Unexpected Reactivity Switch in the Statistical Copolymerization of 2

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Unexpected Reactivity Switch in the Statistical Copolymerization of 2 Oxazolines and 2-Oxazines Enabling the OneStep Synthesis of Amphiphilic Gradient Copolymers Ondrej Sedlacek, Kathleen Lava, Bart Verbraeken, Sabah Kasmi, Bruno G. De Geest, and Richard Hoogenboom J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b02607 • Publication Date (Web): 28 May 2019 Downloaded from http://pubs.acs.org on May 28, 2019

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Journal of the American Chemical Society

Unexpected Reactivity Switch in the Statistical Copolymerization of 2-Oxazolines and 2-Oxazines Enabling the One-Step Synthesis of Amphiphilic Gradient Copolymers Ondrej Sedlacek,† Kathleen Lava,† Bart Verbraeken,† Sabah Kasmi,‡ Bruno G. De Geest‡ and Richard Hoogenboom†,* †Supramolecular

Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4, B-9000 Ghent, Belgium. ‡Department of Pharmaceutics, Ghent University, B-9000 Ghent, Belgium ABSTRACT: Poly(2-oxazoline)s and, more recently also poly(2-oxazine)s, represent an emerging class of polymers with a broad range of applications. Surprisingly, to date, the statistical copolymerization of these two cyclic imino ether monomers has not yet been reported. Herein, we demonstrate that the statistical copolymerization of 2-oxazines with 2-oxazolines can lead to the formation of amphiphilic gradient copolymers in a single step. These gradient copolymers combine the high structural modularity of poly(2oxazoline)s with the excellent biological properties of poly(2-oxazine)s, especially poly(2-methyl-2-oxazine). The copolymerization was found to proceed in a non-expected way with the relative incorporation rates of the monomers being opposite to the reactivity observed for the corresponding homopolymerizations. In fact, the statistical copolymerizations lead to faster incorporation of the 2oxazine followed by a gradual transition towards the 2-oxazoline. The self-assembly properties of the prepared amphiphilic poly[(2methyl-2-oxazine)-grad-(2-butyl-2-oxazoline)] (PMeOzi-grad-PBuOx) as well as the thermoresponsive poly[(2-methyl-2-oxazine)grad-(2-propyl-2-oxazoline)] (PMeOzi-grad-PPrOx) confirmed their potential as stimuli-responsive non-ionic surfactants for various applications. Finally, the non-cytotoxic character and cellular uptake of PMeOzi-grad-PBuOx copolymers was confirmed in vitro in SKOV3 cells.

INTRODUCTION Amphiphilic polymer surfactants have been established for widespread areas of applications, ranging from emulsifiers or dispersing agents to advanced drug delivery systems, catalysts or nanoelectronics.1-4 They are mostly represented by block copolymers whose amphiphilic nature leads to self-assembly in aqueous solution, leading to the formation of nanoparticles with size and morphology (e.g., micelles, polymerosomes or nanogels) depending on the polymer type, length and the ratio of the hydrophobic/hydrophilic blocks. Apart from block copolymers, amphiphilic gradient copolymers are extremely versatile, as they can be prepared in a single step by the statistical living/controlled copolymerization of monomers with different reactivity, leading to composition drift along the polymer chain in the formed copolymer.5-7 Based on the difference in monomer reactivity, the obtained architectures range from nearly random copolymers to steep-gradient copolymers with block-like structure. Unlike the block copolymers, which form micelles with a uniform core density, the block-like gradient copolymers form a unique type of micelle, with the outer part of the core denser than the core center due to the chain back-folding.8-10 This feature might be beneficial for, e.g., drug delivery systems to slow down the nonspecific release of hydrophobic drug from the micelle.

Figure 1. Statistical cationic ring-opening co-polymerization (CROP) of 2-methyl-2-oxazine (MeOzi) with 2-n-propyl-2oxazoline (PrOx) and 2-butyl-2-oxazoline (BuOx). (A) Copolymerization scheme. (B) Mechanism of the CROP of cyclic imino ethers initiated by methyl p-toluenesulfonate and terminated by potassium hydroxide.

Polymers based on cyclic imino ethers (CIE) represent an emerging class of materials with a broad range of applications.11-12 The CIE monomers contain an 2-endo-imino group in the ring and can be polymerized by living cationic ringopening polymerization (CROP) initiated by strong alkylating agents (Figure 1).13 The formed iminium cation constitutes a highly reactive electrophilic chain-end that reacts with another

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CIE monomers via ring-opening mechanism including rearrangement of the imino group into a more thermodynamically stable tertiary amide. Finally, the termination with an appropriate nucleophile can be used to introduce chain-end functionality. This CROP results in the controlled synthesis of functional polymers isostructural to proteins and peptides. The polymerization of three types of CIE by CROP have been reported, namely the five-membered 2alkyl-2-oxazolines (AOx),14-16 the six-membered 2-alkyl5,6‐dihydro‐4H‐1,3‐oxazines (herein referred simply as 2alkyl-2-oxazines, AOzi)17-19 and, more recently, the sevenmembered 2-alkyl-4,5,6,7,-tetrahydro-1,3-oxazepines.20 The first type (AOx) is most commonly investigated while the AOzi are gaining more and more interest in recent years. The physical properties of the CIE-based polymers mainly depend on the substituent in the 2-position of the monomer ring.21 Polymers with the shortest 2-substituent, such as poly(2methyl-2-oxazoline) (PMeOx), poly(2-ethyl-2-oxazoline) (PEtOx) or poly(2-methyl-2-oxazine) (PMeOzi), are watersoluble, biocompatible, non-immunogenic and, therefore, very well suited for biomedical applications, with biological properties often outperforming those of poly(ethylene oxide) (PEO).22-25 A recent report showed that the anti-fouling properties of PMeOzi are even superior to other hydrophilic polymers (including PEO and PMeOx).26 Polymers with medium-sized 2-substituent, such as poly(2-n-propyl-2oxazoline) (PPrOx) and poly(2-ethyl-2-oxazine), exhibit lower critical-solution temperature (LCST) behavior in water and can be utilized in stimuli-responsive materials.27 Polymers with longer 2-substituent, e.g., poly(2-n-butyl-2-oxazoline) (PBuOx), are hydrophobic and well suited as hydrophobic block for the synthesis of amphiphilic copolymers. The broad library of different reported CIE monomers represents a versatile toolbox for the synthesis of advanced polymer architectures, including amphiphilic copolymers.13 As a result, a series of reports have appeared on micellar drug delivery systems based on PAOx- or PAOzi-based block copolymers.28-29 However, despite many reports on homopolymerization and sequential polymerization of both AOx, and AOzi, as well as copolymerization between two different AOx, the statistical copolymerization of AOx with AOzi has not yet been reported. Herein, we report for the first time the statistical copolymerization of AOx (PrOx and BuOx) with the hydrophilic MeOzi providing a one-step approach towards amphiphilic gradient copolymers with tunable hydrophobicity of the hydrophobic PAOx block, which has been demonstrated to be key for high drug loading in PAOx block copolymer micelles.28-29 Gradient copoly(2-oxazoline)s have been mostly limited to copolymers of hydrophilic AOx with 2-phenyl-2oxazoline and its derivatives, leading to a rather hydrophobic block with a high glass transition temperature (Tg).30-31 The here reported gradient copolymerization between AOx and MeOzi is advantageous as it allows copolymerization of different lower Tg alkyl-substituted CIE monomers and tuning of the hydrophobicity. In an aqueous environment, the resulting gradient copolymers assemble into nanoparticles due to the hydrophobic character of the AOx units. Then, we can combine the superior antifouling properties of the PMeOzi-rich shell with the modularity of the hydrophobic or thermoresponsive PAOx core to provide architectures with great potential in biomedical sciences (e.g., drug delivery systems).

RESULTS AND DISCUSSION First, the homopolymerization of the selected monomers (MeOzi, PrOx and BuOx) was examined, initiated with methyl p-toluenesulfonate (MeOTs) at an initial monomer-to-initiator ratio of [M]0:[I]0 = 100 in acetonitrile at 140 °C using a microwave reactor, our standard conditions. All polymerizations followed pseudo-first order kinetics, with the apparent propagation rate constants kp being in line with reported values under the applied conditions (Figure 2A, 3A, S1).13,17 The slower propagation of MeOzi is generally attributed to the increased steric hindrance of the 6-position in the cationic oxazinium ring by the hydrogens in the 5-position resulting from its non-planar structure leading to the slower SN2 substitution by the next monomer.32 The copolymerizations of different alkyl-substituted AOx proceed mostly in the way close to „ideal copolymerization“ (r1 x r2 ≈ 1, where r1 = kp11/kp12, r2 = kp22/kp21),33 where the relative rates of monomer consumption are independent of living chain-end and the gradient formation is based on the different monomer reactivity. Then, the monomer consumption proceeds via pseudo-firstorder kinetics and monomer reactivity ratios can be estimated from a single copolymerization kinetic plot.34 Based on the homopolymerization data, we anticipated that in the copolymerization of 2-oxazolines with MeOzi the former will be incorporated faster than the latter.

Figure 2. (A) Homopolymerization kinetics for the polymerization of MeOzi, PrOx, and BuOx; (B) Copolymerization kinetics of MeOzi with PrOx; (C) Copolymerization kinetics of MeOzi with BuOx. All polymerization were performed in acetonitrile at 140 °C initiated by MeOTs, [M]0 = 4 M, [M]0:[MeOTs]0 = 100. (D) Gradient microstructure of the copolymers obtained from a 1:1 monomer feed.

Surprisingly, we observed a complete reversal in the relative reactivity of the AOx/MeOzi pair in the copolymerizations compared to their respective homopolymerizations (Figure 2B, C), with MeOzi being the faster-incorporated monomer. To shed light on this unexpected behavior, we performed a kinetic investigation of the process. The copolymerization of MeOzi with PrOx, respectively BuOx proceeds in a non-ideal way, with the logarithm of monomer consumption not following the


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Journal of the American Chemical Society linear kinetics. Furthermore, increasing content of AOx in the monomer feed accelerates the copolymerization rate. The reactivity ratios were determined by analyzing the reaction mixtures at different monomer feed ratios at low conversions using the extended Kelen-Tudos model (Figure S4, S5),35 confirming that MeOzi is the more reactive monomer (rMeOzi = 4.44 - 4.64) compared to AOx (rPrOx = 0.19, rBuOx = 0.15) and indicating the non-ideal character of the copolymerization process (rMeOzi rPrOx = 0.84 and rMeOzi rBuOx = 0.70). The microstructure composition along the polymer chain was predicted for a 1:1 monomer feed ratio using the Skeist model (Figure 2D)36 revealing that the initial part of the copolymer is rich in MeOzi, while the last ca. 15 % of the copolymer chain resembles a homopolymer of AOx. Furthermore, the gradient of the MeOzi/PrOx pair was slightly steeper than that of the MeOzi/BuOx pair.

Figure 3. Overview of the reactive chain-end conformations and rate constants (kp) of homo-propagation (A) and cross-propagation (B) in the copolymerization of 2-oxazines with 2-oxazolines.

Combining both homopolymerization rate constants and copolymerization parameters, the cross-propagation rate constants for all monomer pairs were calculated (Figure 3, S1). The slowest step is the ring-opening of the oxazinium chain-end by the oxazoline monomer and vice versa, i.e., the fastest reaction is the opening of the oxazolinium chain-end by the oxazine monomer. This can be explained by the higher nucleophilicity of the oxazine nitrogen compared to the oxazoline nitrogen as may be derived from the higher initiation rate constant for 2-oxazines compared to 2-oxazolines as reported by Saegusa,32 making the 2-oxazine the more reactive monomer in all cases. On the other hand, the steric hindrance of the 6-position in the non-planar oxazinium chain-end ring results in its lower reactivity towards the monomers compared

to the nearly planar oxazolinium ring (Figure 3). This also explains the slow homopolymerization of MeOzi, where the high monomer reactivity is overweighed by the low reactivity of the oxazinium chain-ends. In the copolymerization with 2oxazolines, however, the stronger nucleophilicity of MeOzi plays the major role. Furthermore, the slower incorporation of BuOx compared to PrOx may be attributed to enhanced steric hindrance for attack on the more sterically hindered oxazinium chain end. Given the synthetic versatility and structural modularity of PAOx and PAOzi, understanding of these general rules will help us in the future to design a whole family of new hybrid gradient copolymers with a wide range of applications. The one-step copolymerization of MeOzi with AOx provides an elegant approach for the preparation of amphiphilic gradient copolymers. To exploit the excellent biocompatibility and antifouling properties of PMeOzi, we synthesized a series of amphiphilic gradient copolymers of PMeOzi with the hydrophobic PBuOx, respectively the thermoresponsive PPrOx. The resulting copolymers with a degree of polymerization of 100 were well defined (dispersity Ɖ < 1.25), differing just in the MeOzi content (FMeOzi). Their self-assembly was studied in phosphate buffer saline (PBS, pH = 7.4) by dynamic light scattering (DLS) and turbidimetry. In the series of PMeOzi-grad-PBuOx copolymers (MB1-4), FMeOzi ranged from 53 % (MB1) to 81 % (MB4) (Figure 4, Table S3). The hydrophobic character of the BuOx units entails the copolymers amphiphilic character, resulting in the formation of micellar nanoparticles with a size of 25 - 35 nm in PBS (Figure S8). In the copolymer with the lowest BuOx content (MB4), however, the micelles coexisted with single chain unimers. The critical micelle concentration (cmc) of copolymers MB1 – MB3 was measured by fluorescence spectroscopy in presence of pyrene and the obtained values (cmc = 6.0 – 7.2 mg L-1) were in a similar range as reported for di-block copolymer micelles (Figure S9). These polymers are, therefore, promising candidates for biomedical applications as delivery systems for hydrophobic drugs, as the spontaneous formation of these gradient copolymers is more reproducible and more straightforward than the two-step preparation of the currently used block copolymers. For this application as drug delivery vehicles, the non-cytotoxic character of the synthesized polymer carriers is crucial. This was confirmed by an in vitro experiment on human ovarian cancer SKOV3 cells (Figure 4B). The copolymers were incubated with cells for 48 h at 37 °C, after which the cell viability was assessed by MTT assay. All copolymers were found to be non-cytotoxic up to a concentration of 1 mg mL–1, proving their excellent in vitro biocompatibility. Furthermore, the in vitro uptake by SKOV3 cells of micelles loaded with the hydrophobic fluorescent dye octadecylrhodamine was measured by flow cytometry SKOV3 cells (Figure 4C). All copolymer micelle formulations readily increased cellular uptake of the fluorescent dye, confirming their biomedical potential.



Figure 4. Amphiphilic PMeOzi-grad-PBuOx copolymers MB1-4: (A) Chemical structure. (B) In vitro cytotoxicity of copolymers tested on SKOV3 cells. In vitro uptake of copolymers labeled with octadecylrhodamine (R18) by SKOV3 cells after 16 h of incubation (C) measured by FACS at different polymer concentrations and (D) visualized by confocal microscopy (red color). Cells were stained with phalloidin (membranes, green color) and Hoechst (nuclei, blue color). Scale bars are 15 µm.

Finally, the surfactant properties of PMeOzi53-grad-PBuOx47 (MB4) were demonstrated by encapsulation of curcumin, a compound widely studied for its anti-cancer properties with an extremely low water solubility (Figure 5). Nanoparticles were obtained containing 9 wt % of curcumin, with an encapsulation efficiency of 81 %. DLS analysis revealed a slight increase in the nanoparticle size (Dh = 37 nm) upon drug loading. These results further suggest the strong potential of the prepared amphiphilic PMeOzi-grad-PBuOx copolymers for biomedical applications. The detailed physical and biological study of the drug-loaded nanoparticles is, however, beyond the scope of this article focusing on the gradient copolymerization of AOx and AOzi and will be studied in the near future.

Figure 5. (A) Encapsulation of curcumin by MB1 copolymer, (B) hydrodynamic diameter of MB1 in MeOH, PBS and curcumin loaded MB1 in PBS (cpol = 2 mg mL−1).

In the series of thermoresponsive PMeOzi-grad-PPrOx copolymers (MP1-7), the lower critical solution temperature (LCST) behavior was found to be strongly dependent on the copolymer composition (FMeOzi = 22 - 89 %) (Figure 6, S10-13, Table 1, S5). Copolymers with a lower MeOzi content (MP1, FMeOzi < 22 %) exhibited one cloud point temperature and complete precipitation at ca. 30 °C, suggesting that the copolymer is not amphiphilic enough to induce micellization. The thermoresponsive properties are rather similar to those of homopolymers. On the other hand, the copolymer with a high MeOzi content (MP7, FMeOzi = 89 %) is hydrophilic and does not exhibit any thermoresponsive behavior within the investigated temperature range (15 – 85 °C). With intermediate MeOzi content (MP2-7, FMeOzi = 31 - 61 %), the nanoscale selfassembly of the copolymers became apparent as they exhibit two distinct cloud point temperatures, both increasing with the FMeOzi value (Figure 6C). At low temperatures, the copolymers are hydrophilic and are present as individual polymer chains with a hydrodynamic diameter of Dh ≈ 6 nm. Once they reach the first cloud point temperature Tcp1, they self-assemble into micellar nanoparticles with a Dh of 25 - 35 nm. At this point, the PrOx units lose their hydration layer and become hydrophobic, leading to self-assembly into micelles with a PPrOx-rich core and a PMeOzi-rich shell. Similar behavior was observed for thermoresponsive PAOx block copolymers.27 With further increase in temperature, the nanoparticles remain stable until they reach the second cloud point temperature Tcp2, indicated by aggregation and macroscopic precipitation of the polymer as observed by both DLS (increase on Dh) and turbidimetry. This behavior is rather unusual for amphiphilic (block) copolymers consisting of a permanently hydrophilic block and a thermoresponsive block and can be explained by the presence of a small amount of PrOx units in the micellar


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Journal of the American Chemical Society PMeOzi shell, inducing thermoresponsive properties. Particularly interesting from the point of application is the copolymer MP3 (FMeOzi = 36 %) that has cloud point temperature values of Tcp1 < 37 °C < Tcp2 (Figure 6D). At room temperature the polymer is molecularly dissolved while at body temperature (ca. 37 °C) it self-assembles into micellar nanoparticles making it interesting as stimuli-responsive delivery system for hydrophobic drugs or radiotherapeutics. This double-LCST behavior can be further exploited to design advanced stimuli-responsive architectures.

rates of monomer incorporation being opposite to those obtained from homopolymerization kinetics. This general trend was explained based on detailed kinetic investigation revealing that a combination of electronic and steric effects reverses the monomer incorporation in the statistical copolymerization compared to the homopolymerizations, which can serve as a basis for the design of new class of poly(2-oxazine/2-oxazine) materials. Finally, the application potential of these compounds was demonstrated by the single-step synthesis of two different types of amphiphilic gradient copolymers that self-assemble into the micellar nanoparticles in an aqueous environment. While PMeOzi-grad-PBuOx polymers exhibit behavior similar to the amphiphilic block copolymers,37 thermoresponsive PMeOzi-grad-PPrOx assemble into the nanoparticles upon heating. Considering the excellent anti-fouling properties of hydrophilic PMeOzi, these polymers represent attractive materials for biomedical research.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx: Detailed experimental procedures and characterization of the synthesized (co)polymers.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions

Figure 6. Thermoresponsive behavior of PMeOzi-grad-PPrOx copolymers in PBS (c = 2 mg mL−1). (A) Chemical structure. (B) Schematic illustration of the double-LCST behavior. (C) Dependence of the cloud-point temperatures of the polymer composition. (D) Dependence of the MP3 hydrodynamic diameter on temperature.

Table 1. Characteristics of thermoresponsive PMeOzi-gradPPrOx copolymers. Polymer

FMeOzi (%)a

Mn (kg mol-1)b

Ɖb

Tcp1 (°C)c

Tcp2 (°C)d

MP1

22

10.1

1.08

29.5

30.7

MP2

31

10.0

1.11

32.0

34.3

MP3

36

10.4

1.11

33.0

40.3

MP4

45

10.7

1.16

37.0

50.8

MP5

51

10.5

1.17

41.0

60.7

MP6

61

10.4

1.20

47.0

74.4

MP7

89

7.81

1.17

-

aMeOzi

1H

fraction in copolymer determined by NMR. by SEC-MALS using DMA/LiCl as an eluent. cCloud-point temperature determined by DLS as onset of scattering intensity increase. dCloud-point temperature determined by turbidimetry. bDetermined

CONCLUSIONS In summary, we report for the first time the cationic ring copolymerization of 2-oxazolines with 2-oxazines as a facile one-step route towards amphiphilic gradient surfactants. The copolymerization proceeds in a non-expected way with relative

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes R.H. is one of the founders of Avroxa BVBA that commercializes poly(2-oxazoline)s as Ultroxa®. R.H. and B.V. are listed as inventors of patent application WO2018002382A1 that is based on parts of this work. The other authors have no conflicts to declare.

ACKNOWLEDGMENT This work is dedicated to Prof. Ulrich S. Schubert on the occasion of his 50th birthday. This work was financially supported by FWO and Ghent University. O.S. thanks the funding from the FWO and European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 665501.

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