radical polymerization (ATRP), [135, 136] reversible addition- fragmentation chain transfer polymerization (RAFT), [137, 138] and nitroxide- mediated polymerization (NMP). [139] Nonetheless, these polymerization methods are expensive in comparison with other approaches. In this thesis, we present the easy and cheap production of polymeric surfactants by the incorporation of hy- drophilic pendant groups onto a hydrophobic polyketone via the Paal-Knorr reac- tion. In addition, these amphiphilic polymers were used as building blocks for the production of smart colloids via the electrostatic self-assembly method, since they display pH stimuli-responsive properties (weak polyelectrolytes).
1.3 Polyketones
Aliphatic polyketones are a relatively new type of polymer. They can be synthesized in three different methods. In two of them, the copolymerization of CO with olefins is promoted by γ-rays or radicals. [140] They have not found widespread utilization owing to need harsh reaction conditions and display poor control of the structure and properties of the resultant polymers. For instance, the first attempted copolymeriza-
1.3. POLYKETONES 9 O
O R
R
R = H, CH3 O
O
O R
R
Figure 1.2: Scheme of aliphatic polyketone.
tion of CO with ethylene was achieved by Farbenfabriken Bayer using free-radical polymerization. It yields a random aliphatic polyketones by using extreme reaction conditions (230○C and 2000 atm). [141] On the other hand, a third approach, which uses a catalyst that is based on transition metal compounds, gives as result a copoly- mer with a precise alternating structure. The first alternating aliphatic polyketones was reported by Reppe and Magin by using milder condition (230○C and 200 atm) and K2[Ni(CN)4] as catalyst. [142] There was no technological breakthrough until a homogeneous palladium-based catalytic system was developed by Shell researchers of the 20thcentury. The economic requirements for industrial-scale production were achieved by using this system, which was 6 (kg of polymer) (g of Pd)-1h-1 under mild conditions (90○C, 4-5 MPa). [143]
The copolymerization of CO and ethylene using palladium derivatives as catalyst yield the synthesis of semicrystalline polyketones which are the simplest members of these type of polymers. They possess a relatively high melting point close to the thermal decomposition temperature. In addition, they are insoluble in common organic solvents and dissolve in highly polar and acid solvents. Overall, it makes their processing complicated. In order to improve the processability of polyketones, propylene is incorporated as a monomer at a desired molar ratio (Figure 1.2). The ter- polymerization brings disorder to the molecular packaging and, therefore, decreases the melting point of the polymer.
Polyketones exhibit important properties that make them interesting polymers.
Their properties include:
- good chemical resistance towards solvent, acid and bases;
- impermeability to hydrocarbons;
- low costs (e.g. carbon monoxide is a low-cost and easily accessible monomer);
- relatively high reactivity, with the reactive carbonyl groups endowing the poly- mer with photo-and biodegradability.
Due to the presence of the carbonyl groups, polyketones can be utilized as pre- cursors for the production of functional polymers via the chemical modification ap-
proach. Polyketones can readily be converted into a variety of polymers that contain functional groups such as bisphenols, [144] alcohols, [145] and ketals, [146] among others. Recently, the synthesis of pyrroles via the Paal-Knorr reaction has attracted special attention due to the absence of any catalysts and, more generally, of demand- ing reaction conditions.
1.3.1 The Paal-Knorr reaction
The Paal-Knorr condensation is a straightforward reaction for the synthesis of func- tional polymers. The mechanism of this reaction consists of the pyrrole ring forma- tion via the double condensation reaction of a primary amine group with the polyke- tone 1,4 di-ketone group (Figure 1.3). [147] Generally, the first step of the Paal- Knorr reaction is the addition of an amine to one of the carbonyl groups forming a hemiaminal (molecule number 2 in Figure 1.4). Afterwards, the second carbonyl is attacked by the nitrogen, resulting in the cyclization of the hemiaminal. Finally, the ring undergoes dehydration to yield the pyrrole ring (Figure 1.4). [148] In our research group, polyketones chemically functionalized via the Paal-Knorr reaction, have been applied to the production of self-healing materials, [149–151] emulsions with adhesive properties, [152, 153] coating materials, [154] and water-insoluble resins. [155, 156] This reaction shows several advantages such as the absence of solvent and catalysts, it is carried out in one-pot, it yields water as the single by- product, and it displays fast kinetics and relatively high yields. In particular, up to 80% carbonyl conversion (an upper limit due to the reversibility of the reaction and statistical reasons), the amine conversion is practically quantitative, thus avoiding also the need for any purification step of the final product. In addition, the reaction can readily be performed either in the laboratory or on larger scales. The num- ber of primary amines that may be used to functionalize polyketones is factually unlimited, making this easy synthesis a versatile method to prepare polymers with almost any desired pendant functional group. [147, 149, 150, 157] In this thesis, we proved that the Paal-Knorr reaction on a polyketone with different primary amines represents a simple, low cost, and straightforward method to produce polymeric sur- factants. The architecture of these amphiphilic polymers is based on a hydrophobic backbone consisting of aliphatic 1,4-dicarbonyl units and N-substituted pyrrole moi- eties. The hydrophilic pendant groups are provided by the primary amine chosen for the polyketone derivatization. In this thesis the pendant groups also display pH- responsive properties.
1.4 Aim of the thesis
This thesis is focused on the preparation of polymeric surfactants with pH-responsive properties by using an alternating aliphatic polyketone as starting materials and the Paal-Knorr condensation as synthetic route. These polyketones are used as building
1.4. AIM OF THE THESIS 11
O
O R
R
R = H, CH3
+
N
R R
O R
O R
x y
Paal-Knorr reaction
100 0 C
R' NH2
R'
R': Functional group
Figure 1.3: Schematic representation of polyketones chemical modification via the Paal-Knorr reaction.
RNH2 +
O O H3C CH3
R R
1
O HN H3C CH3
R R
2 R
OH N
R R
OH CH3
R HO H3C
3
- H2O
N R R
R HO H3C
4 N
R R
R 5
CH3
CH3
H3C - H2O
Figure 1.4: Schematic representation of the Paal-Knorr reaction (modify and simpli- fied from Amarnath. V and co-workers [148]).
blocks for the fabrication of supramolecular entities with applications in the encap- sulation of hydrophobic molecules, dispersion of dyes, and synthesis of redox-active nanoparticles.
Chapter 2 focusses in the encapsulation of two types of antibiotic (Oxolinic Acid and Flumequine), extensively used in Chilean salmoniculture, in polymeric micelles, using a pH-sensitive polyketone bearing imidazolium pendant groups. The encapsu- lation process was conducted in water, not using organic solvents, which is a valuable addition from a green chemistry perspective.
Chapter 3 describes the dispersion and aggregation of 5,10,15,20-tetrakis- (sulfonatophenyl)-porphyrin in an acid aqueous environment by using amphiphilic polyketone derivatives. Particularly, polymers with higher hydrophobicity (fewer functional groups) could prevent the aggregation of the dye at lower values of pH in comparison to polymers bearing higher incorporation of functional groups. Among several factors, the electrostatic interactions and preferential solvation of the dye by the polymer chain explain the stabilization and dispersion of the dye.
Chapter 4 describes the design of micro- and nano-polymeric complex micelles composed of polyketones bearing different pendant groups: a heteroaromatic amine, a heterocyclic aliphatic amine, and an aliphatic amine containing a hydroxyl group, and the dye 5,10,15,20-tetrakis-(sulfonatophenyl)porphyrin. In addition, it shows the control of the structure of supramolecular entities by changing the pH of the solutions.
Chapter 5 shows a simple, cheap and “green” method for the synthesis of nanopar- ticles of colored organic nanoparticles by the reduction of the tetrazolium salt (TTC) to the corresponding red formazan (TF) in the presence of aromatic polyketones. It demonstrates that aromatic-aromatic interactions between the functional groups of the polymers and the precursor molecule (TTC) are pivotal in the nanoprecipitation process.
Chapter 6 shows the flexibility of the Paal-Knorr reaction in the synthesis of func- tional polymers via the chemical modification of aliphatic polyketones. Two new applications are presented: the synthesis of polymer supported ionic liquids based on aliphatic polyketones and their utilization for the production of cyclic carbonates;
and the application of two amphiphilic polymers, with a pH-responsive character, in the formation of water in oil emulsions.
Chapter 2
Novel polyketone with pendant imidazolium groups as
nanodispersants of hydrophobic antibiotics 1
***
2.1 Introduction . . . 14 2.2 Experimental . . . 16 2.3 Results and discussion . . . 18 2.4 Final remarks . . . 24 2.5 Conclusions . . . 24
Abstract. In this work, we present a new method for the encapsulation of hy- drophobic antibiotics by the use of a pH-sensitive polyketone with amphiphilic char- acteristics. The polymer bears imidazolium groups as weak acids; therefore, its solvophobic properties can be tuned by changing the pH. The antibiotics were sol- uble in aqueous media at values of pH higher than 7.0 and the polymer was soluble in aqueous media at values of pH lower than 5.5. Nanoprecipitates were formed by mixing the polymer solution at pH 5.4 and the antibiotic solutions at pH 10.5. They displayed hydrodynamic diameters between of 45 and 360 nm and positive zeta po- tential values, depending on the constituent concentration. The out-of-equilibrium process occurring upon mixing both solutions produces pH changing, molecular ar-
1This chapter is based on: Araya-Hermosilla, E., Orellana, S. L., Toncelli, C., Picchioni, F., &
Moreno-Villoslada, I. (2015). Novel polyketones with pendant imidazolium groups as nanodispersants of hydrophobic antibiotics. Journal of Applied Polymer Science, 132(32).
13
rangement, and a controlled collapse of the system in the form of nano- and submi- cron particles. The driving forces for the arrangements can be hydrophobic forces, long-range electrostatic interactions, and short range aromatic-aromatic interactions.
Keywords. Functionalization of polymers; antibiotics; nanostructured polymers;
pH-sensitive polymers.
2.1 Introduction
Amphiphilic polymers [158, 159] can form several structures in the nano and micro- metric range, such as unimolecular micelles and multimolecular aggregates. [160, 161] Tunable amphiphilic polymers constitute a new class of materials for practical applications. By tuning the amphiphilic behavior of polymers, stimuli-responsive materials may be obtained. For instance, poly(N-isopropylacrylamide) is recog- nized by its temperature-sensitive solvophobic properties. [162] The collapse of water-soluble polymers and consequent precipitation is an out-of-equilibrium pro- cess [163–165] that may be induced as a response to changes in environmental condi- tions that modifies the hydrophilic properties of the polymers. [166–168] Changes in the solution pH can result in the collapse of weak polyelectrolytes undergoing acid- base equilibrium. [131, 169–172] In this context, copolymers based on acrylic acid and methyl methacrylate have been tested as coatings of pharmaceutical products for oral administration due to their pH-sensitive properties. [173] Polychelatogens may undergo phase separation when their binding sites become saturated with metal ions.
[174] The amphiphilic characteristics of polyelectrolytes may be tuned whether ion pairs or hydrophobic complexes are formed with organic counterions. [90, 93, 175]
These charged organic counterions may induce the collapse of polymeric systems when they experience secondary interactions such as short-range electrostatic inter- actions, hydrogen bonding, and aromatic-aromatic interactions. [51, 157, 176, 177]
The aggregation of polyelectrolyte chains via secondary interactions may result in structures with defined features, such as shape, size, and zeta potential. [51,176–178]
Controlling the solution properties may allow tuning some of these characteristics, and can lead to a controlled polymer collapse. The same principles may be applied to other out-of-equilibrium processes such as the formation of polyelectrolyte deposits upon solvent evaporation. Polyketones constitute an interesting class of polymeric materials. Perfectly alternating copolymers of carbon monoxide and unsaturated hydrocarbon monomers (such as propylene and ethylene) can be easily obtained us- ing palladium derivatives as catalyst. The resulting polymers bear 1,4-dicarbonyl moieties that can be easily functionalized with a primary amine through the Paal- Knorr reaction. [147,149,150,157] Upon this reaction, the 1,4-dicarbonyl groups are transformed into N-substituted pyrrole groups. The pyrrole groups become part of the main chain, providing rigidity and hydrophobicity, whereas different functional
2.1. INTRODUCTION 15
O R
O R Paal-KnorrReaction N
R R
O R
HN N NH2
HN N
PK50 Histamine PK-Im
R = H (50 mol%), CH3 (50 mol%)
Figure 2.1: Schematic representation of the chemical modification of polyketones (PK50) via Paal-Knorr reaction with histamine.
groups may be incorporated in the pendant groups, providing different functionali- ties and physicochemical properties. This reaction is a solvent-free one-pot reaction, which allows fine tailoring of the polymer properties, and yields water as the sole by- product. Alternated (ethene/propene)/CO polyketones have been recently functional- ized with histamine to produce a polyketone with imidazolium pendant groups (PK- Im) [157] (Figure 2.1). This aromatic cationic polymer demonstrated to bind and dis- perse 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin, whereas aliphatic cationic polymers trigger the dye self-aggregation. These results proved the importance of hydrophobic and aromatic- aromatic interactions on the binding properties ob- served. [157]
The formation of nanostructures that contain target molecules is interesting for many purposes. Nano-formulated antibiotics represent an advantage for pharmaceu- tical applications, in terms of bioavailability, distribution, and efficacy. Some antibi- otics possess charged aromatic groups, so that they are prone to undergo aromatic- aromatic interactions. In particular, quinolones are broad-spectra bactericidal com- pounds, for which adequate vehiculization would result in improved environmental and biological security. Their antibacterial mechanism is based on the inhibition of DNA-gyrase, which renders an unstable condensation and configuration of the bacterial DNA during the cell division process. [179] The quinolones oxolinic acid (OA) and flumequine (FLU) (Figure 2.2) are commonly used in aquaculture as pro- phylactics to prevent diseases or as chemotherapeutic agents to control diseases. OA and FLU present a low bioavailability to aquatic animals, which may result in high concentration of antibiotic residues in aquatic environments. [180] Their overuse produces bacterial resistance, the prevalence of diseases, [181] and risks to human health. Both antibiotics are relatively insoluble in water. Therefore, the development of encapsulation methods for OA and FLU may improve both animal and human health treatments, with a minimal environmental impact. Nano-formulated antibi- otics may be incorporated in pellets to feed fishes, which can improve performances
N O HO
O
O O
N F
O HO
O
AO FLU
Figure 2.2: Structure of oxolinic acid (left) and flumequine (right).
such as minimizing loss of drug in the feces, decreasing the dosage frequency, and protecting the encapsulated drug from undesired effects of external conditions. [147]
All these advantages may have positive consequences in aquaculture as well as in human health therapies and environmental issues. Current techniques to encap- sulate hydrophilic drugs within polymeric micro- and nanostructures involve dou- ble emulsion methods, [182] layer-by-layer (LbL) assembly, [183, 184] inclusion in polymer matrices such as polymer nanoaggregates, and in polymer micelles. [185]
In most cases, these methods require the use of organic solvents to dissolve low water-soluble components, [186–189] which would further assemble upon solvent displacement and evaporation. Given that PK-Im may produce aromatic-aromatic interactions with aromatic counterions, and the need to generate nanoparticles con- taining antibiotics, the aim of this study is to explore the ability of PK-Im to bind and disperse OA and FLU in an out-of-equilibrium process where organic solvents are not used and in which, upon acid-base neutralization reaction, molecules change their hydrophilic/hydrophobic properties and rearrange in order to produce nanos- tructures. It will be shown that nanoprecipitates composed of this polymer and target antibiotics can be formed by controlling the mixture process, so that a final pH close to neutrality is achieved. Dynamic light scattering and UV-vis spectroscopy will be used to observe these processes.
2.2 Experimental
Reagents
Histamine (CAS 51-45-6) (Sigma-Aldrich), OA (CAS 14698-29-4) (Sigma-Aldrich), and FLU (CAS 42835-25-6) (Sigma-Aldrich) were commercially available and used as received.
2.2. EXPERIMENTAL 17 Equipment
Distilled water was deionized by a Simplicity Millipore deionizer. The pH was controlled on an UltraBasic Denver Instrument pH meter. UV-vis measurements were performed in a Hekios c spectrophotometer. Size and zeta potential measure- ments were performed in a zetasizer Nano-ZS (Malvern Instruments) with backscat- ter detection (173○), controlled by the Dispersion Technology Software (DTS 6.2, Malvern).
Procedures
PK-Im Synthesis. The alternating aliphatic polyketone (PK50), precursor of PK- Im, was synthesized according to Mul, et al. [190] using carbon monoxide and both ethylene and propylene as hydrocarbons. The resulting terpolymer presents a total olefin content of 50% of ethylene and 50% of propylene (PK50, Mw 5350 Da). Its formula weight considering the diketone moieties was calculated to be 126 g/mol.
The synthesis of PK-Im was carried out by means of the Paal-Knorr reaction of PK50 with histamine, according to the scheme shown in Scheme 1. About 40 g of the polyketone PK50 was weighed into a glass reactor fitted with a mechanical stirrer and a reflux condenser. The reactor was heated to a temperature of 110○C and histamine was added dropwise during the first hour. Stoichiometric amounts of amine and dicarbonyl moieties have been used. The reaction was allowed to proceed for another 3 hours. The reflux condenser was removed in the last hour to allow the produced water to evaporate. The obtained dark brown product was ground into small particles (freezing the material if necessary). The obtained particles were washed with Milli- Q water, and then freeze-dried for 24 h. The resulting polymer was characterized spectroscopically and by elemental analysis, presenting 1,4- dicarbonyl conversion of 71.1%, and a calculated formula weight per mole of ionizable amine groups of 266 g/mol. Details on the polymer characterization and formula weight calculation can be found elsewhere. [157]
Solubility Studies. Aqueous stock solutions of PK-Im, OA, and FLU were pre- pared at concentrations of 1 10−2and 1 ⋅ 10−3M, respectively. In order to dissolve the reactants, the pH of the stock solutions was adjusted to 5.4, 10.5, and 11 for PK- Im, OA, and FLU, respectively. Then, the solubility of the pristine antibiotics as a function of the pH was searched by turbidimetry in a UV-vis spectrometer using a quartz cuvet with a path length of 1 cm, and registering the signal at 400 nm, where OA and FLU do not absorb. The solubility test was conducted diluting the antibiotic stock solutions to 1 ⋅ 10−3, 2 ⋅ 10−4, and 1 ⋅ 10−5and successively lowering the pH with the aid of aqueous HCl. The obtained data were plotted with the OriginPro 8 software, and non-linear fitting was done using the Boltzman sigmoidal function.
The solubility of PK-Im was also studied. Solutions of the polymer were prepared at concentrations of 1 ⋅ 10−3, 1 ⋅ 10−4, and 1 ⋅ 10−5M and pH 3. DLS responses (intensity
distributions) were registered at 25○C for these solutions after adjusting the pH from 3 to 7. A laser beam operating at 633 nm was used and detection was done at a fixed angle of 173○. Each measurement was done in triplicate. A multimodal analysis was used for data treatment. Results were considered valid under the DTS 6.2 software criteria.
Nanoprecipitate Formation. Nanoprecipitates of PK-Im, PKIm/OA, and PK- Im/FLU were obtained by adjusting physicochemical parameters of the correspond- ing stock solutions. The pH and concentration of the stock solutions allow producing different mixtures whose final pH value was close to neutrality. The mixing protocol consisted on pouring dropwise under vigorous stirring an amount of the antibiotic solution into the polymer solution until the desired concentration was reached. The final concentration and pH values of the resulting mixtures are listed in support in- formation Tables 2.1 and 2.3. The mixtures were controlled by DLS as described above.
2.3 Results and discussion
Solubility of OA, FLU, and PK-Im. The solubility of OA and FLU in water was tested at three different concentrations (1 ⋅ 10−3, 2 ⋅ 10−4, and 1 ⋅ 10−5M) as a func- tion of pH. The relative intensity of scattering at different pH values is plotted in Figures 2.3 and 2.4 . Fitting the data to a Boltzmann sigmoidal curve and further derivation allows finding the transition pH at which the antibiotics precipitate at the different concentrations, related to their acid-base equilibrium for which the car- boxylic group is responsible (Figure 2.2). It can be seen that OA needs more basic conditions to be dissolved than FLU. At a concentration of 1 ⋅ 10−3M OA is soluble from pH 9.0, and 8.6 at a concentration of 2 ⋅ 10−4M. At a concentration of 1 ⋅ 10−5, OA was soluble at all the pH tested. FLU shows a solubility at values of pH higher than 7.6 at a concentration of 1 ⋅ 10−3M, and higher than 7.0 at a concentration of 2 ⋅ 10−4 M, while at a concentration of 1 ⋅ 10−5 M FLU was soluble at all the pH tested.
The solubility of PK-Im at three different concentrations was tracked by DLS in order to follow the collapse of the cationic polymer by increasing the pH and conse- quently deprotonating the imidazolium groups. The results can be seen in Figure 2.5 and Tables 2.1 - 2.3 (support information). At pH 3, the imidazolium groups are highly protonated, and the polymer is more hydrophilic. A low Tyndall effect was found for all the samples, revealed by a low intensity of scattering, which is lower at lower concentrations. Increasing the pH changes the DLS responses, as can be seen in Figure 2.5, where the corresponding correlograms are shown. Monomodal particle size distributions were found. The results show that at low pH such as pH 3, the polymer shows amphiphilic properties and undergoes self-aggregation. When the pH is increased, the imidazolium groups deprotonate, decreasing the repulsive
2.3. RESULTS AND DISCUSSION 19
Figure 2.3: OA precipitation pH at 1 ⋅ 10−3M (a) and 2 ⋅ 10−4M (b).
Figure 2.4: FLU precipitation pH at 1 ⋅ 10−3M (a) and 2 ⋅ 10−4M (b).
electrostatic interaction between segments. Therefore, some parts of the polymeric chains become more hydrophobic and, as a result, the process of aggregation to form a hydrophobic core is enhanced. At pHs between 5.5 and 7.5, an intense Tyndall ef- fect is observed (see Figure 2.5), and aggregates of hydrodynamic diameters ranging in the submicron size are formed. They present a positive zeta potential high enough to allow stability at pHs near neutrality (see support information Tables 2.4 - 2.5).
The corresponding correlograms show monoexponential decays. At pHs higher than 7.5, the deprotonation of the polymer induces its precipitation in the form of macro- precipitates.
PK-Im/OA and PK-Im/FLU Nanoparticles. Nanoprecipitates were obtained by mixing a 10−2 M PK-Im solution at pH 5.4 and 10−3 M OA or FLU solutions at pH 10.5 and 11, respectively. The final pH of the mixtures reached values near neutrality, as can be seen in Tables 2.4 and 2.5 (support information). The final concentration of the reactants can be correlated to the results of solubility described above. Mixtures containing only the antibiotics OA and FLU at a concentration of 2 ⋅ 10−4M precipitated as visible macroparticles at values of pH under 8.6 and 7.0, respectively. On the other hand, in the presence of PK-Im, the mixtures containing the antibiotics at comparable or higher concentrations appeared as nanoprecipitates instead of macroprecipitates, when the pH of the mixtures ranged between 6.7 and 7.5, as can be seen in Figure 2.6, where results obtained by DLS are shown. DLS results showed monomodal intensity distributions. During the mixing process, the collapse of the polymer is accompanied by the co-precipitation of the antibiotic. The size of the precipitates takes values at the submicron scale, which indicates that the antibiotic may be embedded in the polymeric matrix.
The results concerning size and zeta potential of the nanoparticles have been plot- ted in Figure 2.6 as a function of the antibiotic/ polymer ratio. For OA the resulting pH ranged between 6.7 and 7.5, and the respective particle size ranged between 49.4 and 71.8 nm, whilst the zeta potential ranged between 26.3 and 34.5 mV (Figure 2.6 left). The nanoparticles found present smaller hydrodynamic diameters than those corresponding to the pristine polymer, indicating a more compact configuration of the aggregates due to higher hydrophobicity at the core of the nanoparticles. The size of the nanoparticles decreases as the antibiotic/polymer ratio increases. The zeta po- tential, in contrast, decreases as the antibiotic/polymer ratio increases, which may produce instability of the nanoprecipitates. In order to observe this, the stability of the nanoparticles was analyzed by observing the size and zeta potential evolution as a function of time. Figure 2.7 shows that stable nanoparticles are found under a high excess of the polymer, which ensures the presence of enough imidazolium functional groups at the particle surface to stabilize the particle by charge repulsion, so that size and zeta potential changes in time are moderate.
For FLU, the pH ranged between 6.8 and 7.7, the particle size ranged between 37.9 and 361.7 nm, and the respective zeta potential ranged between 27.0 and 21.7
2.3. RESULTS AND DISCUSSION 21
Figure 2.5: Correlograms obtained by DLS of PK-Im at concentration of 1 ⋅ 10−3(a), 1 ⋅ 10−4(b), and 1 ⋅ 10−5M (c) at different values of pH.
Figure 2.6: Size () and zeta potential (_) of particles composed of OA and PK-Im (left) and FLU and PK-Im (right) as a function of their relative concentration.
Figure 2.7: Size (left) and zeta potential (right) of OA/PK-Im nanoparticles as a function of time for OA/PK-Im molar ratios of 0.3 (), 0.4 ( ), 0.5 (N), 0.6 (H), 0.7 (_), 0.8 (J), 0.9 (I), 1.0 ().
2.3. RESULTS AND DISCUSSION 23
Figure 2.8: Size (left) and zeta potential (right) of FLU/PK-Im nanoparticles as a function of time for FLU/PK-Im molar ratios of 0.3 (), 0.4 ( ), 0.5 (N), 0.6 (H), 0.7 (_), 0.8 (J).
mV (Figure 2.6 right). It can be considered that the zeta potential is low to guarantee the stability of the nanoparticles. In fact, at FLU/PKIm ratios higher than 0.6, the sharp increase in nanoparticle size indicated higher aggregation of molecules result- ing in bigger particles. Note that up to a FLU/PK-Im ratio of 0.6, and as in the case of OA, the particle size is significantly lower than that for corresponding concentra- tions of the pristine polymer, indicating a shrinking effect produced by the antibiotic.
However, at FLU/PK-Im over 0.6, the pH obtained is high enough (higher than 7.3) to allow FLU to dissolve in water, due to its higher hydrophilicity, and the particle size of the precipitates exponentially increases, possibly due to the collapse of the polymer alone. Stability analyses shown in Figure 2.8 reflect that the size of the precipitates keeps increasing over time for the samples richer in the antibiotic. Thus, stable submicron particles can be synthesized at an OA/PK-Im of 0.3, reaching a pH of 6.7, showing 72 nm of hydrodynamic radius (PDI 0.5), and a zeta potential of 35 mV. Similarly, at a FLU/PK-Im of 0.3, the pH was 6.8, and stable submicron parti- cles were synthesized showing 40 nm of hydrodynamic radius (PDI 0.4), and zeta potential of 27 mV.
Precipitation upon mixing two solutions of different pH values is an out-of- equilibrium process. Kinetically, the slowest movements may be ascribed to confor- mational changes and translational movements of the polymer chains. The diffusion of the antibiotics is a faster process so that, in order, the molecules can co-precipitate;
the antibiotics should bind the polymer by means of both long-range electrostatic in- teractions and short-range secondary interactions, such as short-range electrostatic and aromatic-aromatic interactions before nanoprecipitation is complete. Short- range electrostatic interactions may produce ion pairs that stabilize the respective charges of both imidazolium and antibiotic ions and render the ensemble hydropho- bic characteristics. Thus, even if most of the imidazolium groups undergo deproto-
