Controlled reversible aggregation of thermoresponsive polymeric nanoparticles by interfacial Diels-Alder reaction

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Abstract

Hypothesis

Stimuli-responsive polymers, with properties changing upon different environmental factor variations such as pH, light, mechanical stress or temperature, and polymeric nanoparticles have found many important applications in fields such as drug delivery, biosensing and environmental research.

Experiments

In this work, thermoresponsive nanoparticles that are able to react by Diels-Alder reaction (a diene-dienophile cycloaddition) were studied. Thermoresponsive polymers were synthesized from a biocompatible polymer (poly(lactic-co-glycolic) acid, PLGA) and diene (furan) or dienophile (maleimide) derivatives. Emulsification-evaporation method was then used as a fast and reproducible method to obtain highly monodispersed polymeric nanosuspensions able to react by Diels-Alder (DA) reaction.

Findings

The nanoparticles’ size decreased down to 25 nm by increasing the emulsification’s time. The possible direct and retro-Diels-Alder (rDA) reactions between furan- and maleimide-functionalized nanoparticles were studied by dynamic light scattering and electron microscopy. For the first time, the reactivity of the interfacial DA and rDA reactions between functionalized-nanoparticles was studied, leading to the determination of the activation energy, the enthalpy of activation and the entropy of activation.

Introduction

The number of studies aiming at designing new smart materials keeps increasing since the beginning of 20th century because they play an important role in the development of new advanced technologies [1], [2], [3]. Today, we can find smart materials in all areas of activity [4], [5], [6]. Recently, chemical stimuli have been studied for various applications, such as the production of pH-responsive materials to control drug delivery and separation processes [7], [8]. The presence of specific molecules, containing polar groups or able to form hydrogen bonds for instance, can also modify the properties of materials [9], [10], [11], [12]. Besides, physical stimuli have also gained a lot of interest because they can be remotely applied. Indeed, electro- or magneto-active materials can be used to elaborate sensors or robotic muscles for instance [13], [14], [15], [16]. Photo-sensitive polymers can change their properties in response to light irradiation at a given wavelength and intensity. They are broadly used for bio-patterning and photo-triggered drug delivery [16]. Another highly-studied physical stimulus consists in the variation of the environmental temperature. This method is used to elaborate self-healing materials (composites) thanks to weak (H-bonds) or covalent interactions [17], [18], [19], [20]. In this study, we are especially interested in smart materials that respond to a thermal stimulus. Few reactions can actually be classified as thermoreversible reactions, in the sense that they lead to the formation of covalent bonds that are reversible only via a thermal stimulus. To the best of our knowledge, they concern alkoxylamine bonds [21], [22], the reversible formation of thiazolinediones[23] and the formation of Diels-Alder adducts [24], [25]. The latter reaction, classified as a click-reaction, [26], [27] corresponds to the cycloaddition between a 1.3 conjugated-diene and a dienophile (a molecule containing a double or triple bond) [24] and is the most studied. This reaction is the topic of this study: since most of thermosensitive particles presented in the literature were made from chitosan derivatives or poly(N-isopropylacrylamide) [28], [29], [30], [31], [32], we prove that the Diels-Alder (DA) chemistry can also be applied to obtain thermoresponsive nanoparticles (NPs). The DA reaction is facilitated when the energetic gap between the electronic orbitals of the diene (HOMO) and the dienophile (LUMO) are low and, thus, it decreases the reaction temperature and time [24]. Furan-maleimide pair has a low HOMO/LUMO gap and this is why it was mainly studied in the literature [25], [33], [34], [35], [36], [37]. We thus first investigated the reactivity of nanoparticles produced from furan- and maleimide-functionalized poly(lactic-co-glycolic) acid (PLGA), a biocompatible polymer allowing us to target a wide range of applications, and the reversibility of the DA reaction. Indeed, the interfacial retro-Diels-Alder (rDA) reaction has been less studied than the direct DA reaction [34], [37] and has never been studied yet at the surface of polymeric particles. The main advantages of this work are (i) to not depend on inorganic materials that are potentially toxic and rarely degradable, (ii) to work with few amounts of chemicals (more economically viable) and (iii) to be able to reach a wide range of nanoparticles’ size by tuning the process parameters.

Section snippets

Materials

N-(2-Aminoethyl)maleimide was obtained from N-(2-Aminoethyl)maleimide trifluoroacetate salt (Sigma) by liquid-liquid extraction.

Poly(lactic-co-glycolic acid) 50:50 (PLGA) (Resomer® RG504 H, Sigma, 60 kg mol−1) was functionalized by furfurylamine (Sigma) and N-(2-Aminoethyl)maleimide (Sigma) in order to obtain furan- and maleimide-derivatives. Dicyclohexylcarbodiimide (DCC, Alfa Aesar) and N-Hydroxysuccinimide (NHS, Sigma) were used to activate the PLGA. Dichloromethane (DCM, Sigma), ethyl

Size-exclusion chromatography

Molecular weights and molecular weight distributions were determined using a SEC system equipped with a Shimadzu LC‐20 AD.

pump, a Shimadzu SIL-20AHT automatic injector, a Shimadzu DGU-20A in-line deaerator, three PLgel B columns in row (separation: 500 g mol-1 – 10 Mg mol-1, 10 µm, 300 mm length x 7.5 mm ID) and a Shimatzu RID-10A refractive index detector. The measurements were operated at 30 °C, thanks to a Shimadzu CTO-10AC oven, using toluene as eluent at a flow rate of 1 mL min-1 as using

Transmission electron microscopy

To analyze the morphology and shape of the particles, cryo-transmission electron microscopy (cryo-TEM) experiments were performed. A 5 μL drop of the NPs suspension was deposited onto a lacey-holey carbon film (Ted Pella) freshly glow discharged (Elmo, Cordouan Technologies). The grid was frozen in liquid ethane cooled by liquid nitrogen in a home-made environment-controlled machine. The grids were mounted onto a Gatan 626 cryoholder and observed in a Tecnai G2 (FEI-Eindhoven) operating at

Production of biodegradable, thermoresponsive nanoparticles

In previous works, our team studied the influence of various process parameters on the polymeric (nano)particles size, leading to the conclusion that, in order to obtain small nanoparticles, the process time has to be long [41], [42], [43]. These results were confirmed in this work with two nanoemulsions (Fig. 2):

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    FUR: with furan-functionalized PLGA in the dispersed phase

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    MAL: with maleimide-functionalized PLGA in the dispersed phase

Indeed, increasing the elongational-flow micromixing time from

Conclusion

In this work, the interfacial Diels-Alder reaction as a way to reversibly bond two nanoparticles, produced from DA-reactive polymers, has been investigated. Assemblies made of maleimide- and furan-functionalized PLGA nanoparticles were obtained and their reversible aggregation was investigated, for the first time, under mild conditions (in water and with reaction temperatures under 373 K). The reactivity of these DA-reactive NPs’ surfaces was then quantified by determining the kinetics and

CRediT authorship contribution statement

Madeline Vauthier: Conceptualization, Investigation, Project administration, Formal analysis, Validation, Writing – original draft, Writing – review & editing, Supervision. Christophe A. Serra: Validation, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to thank Marc Schmutz, Catherine Foussat and Mélanie Legros for the access to the ICS characterization platforms. This work of the Interdisciplinary Institute HiFunMat, as part of the ITI 2021–2028 program of the University of Strasbourg, CNRS and Inserm, was supported by IdEx Unistra (ANR-10-IDEX-0002) and SFRI (STRAT’US project, ANR-20-SFRI-0012) under the framework of the French Investments for the Future Program.

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