Synthesis and self-assembly of a novel block copolymer poly(N-vinylcaprolactam)-b-poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PNVCL-b-PHBHV) by the combination of RAFT/MADIX and click chemistry techniques

Synthesis and self-assembly of a novel block copolymer poly(N-vinylcaprolactam)-b-poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PNVCL-b-PHBHV) by the combination of RAFT/MADIX and click chemistry techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and self-assembly of a novel block copolymer poly(N-vinylcaprolactam)-b-poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PNVCL-b-PHBHV) by the combination of RAFT/MADIX and click chemistry techniques Rodolfo Minto Moraes, Layde Teixeira Carvalho, Gizelda Maria Alves, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6328656/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Polymer Bulletin → Version 1 posted 9 You are reading this latest preprint version Abstract Amphiphilic copolymers have gained significant attention in the field of drug delivery systems (DDS). The key feature that makes them promising for such application is their ability to self-assemble into micelles in aqueous media. In this context, this work describes the synthesis of a novel amphiphilic well-defined block copolymer, poly( N -vinylcaprolactam)- b -poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PNVCL- b -PHBHV), using a strategy that combines reversible addition-fragmentation chain-transfer macromolecular design via interchange of xanthates (RAFT/MADIX) polymerization and click chemistry reaction. Initially, azido-terminated PNVCL homopolymers (PNVCL-N 3 ) were synthesized through RAFT/MADIX polymerization of the N -vinylcaprolactam (NVCL) monomer, mediated by a chain transfer agent (CTA) bearing an azide group. Meanwhile, the alkyne-terminated PHBHV (alkyne-PHBHV) was prepared by the transesterification reaction between PHBHV and propargyl alcohol. Then, the 1,3-cyclo addition reaction between azide and alkyne (CuAAC) was used to obtain the block copolymer PNVCL- b -PHBHV. Different size chains of PHBHV were evaluated as also their influence on the capacity of micelles formation. The chemical structures of all (co)polymers were assessed by Fourier-Transform Infrared spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance spectroscopy ( 1 H NMR) analysis, while their molar masses were determined by Size Exclusion Chromatography (SEC). Differential Scanning Calorimetry (DSC) measurement showed that the PNVCL- b -PHBHV have lower degree of crystallinity than PHBHV. Additionally, it was observed that the critical micelle concentration (cmc) of the block copolymers in aqueous solution decreased as the length of the hydrophobic block increased, whereas the size of the polymeric micelles grew with a higher proportion of hydrophobic segments. Poly(N-vinylcaprolactam) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Amphiphilic Thermoresponsive Block Copolymer RAFT/MADIX Click Chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. INTRODUCTION Amphiphilic and stimuli-responsive block copolymers have been attracting an extensive scientific interest due to their unique properties and versatile applications. Amphiphilic block copolymers are macromolecules composed of different polymer blocks with distinct hydrophilic and hydrophobic segments. Such materials have the ability to self-assemble in aqueous solution into various nanostructured aggregates including micelles and vesicles [1-3]. This feature contributes to the usefulness of these materials in various applications areas including microelectronics [4], nanoreactors [5], catalysis [5] and drug delivery systems [6]. In the context of biomedical applications, stimuli-responsive or so-called “smart” polymers, particularly those used in drug delivery systems, have gained significant attention. This is due to their ability to undergo rapid and reversible changes in response to various external stimuli, such as temperature, pH, and other environmental factors, which allows for controlled and targeted release of therapeutic agents [7]. Thermoresponsive polymers (also called temperature-responsive or thermosensitive polymers) are a class of material that exhibits a phase transition temperature in correspondence of which a drastic change in the solubility occurs [8]. Based on their thermosensitive behavior in solution, these polymers can be classified into two categories, namely the lower critical solution temperature (LCST) behavior and upper critical solution temperature (UCST) behavior [9, 10]. In the first case, the polymer is miscible with the solvent at temperatures below the phase transition temperature, however, two immiscible phases (a diluted polymer phase and a concentrated polymer phase) form above this temperature. On the other hand, polymers exhibiting UCST show the opposite behavior, the phase separation occurs upon cooling below the phase transition temperature [9, 10]. Poly( N -vinylcaprolactam) (PNVCL) is widely investigated as a hydrophilic homopolymer with a lower critical solution temperature (LCST) close to physiological levels. Its LCST varies based on molar mass and polymer concentration, and depends on the chemical composition in the case of copolymers [11, 12]. PNVCL, known for its biocompatibility, low toxicity, and the ability to complex with other molecules, is applied across various domains, particularly in the medical field [13]. Despite extensive research in this area, literature predominantly details the preparation of a wide range of PNVCL-based amphiphilic thermosensitive block copolymers, such as PNVCL- b -poly(ε-caprolactone) [14, 15], PNVCL- b -poly( D,L -lactide) [16, 17], and PNVCL- b -PVAc [18]. Nevertheless, a significant gap exists, with no documented instances of the synthesis of block copolymers that combine PNVCL with PHBHV blocks. This unexplored field offers intriguing potential for developing novel materials with tailored properties and applications. Poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBHV), a derivate from the highly popular poly(3-hydroxybutyrate) (PHB), is a low-cytotoxicity hydrophobic copolymer that belongs to the poly(hydroxyalkanoates) (PHAs) family. The PHBHV is a biocompatible and biodegradable thermoplastic polyesters, as all other polyhydroxyalkanoates (PHAs), product of biosynthesis of a wide variety of both gram-positive and gram-negative bacteria [19]. Among wild microbes, Ralstonia eutropha , also known as Cupriavidus necator or Alcaligenes eutrophus [20-22] has been the most used specie . PHBHV’s properties, such as its biological origin, low cytotoxicity, absorption capacity, piezoelectricity, and thermoplasticity, justify its potential for biomaterial applications, including the fabrication of cardiovascular stents [23], drug release and transport systems [24, 25], absorbable surgical sutures, and medical packaging [26]. However, its intrinsic hydrophobicity property may restrict some of its in vivo applications. Therefore, the elaboration of PHBHV-based amphiphilic copolymers is one of the most interesting alternatives to overcome such drawback, making this polymer viable for these applications. The combination of synthetic organic chemistry and polymer chemistry is an interesting approach to build complex structures. An extremely versatile method and a powerful tool that has been used to constructed polymers with well-defined structures is the combination of “click chemistry” and controlled/living polymerization techniques. Such strategy has been enabled the synthesis of a wide range of block copolymers [27-30] and graft copolymers [29, 31], as well the preparation of advanced macromolecular architectures [32]. Controlled radical polymerization (CRP) techniques, also named reversible deactivation radical polymerization (RDRP) [33], have enabled the synthesis of polymers with well-defined structures and with control of molar mass and molar mass distribution, in addition to enabling the preparation of functional polymers and/or with complex architectures, such as blocks, stars, grafted, microgel, dendritic, among others [34, 35]. Among the various types of CRP, the RAFT technique is considered one of the most versatile, since it exhibits good performance to a wide range of functional groups in monomers, initiators and solvents, besides offering a control over different types of monomers [36]. According to its ability to react with free radicals, the activated vinyl monomers can be classified into two broad categories: the more-activated (MAMs) (e.g., styrene, acrylonitrile, vinyl pyridine and (meth)acrylates) and the less-activated (LAMs) (e.g., vinyl esters and N -vinylamides) monomers. Different classes of RAFT agents must be employed to promote the controlled polymerization of MAMs and LAMs. The LAMs are highly reactive and behave as poor homolytic groups, therefore, the O -alkyl xanthates or N -alkyl- N -aryldithiocarbamates, which form less-stable intermediate radicals, are more suitable CTAs for the RAFT polymerization of this type of monomers [37]. A very interesting feature of the RAFT technique is the retention of the α (R group) and ω (Z functional thiocarbonylthio group) groups of the CTA in the polymeric chains. The incorporation of the thiocarbonylthio group at the end of the polymeric chains enables this material to act as a RAFT macroagent in the polymerization of other suitable monomer leading to the formation of block copolymers by chain extension [38]. Besides linear block copolymers, other architectures can be formed, including star, branched or hyperbranched polymers. Moreover, since the RAFT agent is retained in the polymer structure, polymers with the desired end-functionality can be also obtained simply by choosing the appropriate RAFT agent. Subsequently, the end-functionalized polymers can be coupled with other polymers leading to a copolymerization. Generally, the in situ synthesis of end-functional polymers is conducted by using the R-group of the CTA, as the Z groups are quite unstable to nucleophiles [39]. "Click Chemistry" was a concept introduced in 2001 by Sharpless that described several types of thermodynamically favorable reactions for coupling two molecules in a very simple, fast, regioselective way and with high yields, conducting under mild experimental conditions [40]. Among the numerous techniques that are included in the “Click Chemistry” concept, the 1,3-dipolar cycloaddition reaction between azides and alkynes catalyzed by Cu(I) species (Copper(I)-catalyzed Azide-Alkyne Cycloaddition - CuAAC) has been one of the most used due to its high yield and selectivity [41]. As shown in the literature, a wide variety of both PHBHV-based (e.g.: PULL- g -PHBHV [42], dextran- g -PHBHV [43], PHBHV- b -poly(2-methyl-2-oxazoline [44]) and PNVCL-based (e.g.: PNVCL- b -poly(ε-caprolactone) [45], PNVCL- b -poly(D,L-lactide) [16], PNVCL- b -poly(oligo(ethylene oxide) methyl ether methacrylate [30] copolymers have been successfully synthesized by the CuAAC reaction. Up to now, as far as we are aware, there have been no reports in the literature regarding the synthesis of block copolymers comprising both PNVCL and PHBHV segments. In this work, we performed the synthesis of the well-defined amphiphilic block copolymer, PNVCL- b -PHBHV, using the combination of the RAFT/MADIX and click chemistry techniques. A ω-azido-functionalized xanthate agent, namely, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was first synthesized and subsequently used to mediate the RAFT/MADIX polymerization of NVCL to give rise to the well-defined PNVCL homopolymer comprising an azido-end group (PNVCL-N 3 ). Meanwhile, alkyne-terminated PHBHV (alkyne-PHBHV) was prepared by the transesterification reaction between a PHBHV with high molar mass and propargyl alcohol using dibutyltin dilaurate as catalyst. PHBHV- b -PNVCL block copolymers were then prepared by Copper(I)-catalyzed Azide Alkyne Cycloaddition ( CuAAC – “click chemistry’’). These (co)polymers were characterized by Size Exclusion Chromatography (SEC), Fourier-Transform Infrared spectroscopy (FTIR), Proton Nuclear Magnetic Resonance spectroscopy ( 1 H NMR) and Differential Scanning Calorimetry (DSC) measurements, and the effect of PHBHV chain length on the amphiphilicity of the copolymers besides their ability of micelles formation were evaluated. 2. EXPEMIMENTAL SECTION 2.1 Materials N -vinylcaprolactam (NVCL, kindly supplied by BASF) was distilled under reduced pressure. Poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBHV containing 8.6% of HV, M n SEC = 114873 g mol -1 and Đ = 2.86, kindly supplied by PHB Industrial S/A) was dispersed in a large amount of deionized water, recovered by filtration, washed with cold ethanol and, subsequently, with cold diethyl ether, and dried under vacuum. 2,2’-azobis(isobutyronitrile) (AIBN) was recrystallized from methanol. 2-bromoethanol (BE, Aldrich, 95%), sodium azide (NaN 3 , Synth, 99%), 2-chloro-2-phenylacetyl chloride (Aldrich, 90%), potassium ethyl xanthogenate (KEX, Aldrich, 96%), dibutyltin dilaurate (DBTD, 95%, Synth), propargyl alcohol (99%, Aldrich), copper bromide (CuBr, Aldrich, 98%), N,N,N′,N′′,N′′ -pentamethyldiethylenetriamine (PMDTA, Aldrich, 99%), sodium hydrogen carbonate (NaHCO 3 , Synth, 99.7-100%), ammonium chloride (NH 4 Cl, Synth, 100%), anhydrous magnesium sulfate (MgSO 4 XH 2 O, Synth, 98%) and 1.3.5-trioxane (Aldrich, ≧99%) were used as received. Triethylamine (TEA, Aldrich, 99%), toluene (Synth, 99.5%), tetrahydrofuran (THF, Synth, 99%) and 1.4-dioxane (Synth, 99%) were dried and fractionally distilled from sodium. Dimethylformamide (DMF, Synth, 99.8%) was distilled under reduced pressure in the presence of calcium hydride (CaH 2 ). Hexane (Synth, 98.5%), ethyl acetate (Synth, 99.5%), diethyl ether (Synth, 98%) and dichloromethane (DCM, Synth, 99.5%) were used without purification steps. The water used in the entire process was deionized. 2.2 Methods 2.2.1 Synthesis of the PNVCL-N 3 Synthesis of RAFT agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate The RAFT agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was synthesized in three steps. The synthetic route is shown in Scheme 1. a) Synthesis of 2-azidoethanol In a 100 mL round-bottom flask, 40 mmol of 2-bromoethanol was diluted in 20 mL of deionized water. The result solution was placed in an ice bath and kept under vigorous stirring. Meanwhile, 4 g of NaN 3 was added in small portions in the flask. Then, the system was removed from the ice bath and left under stirring for 2 hours at room temperature. Then, 2.5667 g of NaN 3 was added to the mixture, and the flask was immersed in a glycerin bath previously heated at 80 °C and maintained at this temperature, under stirring, for 24 hours. The product was then extracted with ether (4x30 mL). The combined organic extracts were dried over anhydrous MgSO 4 , and concentrated under reduced pressure at room temperature. b) Synthesis of 2-azidoethyl-2-chloro-2-phenylacetate A solution of 2-chloro-2-phenylacetyl chloride (4.20 g, 20 mmol) in 35 mL of dry THF was prepared in a dried 100 mL round-bottom flask equipped with a magnetic stirring bar under nitrogen at room temperature. A mixture of 2-azidoethanol (1.39 g, 16 mmol) in 8 mL of dry THF and TEA (3.24 g, 32 mmol) in 7 mL of dry THF was then added dropwise to the above solution mixture that was cooled in an ice bath. Then, the reaction mixture was stirred for 24 hours at room temperature. The precipitated by-product (i.e., Et 3 N.HBr), was removed by filtration and THF was evaporated. After that, the concentrated mixture was dissolved in 20 mL of DCM and washed thoroughly with saturated sodium bicarbonate (4x30 mL). The organic layer was further washed with water (3x30 mL), dried over anhydrous MgSO 4 overnight, and filtered. Finally, the filtrate was concentrated under reduced pressure at room temperature. c) Synthesis of 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate In a dried 500 mL round-bottom flask equipped with a magnetic stirring bar under nitrogen at room temperature, a solution of potassium ethyl xanthogenate (3.63 g, 22 mmol) in 330 mL of dry THF was prepared. Then, a solution of 2-azidoethyl-2-chloro-2-phenylacetate (3.48 g, 14.5 mmol) in 20 mL of dry THF was added dropwise to the previous solution during stirring under nitrogen flow. The reaction mixture was then stirred for 24 hours at room temperature. The THF was evaporated under vacuum at room temperature. Then, the concentrated mixture was dissolved in 150 mL of ether and washed consecutively with saturated NH 4 Cl solution (4x50 mL), saturated NaHCO 3 , solution (4x50 mL), and water (4x50 mL). The organic layer was dried over anhydrous MgSO 4 , and filtered. The filtrate was concentrated under reduced pressure at room temperature. RAFT/MADIX polymerization of NVCL Well-defined azido-end capped PNVCL (PNVCL-N 3 ) was synthesized by RAFT/MADIX polymerization of NVCL mediated by the azido-functionalized xanthate chain transfer agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate at 70 °C, using AIBN as the initiator and 1.4-dioxane as solvent (Scheme 2). This reaction was performed with the feed molar ratio [NVCL] 0 :[CTA] 0 :[AIBN] 0 = 300:1:0.25. In a dried and nitrogen purged round-bottom flask, 39.0 mg of 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, 5 g of NVCL, 4.9 mg of AIBN and 101.1 mg (1.12 mmol) of 1,3,5-trioxane (internal standard) were dissolved in 2.5 mL of 1,4-dioxane. A homogeneous solution was obtained after stirring, and degassed under nitrogen for 30 min. The flask was immersed in an oil bath preheated at 70 °C. In order to optimize the NVCL RAFT/MADIX polymerization, the NVCL polymerization rate, and the evolution of the number average molar mass ( M n) and the dispersity (Đ) of the polymer were monitored by 1 H NMR and HPLC for 24 hours, respectively. The material was reproduced under the same conditions, but on a larger scale (all quantities were increased by a factor of 10) and for only 6 hours. This reaction was stopped by cooling the mixture in an ice bath. The resulting polymer was purified by precipitation in cold diethyl ether, recovered by filtration, dried under vacuum at room temperature for 24 hours. 2.2.2 Preparation of the alkyne-PHBHV PHBHV comprising an alkyne-end group (alkyne-PHBHV) was obtained via transesterification reaction between a high-molecular-weight PHBHV ( M n SEC = 114873 g.mol -1 and Đ = 2.86) and propargyl alcohol. The transesterification reaction was catalyzed by dibutyltin dilaurate (DBTD) at 110 °C, using toluene as solvent (Scheme 3). To obtain alkyne-PHBHV macrochains with two different lengths, a transesterification reaction (ReT (1)) was performed to evaluate the effect of time on the reduction of molar mass of the alkyne-PHBHV. During this first transesterification reaction samples were withdrawn at pre-established times (2, 4, 6, 8, 10 and 12 hours). In a 125 mL dried round-bottom flask equipped with a magnetic stirring bar under nitrogen flow, 5 g of high molar mass PHBHV was solubilized in 60 mL of toluene at 130 °C in a glycerin bath. After complete solubilization of the polymer, the reaction temperature was decreased to 110 °C and a solution of propargyl alcohol and dibutyltin dilaurate (DBTD) in 15 mL of toluene was immediately added into the flask. The polymers contained in the aliquots were precipitated in cold hexane and their M n was monitored by SEC (Table S1 in Supporting Information). Subsequently, two other reactions were conducted under the same conditions, but on a larger scale (all quantities were increased by a factor of 4), using different reaction times: 12 hours (alkyne-PHBHV (1)) and 7 hours (alkyne-PHBHV (2)). The polymers were precipitated in cold hexane, and a selective precipitation method of cold mixture of hexane and ethyl acetate (15/85 %, v/v) was used to improve the purification and to reduce the polymer dispersity ( Đ ). In brief, the material recovered from the alkyne-PHBHV (1) reaction was dissolved in DCM and precipitated three times in the cold solvent mixture. On other side, the material obtained from the alkyne-PHBHV (2) reaction was precipitated four times in this cold solvent mixture. Finally, the polymers were dried under vacuum at room temperature for 24 hours. 2.2.3 Synthesis of the PNVCL- b -PHBHV block copolymers by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction PNVCL- b -PHBHV block copolymers were synthesized by click chemistry using CuBr as catalyst, N , N , N ′, N ′′, N ′′-pentamethyldiethylenetriamine (PMDTA) as nitrogenous base and DMF as solvent (Scheme 4). The CuAAC reactions were performed in the presence of a slight excess of PNVCL-N 3 , using the [alkyne-PHBHV] 0 :[PNVCL-N 3 ] 0 :[CuBr] 0 :[PMDTA] 0 molar ratio = 1:1.25:4:4. The ratio of DMF volume relative to the polymer weight used in these reactions was 10.0 mL g -1 . In a dried and nitrogen purged round-bottom flask, a solution containing 0.6 g of alkyne-PHBHV and 1.1 g of PNVCL-N 3 in dry DMF was prepared and kept under stirring at room temperature. Meanwhile, solutions of 73.9 mg of PMDETA and 61.8 mg of CuBr in DMF were prepared in two others dried and nitrogen purged round-bottom flask. After 30 minutes under stirring, the PMDTA and CuBr solutions were added dropwise into the polymer mixture that was cooled in an ice bath. After that, the flask was immersed in an oil bath preheated at 60 °C and kept for 120 hours. To eliminate the unreacted PNVCL-N 3 , the mixture was dialysis against water. Then, the reaction product was extracted with DCM. To eliminate residual copper salts, the organic layer was passed through a short aluminum oxide column. Finally, the polymer was precipitated in cold diethyl ether and dried under vacuum at room temperature for 24hours. Polymeric particles preparation The particles were prepared via the nanoprecipitation technique. 12.5 mg block copolymer was dissolved in 5 mL THF, thereafter, 1 mL of this polymeric solution was added dropwise into 45 mL of ultrapure water under stirred at 25 °C. The solvent THF was then removed using a rotary evaporator. Subsequently, the obtained dispersion was transferred into a 50 mL volumetric flask, followed by dilution to the calibration mark with water. 2.3 Characterization Polymer Characterization The number average molar masses ( M n) and dispersities ( Đ ) were determined by SEC (Waters 1515) using chloroform or THF with triethylamine (0.3% v/v) as the solvent, at 35 °C, with a flow rate of 1.0 mL min −1 on three Phenogel columns (10 3 , 10 4 and 10 6 Å) connected in series to a 2414 differential refractive index detector. A high-performance liquid chromatography (HPLC, Shimadzu) was also used to determine the M n and Ð using THF with triethylamine (0.3% v/v) as the solvent, at 35 °C with a flow rate of 0.8 mL min −1 on two Phenogel columns (10 4 and 10 6 Å) connected in series to a differential refractive index detector (Sil−20A Shimadzu). The columns were calibrated using polystyrene (PS) standards. The 1 H NMR spectra were obtained in a Varian Mercury-300 NMR spectrometer (300 MHz) at room temperature using deuterated dimethylsulfoxide (DMSO- d 6 ) or chloroform (CDCl 3 ) as solvents, and the chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as the internal standard. FTIR spectra were collected using a Shimadzu IRPrestige-21 spectrometer using the KBr disk method. The (co)polymers thermal properties were evaluated using a TA Instruments Q20 DSC under nitrogen atmosphere. The instrument was calibrated with Indium before use. The samples were first heated from −80 to 180 °C at a 10 °C min −1 heating rate, followed by quenching to −80 °C. Then, the samples were re-heated to 220 °C at a rate of 10 °C min −1 . X-ray diffraction patterns were obtained by a PAN-analytical Empyrean ACMS 101 (Malvern, UK) diffractometer at room temperature. The diffractometer was used with a monochromatic radiation beam of the CuKα. Experiments were conducted with a scan range from 10 to 90° (2θ), a step size of 0.01° (2θ), and a counting time of 10 s per step. Micelles characterization Critical micelle concentrations (cmc) of the block copolymers were determined by fluorescence measurements at 390 nm emission wavelength (Varian Cary Eclipse fluorescence spectrophotometer) using pyrene as a probe. A pyrene solution in acetone was added into a series of volumetric flasks in such an amount that the final concentration of pyrene in each solution was 6.10 -7 mol L -1 . The acetone was then allowed to completely evaporate. The polymer solution, previously prepared with the aid of an ultrasound bath (30 min.), was added into the volumetric flasks and diluted to the calibration mark using deionized water to obtain different copolymer concentrations ranging from 10 -4 to 0.5 mg mL -1 . The samples were stored at room temperature overnight to equilibrate micelles and pyrene. The size distribution of micelles (prepared by the nanoprecipitation technique) was determined by dynamic light scattering (DLS) using a Malvern Nano ZS instrument. 3. RESULTS AND DISCUSSION 3.1 Synthesis of the PNVCL-N 3 3.1.1 Synthesis of RAFT agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate The PNVCL- b -PHBHV block copolymers were synthesized via the combination of RAFT/MADIX and click chemistry techniques as shown in the Scheme 4. In the first step, the xanthate agent comprising azide group, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was synthesized and, subsequently, used to mediate the RAFT/MADIX polymerization of NVCL to the preparation of well-defined azido-terminated poly( N -vinylcaprolactam) homopolymers (PNVCL-N 3 ). The 1 H NMR spectra of 2-azidoethanol, 2-azidoethyl-2-chloro-2-phenylacetate and 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate are shown in Fig. 1. The resonance signals of the methylene (-CH 2 -, a e b ), and hydroxyl (-OH, *) protons were observed at 3.44 and 3.76, and 2.6 ppm, respectively, confirming the achievement of 2-azidoethanol [46]. As for 2-azidoethyl-2-chloro-2-phenylacetate, the displacement of the methylene (- CH 2 CH 2 N 3 , b ) signal to 4,31ppm, and the appearance of the methane (CH) signals of the aliphatic chain ( c ) and the aromatic ring ( d ) at 5,40 and 7,46 ppm, respectively, confirmed the synthesis of this second intermediate product. Finally, the shift of the signal of the methylene (-CH 2 -, b ) protons to 4.60 ppm, and the appearance of two new peaks of the methylene (-OCH 2 CH 3 , e ) and methyl (-CH 3 , f ) groups at 4.32 and 1.43 ppm, respectively, confirmed the synthesis of the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate. The production process yields for 2-azidoethanol, 2-azidoethyl-2-chloro-2-phenylacetate and 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate products were approximately 82, 95 and 93%, respectively. 3.1.2 Synthesis of well-defined azido-terminated poly( N -vinylcaprolactam) homopolymers (PNVCL-N 3 ) The livingness of the RAFT/MADIX polymerization of NVCL in the presence of the RAFT agent 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate was evaluated by the polymerization kinetics using a [NVCL]:[CTA]:[AIBN] molar ratio of 300:1:0.25. The polymerization of NVCL proceeded slowly, reached a conversion limit of approximately 32.5% (Fig. S1(a) in Supporting Information). Although an induction period is often observed in RAFT/MADIX systems [47], such a period was not clearly observed in our system. The low chain transfer efficiency due to the steric hindrance caused by the caprolactam ring may be the reason for the low conversion rate. Similar findings were reported by Peng et al. (2015) [48] during the RAFT copolymerization of NVCL with methacrylic acid N-hydroxysuccinimide ester (MNHS) mediated by the CTA methyl 2-(ethoxycarbonothioylthio)propanoate, where conversion values below 50% were attributed to high steric hindrance caused by the 7-membered group. Despite the linear increase in the number-average molar mass ( M n SEC ) of the PNVCL-N 3 polymers with monomer conversion, the dispersity ( Ð ) of the polymer also slightly increased with the reaction time (Fig. S1(b) in the Supporting Information), suggesting that side reactions occurred under the experimental conditions, potentially due to the low thermal stability of the xanthate end group and/or impurities. Therefore, to keep the polymer dispersity ( Ð ) below 1.5, the NVCL conversion had to be limited to approximately 20% (reaction time = 6 hours). SEC chromatogram of PNVCL-N 3 (1) is depicted in Figure S2 in the Supporting Information . The number average molar masses ( M n) and dispersity ( Đ ) values obtained for this polymer are presented in Table 1. Table 1 Related data on PNVCL-N 3 synthesized by the RAFT/MADIX polymerization of NVCL mediated by the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate. Sample [NVCL] 0 :[CTA] 0 :[AIBN] 0 molar ratio Conv. a (%) M n theo b (g.mol -1 ) M n NMR c (g.mol -1 ) M n SEC d (g.mol -1 ) Đ d PNVCL-N 3 (1) 300:1:0.25 19.8 8595 8344 3298 1.45 Reaction time = 6h; a Determined by 1 H NMR; b M n theo. = ([NVCL]/[CTA] . MM NVCL . conv) + MM CTA ; c determined by 1 H NMR by using the average peak area of methine protons from PNVCL backbone at 4.30 ppm and that one of the methine protons of the aromatic ring from the CTA present in the end of the PNVCL chain at 7.2 ppm; d determined by SEC in THF and TEA (0.3% v/v) at 35 °C. The experimental molar mass determined by NMR ( M n NMR ) agreed with its theoretical molar mass ( M n theo ) (Table 1). However, due to the use of PS as a calibration standard in the SEC analysis, there was a significant discrepancy between them and that one obtained by SEC ( M n SEC ). Nevertheless, the sample exhibits monomodal molar mass distribution and a relative low dispersity ( Đ < 1.5), as desired. FTIR spectrum of the PNVCL-N 3 (1) presented in Fig. 2 showed absorption bands at 1631 cm -1 due to the axial deformation of the carbonyl (C=O) of the caprolactam ring; at 2930 and 2858 cm -1 relative to the C-H stretch; at 1442 cm -1 referring to the -CH 2 - angular deformation and at 1480 cm -1 relative to the C-N axial deformation. The shoulder at around 3250 cm −1 is assigned to N-H stretching vibration. The broad absorption band in the region 3200–3700 cm −1 has arisen from the moisture adsorbed by the sample. The -N=N + =N - band present at around 2100 cm -1 indicates the functionalization of the polymer with azide groups from the CTA. As seen in 1 H NMR spectrum of PNVCL-N 3 (1) from Fig. 3, the sample gave the typical resonance signals of PNVCL at δ (ppm) = 4.35 (1H, –NCH– α position, c ), 3.05 (–NCH 2 –, d ), 2.25 (–C(=O)CH 2 –, h ) and 1.0–2.0 (6H, –CH 2 –, e and g of the caprolactam ring, and –CH 2 – of the backbone chain of the polymer, i ). The resonance signals located at 7.23 and 5.0 ppm refer to the methine (5H, =CH-, k ) groups of the aromatic ring, and the methylene (-C(=O)O CH 2 CH 2 N 3 , l ) group of the CTA, respectively. The peak located at 4.6 ppm is relative to the methylene (– CH 2 CH 3 , b ) protons of the xanthate group. The other protons from CTA ( a , j and m ) presented their signals overlapped by the characteristic peaks of the PNVCL. 3.2 Synthesis of the alkyne end-capped PHBHV (alkyne-PHBHV) Alkyne end-capped PHBHV were prepared through a transesterification reaction between PHBHV ( M n SEC = 114873 g mol -1 and Đ = 2.86) and propargyl alcohol, in toluene, at 110 °C, using dibutyltin dilaurate (DBTD) as catalyst. SEC chromatograms obtained for the alkyne-PHBHV are shown in Fig. S4 in the Supporting Information . The M n and Ð values for these materials are presented in Table 2. Table 2 Related data on alkyne-PHBHV prepared by transesterification reaction between a high molar mass PHBHV and propargyl alcohol. Sample t (h) M n NMR a (g.mol -1 ) M n SEC b (g.mol -1 ) Ð b Alkyne-PHBHV (1) 12 5687 4536 1.45 Alkyne-PHBHV (2) 7 9807 9403 1.47 a Determined by 1 H NMR by using the average peak areas of the methine and methyl protons from PHBHV backbone at 5.25 and 0.85 ppm, respectively, and methylene protons from the polymer chain termination at 4.68 ppm; b determined by SEC in THF and TEA (0.3 % v/v) at 35 °C. As expected, it was observed a decrease in the M n SEC of the PHBHV after its transesterification reaction with propargyl alcohol. Moreover, the alkyne-PHBHV (2) showed higher M n than alkyne-PHBHV (1), as expected, and both materials showed low dispersity values ( Ð ≤ 1.5) and monomodal SEC chromatograms. Fig. 4 and 5 show the FTIR and 1 H NMR spectra of the alkyne-PHBHV (1), respectively. FTIR spectra of alkyne-PHBHV exhibited the characteristic strain bands of PHBHV, such as axial strain of the carbonyl (C=O) at 1722 cm -1 , ester C-O at 1275 cm -1 , O-H at 3431 cm -1 and C-C at 976 cm -1 . The spectra also showed the bands of symmetric angular deformation in the plane of the methyl (-CH 3 ) groups at 1380 cm -1 and the typical band of the chain spiral conformation at 1222 cm -1 . The bands in the range between 2830 and 3070 cm -1 correspond to the C-H axial strains of methyl and methylene groups, and the bands at 1130 and 1180 cm -1 refer to the symmetric and asymmetric stretches of the -C-O-C- group, respectively. Moreover, the axial deformation vibration band of the ≡C-H appears at 3255 cm -1 . In 1 H NMR spectrum of the alkyne-PHBHV it was possible to attribute the chemical shifts that characterize the PHBHV structure, at: δ (ppm) = 5.10-5.32 (-CH- of HB and HV, a and d respectively), 2.40-2.67 (-CH 2 - of HB and HV, c and g , respectively), 1.5-1.7 (-CH CH 2 CH 3 of HV, e ), 1.20-1.35 (-CH CH 3 of HB, b ) and 0.79-0.95 (-CHCH 2 CH 3 of HV, f ). Furthermore, it was possible to observe the resonance signals of the adjacent protons to the hydroxyl-terminal group characteristic of the PHBHV molecule at 4.16 ppm (- CH (CH) 3 OH, a' ), and the protons from the propargyl alcohol molecule coupled to the end of the PHBHV chain at 4.67 ppm (HC≡C- CH 2 -, h ) and 2.36 ppm ( HC ≡C-CH 2 -, i ). 3.3 Coupling reaction between PNVCL-N 3 and alkyne-PHBHV homopolymers via CuAAC Finally, PNVCL-N 3 and alkyne-PHBHV were reacted to give the corresponding PNVCL- b -PHBHV block copolymers. Aiming to make all the coupling reactions completely, they were carried out by using an excess of the PNVCL-N 3 homopolymer. After the click reactions, the unreacted PNVCL-N 3 was removed using dialysis method. PNVCL- b -PHBHV block copolymers were characterized by SEC as shown in Fig. 6. Table 3 shows the M n and Đ values obtained for these copolymers synthesized with different lengths of PHBHV hydrophobic segment. Table 3 Related data on PNVCL- b -PHBHV block copolymers synthesized by CuAAC Sample M n SEC a (g.mol -1 ) Đ a PNVCL- b -PHBHV (1) 6642 1.47 PNVCL- b -PHBHV (2) 11190 1.48 Using the [alkyne-PHBHV] 0 :[PNVCL-N 3 ] 0 :[CuBr] 0 :[PMDTA] 0 = 1:1.25:4:4; a determined by SEC in THF and TEA (0.3 % v/v) at 35 °C. As seen in Fig. 4, the SEC chromatography of PNVCL- b -PHBHV shifted toward high molecular weight direction as compared to that of PNVCL-N 3 and alkyne-PHBHV, while neither PNVCL-N 3 nor alkyne-PHBHV traces were found. Furthermore, relatively low Đ values were observed for both samples. Therefore, these results suggest the successful coupling between both macrochains, and the absence of residual PNVCL-N 3 and alkyne-PHBHV polymers. Further confirmation of the ‘‘click’’ coupling can be taken from FTIR spectroscopy. In Fig. 7, the IR spectra of PNVCL- b -PHBHV block copolymers are compared to the spectra of starting polymers. As expected, the FTIR spectrum of PNVCL- b -PHBHV showed the characteristic vibration bands of both PNVCL and PHBHV segments including the axial deformation of the carbonyl (C=O) of the caprolactam ring at 1630 cm -1 and the C=O stretching of esters at 1725 cm -1 . The disappearance of the band of the azide group (–N=N + =N - ) at 2100 cm -1 in the spectra of the block copolymers suggest the formation of the desired block copolymers. However, it was not possible to confirm the disappearance of the ≡CH band at 3255 cm -1 because the materials exhibited a broad absorption in the region 3200–3600 cm −1 due to hydroxyl groups and/or moisture adsorbed by the sample and a band of NH stretching vibration at around 3275 cm −1 . In 1 H NMR spectra of PNVCL- b -PHBHV (Fig. 8), it was possible to observe the characteristic peaks of PHBHV ( u , q , t , p , v and s ) and PNVCL ( c , d , h , e , f , g and i ) segments, which were previously detailed. Comparing the spectrum of the alkyne-PHBHV with the PNVCL- b -PHBHV, the methylene protons resonance signal characteristics of the propargyl group of the alkyne-PHBHV at 4.68 ppm (represented by “ h ” in Fig. 5) had clearly shifted to 5.22 ppm (represented by “ o ” in Fig. 8), which is overlapping by the q and u peaks. Moreover, the appearance of a new peak, relative to the methine (CH, n ) protons from the triazole ring at 7.82 ppm, confirms the coupling of PNVCL-N 3 and alkyne-PHBHV segments. Furthermore, these NMR results, in agreement with those obtained by SEC and FTIR analyses, also indicate a high degree of purity for the block copolymers, which is confirmed by the absence of PNVCL-N 3 and alkyne-PHBHV residue. It was not possible to determine the M n of these block copolymer by 1 H NMR because all characteristic peaks of the HV units ( p , q , r and s ) of the PHBHV segment are overlapping by other peaks of the block copolymer. PNVCL–N 3 , alkyne-PHBHV and the PNVCL- b -PHBHV block copolymers were characterized by DSC, as shown in Fig. 9, with the main transition temperature data summarized in Table 4. Table 4 Thermal characterization of PNVCL-N 3 , alkyne-PHBHV and PNVCL- b -PHBHV by DSC Sample Tg PNVCL (°C) Tg PHBHV (°C) Tm 1 PHBHV (°C) Tm 2 PHBHV (°C) ΔHm 1 (J/g) ΔHm 2 (J/g) Xc a,b (%) PNVCL-N 3 (1) 175.4 - - - - - - Alkyne-PHBHV (1) - 3.3 135.7 147.7 21.6 49.8 45.7 Alkyne-PHBHV (2) - 4.7 146.3 157.4 39.2 51.3 47.1 PNVCL- b -PHBHV (1) nd 27.0 nd nd nd nd nd PNVCL- b -PHBHV (2) nd 14.9 nd nd nd nd nd Nd = not determined, a Xc (%) = (ΔHm/ ΔH°m).100; ΔH°m = enthalpy fusion of 100% crystalline PHBHV = 109 J.g -1 [49]; b determined by using Tm 2PHBHV The glass transition temperature (Tg) of the PNVCL, an amorphous material, is commonly reported to be around 147 °C [50-52]. However, its Tg can be influenced by several factors, including molar mass, dispersity, purity [53], and the presence of water in the polymer [51, 52]. In our study, the determined Tg of the dried PNVCL–N 3 was determined to be 175,4 °C, which is consistent with previous findings reported by our group [15] and in the literature, such as those by Usanmaz et al. (2009) [54] and Durkut et al. (2009) [55], who observed values of 177.2, 174.6 and 174.0 °C, respectively. For the PHBHV-alkyne samples, the Tg values were about 4.0 °C, which is close to the T g of PHBHV reported in the literature [56, 57]. Two peaks related to melting events (Tm 1 and Tm 2 ) were observed in the DSC thermograms of PHBHV-alkyne. This thermal behavior referring to the PHBHV fusion agrees with the literature [58-60]. According to Liu et al. (2009) [61], these two melting peaks can be attributed to the polymer melting-recrystallization-remelt process. The second melting point (Tm 2 ), the one with the highest temperature, is generally used as the Tm of the PHBHV. Furthermore, it is important to mention that the two endothermic peaks in the DSC thermogram have also been commonly observed for PHB [62-64]. Comparing the T m and the crystallinity degree (Xc) of both PHBHV-alkyne, the PHBHV-alkyne (2) showed the highest T m and crystallinity degree (Xc) values due to its higher molar mass, as expected. The thermograms of the block copolymers samples showed Tg values at 27.0 and 14.9 °C, corresponding to the PHBHV block segments in PNVCL-b-PHBHV (1) and PNVCL-b-PHBHV (2), respectively. However, the Tg associated with the PNVCL segment was not observed for both PNVCL- b -PHBHV copolymers, possibly due to the dominance of the PHBHV phase in these copolymers, or due to specific conditions of the DSC analysis. Additionally, the DSC thermograms of the block copolymers did not show endothermic peak related to melting, suggesting that these materials have a low degree of crystallinity or exhibit amorphous polymer characteristics. The PNVCL segment covalently bound to the PHBHV segment must have restricted the PHBHV crystallization, compromising the regularity of its crystalline structure. 3.4. Determination of the cmc of the block copolymers The onset of micellization and the critical micelle concentration (cmc) of the PNVCL- b -PHBHV block copolymers were obtained using a fluorescence technique with pyrene as the fluorescence probe. The plot of the I337/I333 intensity ratio (from fluorescence measurement) versus the logarithm of the corresponding concentration of the copolymer in water (in mg mL −1 ) is shown in Fig. 10 . At a certain concentration, the intensity ratio started to increase dramatically due to the incorporation of pyrene into the hydrophobic core of the micelles. The intersection of the baseline and the rapidly rising I337/I333 line is considered as the cmc of the amphiphilic block copolymer [14]. From this plot, CMC values for both samples were close, being 3.89 and 3.09 10 -3 mg mL -1 for PNVCL- b -PHBHV (1) and PNVCL- b -PHBHV (2), respectively. In agreement with the literature for amphiphilic block copolymer [14-16, 65], the cmc decreased as the length of the hydrophobic block increased. This trend is strictly related to the hydrophilicity of the copolymer since a higher length of the hydrophobic segment results in stronger interactions between the hydrophobic chains; therefore, a lower concentration of polymer in water is necessary to induce micellization. 3.5. E ffect of the hydrophobic block length on the hydrodynamic diameter of the polymeric micelles To evaluate the effect of the PHBHV hydrophobic length on the average size of PNVCL- b -PHBHV-based polymeric micelles, micellar solutions, prepared with the PNVCL- b -PHBHV copolymers by the precipitation technique, were analyzed by DLS. The hydrodynamic diameter (Zav) of the polymeric micelles formed from PNVCL- b -PHBHV (1) and PNVCL- b -PHBHV (2) (0.05 mg mL −1 ) was about 173.3 nm (PDI 0.243) and 206.6 nm (PDI 0.372), respectively. In addition, both PNVCL- b -PHBHV micelles exhibited a monomodal size distribution (Fig. 11). As was expected, for the block copolymes synthesized in this study, with a fixed leght for the hydroplilic segment (PNVCL), an increase in the length of the hydrophobic segment of PHBHV led to micelles with larger diameters due to the greater hydrophobic interaction forces and packing density required to accommodate the longer PHBHV blocks within the micelle core. The increased hydrophobic block length results in a larger micelle core to minimize the unfavorable interactions between the hydrophobic PHBHV segments and the aqueous environment. These results are in agreement with the results reported in the literature for amphiphilic block copolymers [14-16, 65] and suggesting that the size of the micelle could be adjustable as a function of the length of the hydrophobic segment in the block copolymer. 4. CONCLUSION A novel block copolymer, PNVCL- b -PHBHV, was successfully synthesized by combining the RAFT/MADIX polymerization and the click chemistry reaction. First, well-defined azido-group functionalized PNVCL homopolymers (PNVCL-N 3 ) was synthesized by RAFT/MADIX polymerization of NVCL mediated by the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate. The RAFT/MADIX polymerization of NVCL showed partial control by the CTA. Therefore, aiming to obtain PNVCL homopolymers with low dispersity, the NVCL conversion had to be limited to values below 20%. Alkyne-terminated PHBHV was prepared by transesterification reaction of the PHBHV with propargyl alcohol. SEC technique confirmed the transesterification reaction of PHBHV leading a material with low molecular mass. The chemical structures of the PNVCL-N 3 and alkyne-PHBHV were successfully confirmed by FTIR and 1 H NMR analyses. Then, PNVCL-N 3 and PHBHV-alkyne macrochains were coupled by the CuAAC technique, which was confirmed by the SEC analyzes. Furthermore, while FTIR analyzes showed the disappearance of the azide function of PNVCL-N 3 , after its reaction with PHBHV-alkyne, the formation of the triazole ring was verified by NMR analyses. The DSC analysis indicated that the PNVCL- b -PHBHV block copolymers had characteristics of amorphous material or very low degrees of crystallinity. The amphiphilic character of the block copolymers and their ability to form micelles were successfully confirmed. The cmc values of these copolymers decreased with increasing the length of the PHBHV segment. Finally, the size of the polymeric micelles was investigated. The average diameter of the polymeric micelles determined by DLS increased with increasing the length of the PHBHV segment. Declarations Credit Authorship Contribution Statement Rodolfo Minto de Moraes: Conceptualization, Methodology, Formal Analysis, Investigation, Validation, Data Curation, Writing-Original Draft Preparation, Writing—Review and Editing. Layde Teixeira de Carvalho: Writing-Original Draft Preparation. Gizelda Maria Alves: Formal Analysis and Data Curation. Simone de Fátima Medeiros: Writing-Original Draft Preparation, Writing—Review and Editing. Amilton Martins dos Santos: Writing—Review and Editing, Supervision, Project administration, Funding Acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper. Conflicts of Interest The authors declare no conflict of interest. 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Karpova SG, Iordanskii AL, Popov AA, Shilkina NG, Lomalin SM, Shcherbin MA, Chvalun SN, Berlin AA (2012) Effect of external influences on the structural and dynamic parameters of polyhydroxybutyrate-hydroxyvalerate-based biocomposites. Russ J Phys Chem B 6:72–80. https://doi.org/10.1134/S1990793112010095. Santos IF, Moraes RM, Medeiros SF, Kular JK, Johns MA, Sharma R, Santos AM (2021) Enhanced ligand-free attachment of osteoblast to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanoparticles. Int J Biol Macromol 189:528–536. https://doi.org/10.1016/j.ijbiomac.2021.08.120. Liu Q-S, Zhu M-F, Wu W-H, Qin Z-Y (2009) Reducing the formation of six-membered ring ester during thermal degradation of biodegradable PHBV to enhance its thermal stability. Polym Degrad Stab 94:18-24. https://doi.org/10.1016/j.polymdegradstab.2008.10.016. Yoshie N, Fujiwara M, Ohmori M, Inoue Y (2001) Temperature dependence of cocrystallization and phase segregation in blends of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Polymer 42:8557-8563. https://doi.org/10.1016/S0032-3861(01)00408-6. Wellen RMR, Rabello MS, Júnior ICA, Fechine GJM, Canedo EL (2015) Melting and Crystallization of poly(3-hydroxybutyrate): effect of heating/cooling rates on phase transformation. Polímeros 25:296-304. https://doi.org/10.1590/0104-1428.1961. Mottin AC, Ayres E, Oréfice RL, Câmara JJD (2016) What Changes in Poly(3-Hydroxybutyrate) (PHB) When Processed as Electrospun Nanofibers or Thermo-Compression Molded Film?. Mater Res 19:57-66. https://doi.org/10.1590/1980-5373-MR-2015-0280. Moraes RM, Carvalho LT, Teixeira AJRM, Medeiros SF, Santos AM (2023) Well-defined amphiphilic poly(ε-caprolactone)- b -poly( N -isopropylacrylamide) and thermosensitive micelles formulation. Iran Polym J 32:1627–1641. https://doi.org/10.1007/s13726-023-01230-4. Schemes Schemes 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.tiff Scheme2.tiff Scheme3.tiff Scheme4.tiff SupportingInformationSIPolymerBulletin.docx Cite Share Download PDF Status: Published Journal Publication published 10 Jul, 2025 Read the published version in Polymer Bulletin → Version 1 posted Editorial decision: Revision requested 11 Jun, 2025 Reviews received at journal 11 Jun, 2025 Reviewers agreed at journal 02 Jun, 2025 Reviews received at journal 23 May, 2025 Reviewers agreed at journal 07 May, 2025 Reviewers invited by journal 03 Apr, 2025 Editor assigned by journal 29 Mar, 2025 Submission checks completed at journal 29 Mar, 2025 First submitted to journal 28 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6328656","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447101447,"identity":"b57b9e32-ad88-4dc9-bf77-b720b96956dd","order_by":0,"name":"Rodolfo Minto 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3","display":"","copyAsset":false,"role":"figure","size":451878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectrum of PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6328656/v1/d4ac102610884bca44e08997.png"},{"id":81308123,"identity":"d5147f7a-97c4-496b-a435-2ad2840f8192","added_by":"auto","created_at":"2025-04-24 15:01:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292068,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of alkyne-PHBHV (1)\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6328656/v1/c28a7eef1a7c308e36af6559.png"},{"id":81308116,"identity":"45231576-a188-4f0b-8737-27941739a1a6","added_by":"auto","created_at":"2025-04-24 15:01:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1067607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectra of alkyne-PHBHV (1)\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6328656/v1/061f5dc7f8f4a4740e1da15b.png"},{"id":81310987,"identity":"dcc89bd3-0228-4dba-82db-0b2e08d83601","added_by":"auto","created_at":"2025-04-24 15:25:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":302909,"visible":true,"origin":"","legend":"\u003cp\u003eSEC chromatogram of \u003cstrong\u003e(a)\u003c/strong\u003e PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1), alkyne-PHBHV (1) and PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1), and \u003cstrong\u003e(b)\u003c/strong\u003e PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1), alkyne-PHBHV (2) and PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV 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9","display":"","copyAsset":false,"role":"figure","size":239280,"visible":true,"origin":"","legend":"\u003cp\u003eDSC thermograms of \u003cstrong\u003e(a)\u003c/strong\u003e PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1), \u003cstrong\u003e(b)\u003c/strong\u003e alkyne-PHBHV (1), \u003cstrong\u003e(c)\u003c/strong\u003e alkyne-PHBHV (2),\u003cstrong\u003e (d)\u003c/strong\u003e PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1) and \u003cstrong\u003e(e)\u003c/strong\u003e PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (2)\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6328656/v1/0bdd18535c81c7e5e69d9f36.png"},{"id":81310047,"identity":"651fe0e6-52b1-437d-bb75-8823e5f4f11b","added_by":"auto","created_at":"2025-04-24 15:17:43","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":380874,"visible":true,"origin":"","legend":"\u003cp\u003ePlots of I337/I333 \u003cem\u003evs\u003c/em\u003e 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Amphiphilic block copolymers are macromolecules composed of different polymer blocks with distinct hydrophilic and hydrophobic segments. Such materials have the ability to self-assemble in aqueous solution into various nanostructured aggregates including micelles and vesicles [1-3]. This feature contributes to the usefulness of these materials in various applications areas including microelectronics [4], nanoreactors [5], catalysis [5] and drug delivery systems [6]. In the context of biomedical applications, stimuli-responsive or so-called \u0026ldquo;smart\u0026rdquo; polymers, particularly those used in drug delivery systems, have gained significant attention. This is due to their ability to undergo rapid and reversible changes in response to various external stimuli, such as temperature, pH, and other environmental factors, which allows for controlled and targeted release of therapeutic agents [7]. Thermoresponsive polymers (also called temperature-responsive or thermosensitive polymers) are a class of material that exhibits a phase transition temperature in correspondence of which a drastic change in the solubility occurs [8]. Based on their thermosensitive behavior in solution, these polymers can be classified into two categories, namely the lower critical solution temperature (LCST) behavior and upper critical solution temperature (UCST) behavior [9, 10]. In the first case, the polymer is miscible with the solvent at temperatures below the phase transition temperature, however, two immiscible phases (a diluted polymer phase and a concentrated polymer phase) form above this temperature. On the other hand, polymers exhibiting UCST show the opposite behavior, the phase separation occurs upon cooling below the phase transition temperature [9, 10].\u003c/p\u003e\n\u003cp\u003ePoly(\u003cem\u003eN\u003c/em\u003e-vinylcaprolactam) (PNVCL) is widely\u0026nbsp;investigated as a hydrophilic homopolymer with a lower critical solution temperature (LCST) close to physiological levels. Its LCST varies\u0026nbsp;based on molar mass and polymer concentration, and\u0026nbsp;depends on the chemical composition in the case of copolymers\u0026nbsp;[11, 12]. PNVCL, known for its\u0026nbsp;biocompatibility, low toxicity, and the ability to complex with other molecules, is applied\u0026nbsp;across various domains, particularly in the\u0026nbsp;medical field [13].\u0026nbsp;Despite extensive research in this area, literature predominantly details the preparation of a wide range of PNVCL-based amphiphilic thermosensitive block copolymers, such as PNVCL-\u003cem\u003eb\u003c/em\u003e-poly(\u0026epsilon;-caprolactone) [14, 15], PNVCL-\u003cem\u003eb\u003c/em\u003e-poly(\u003cem\u003eD,L\u003c/em\u003e-lactide) [16, 17], and PNVCL-\u003cem\u003eb\u003c/em\u003e-PVAc [18]. Nevertheless, a significant gap exists, with no documented instances of the synthesis of block copolymers that combine PNVCL with PHBHV blocks. This unexplored field offers intriguing potential for\u0026nbsp;developing novel materials with tailored properties and applications.\u003c/p\u003e\n\u003cp\u003ePoly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate) (PHBHV), a derivate from the highly popular poly(3-hydroxybutyrate) (PHB), is a low-cytotoxicity hydrophobic copolymer that belongs to the poly(hydroxyalkanoates) (PHAs) family. The PHBHV is a\u0026nbsp;biocompatible and biodegradable thermoplastic polyesters, as all other polyhydroxyalkanoates (PHAs), product of biosynthesis of a wide variety of both gram-positive and gram-negative bacteria [19]. Among wild microbes, \u003cem\u003eRalstonia eutropha\u003c/em\u003e, also known as \u003cem\u003eCupriavidus necator\u003c/em\u003e or \u003cem\u003eAlcaligenes eutrophus\u003c/em\u003e [20-22] has been the most used specie\u003cem\u003e.\u0026nbsp;\u003c/em\u003ePHBHV\u0026rsquo;s properties, such as its biological origin, low cytotoxicity, absorption capacity, piezoelectricity, and thermoplasticity, justify its potential for biomaterial applications, including the fabrication of cardiovascular stents [23], drug release and transport systems [24, 25], absorbable surgical sutures, and medical packaging [26]. However, its intrinsic hydrophobicity property may restrict some of its \u003cem\u003ein vivo\u003c/em\u003e applications. Therefore, the elaboration of PHBHV-based amphiphilic copolymers is one of the most interesting alternatives to overcome such drawback, making this polymer viable for these applications.\u003c/p\u003e\n\u003cp\u003eThe combination of synthetic organic chemistry and polymer chemistry is an interesting approach to build complex structures. An extremely versatile method and a powerful tool that has been used to constructed polymers with well-defined structures is the combination of \u0026ldquo;click chemistry\u0026rdquo; and controlled/living polymerization techniques. Such strategy has been enabled the synthesis of a wide range of block copolymers [27-30] and graft copolymers [29, 31], as well the preparation of\u0026nbsp;advanced macromolecular architectures [32].\u003c/p\u003e\n\u003cp\u003eControlled radical polymerization (CRP) techniques,\u0026nbsp;also named reversible deactivation radical polymerization (RDRP) [33], have enabled the synthesis of polymers with well-defined structures and with control of molar mass and molar mass distribution, in addition to enabling the preparation of functional polymers and/or with complex architectures, such as blocks, stars, grafted, microgel, dendritic, among others [34, 35]. Among the various types of CRP, the RAFT technique is considered one of the most versatile, since it exhibits good performance to a wide range of functional groups in monomers, initiators and solvents, besides offering a control over different types of monomers [36]. According to its ability to react with free radicals, the activated vinyl monomers can be classified into two broad categories: the more-activated (MAMs) (e.g., styrene, acrylonitrile, vinyl pyridine and (meth)acrylates) and the less-activated (LAMs) (e.g., vinyl esters and \u003cem\u003eN\u003c/em\u003e-vinylamides) monomers. Different classes of RAFT agents must be employed to promote the controlled polymerization of MAMs and LAMs. The LAMs are highly reactive and behave as poor homolytic groups, therefore, the \u003cem\u003eO\u003c/em\u003e-alkyl xanthates or \u003cem\u003eN\u003c/em\u003e-alkyl-\u003cem\u003eN\u003c/em\u003e-aryldithiocarbamates, which form less-stable intermediate radicals, are more suitable CTAs for the RAFT polymerization of this type of monomers [37].\u003c/p\u003e\n\u003cp\u003eA very interesting feature of the RAFT technique is the retention of the \u0026alpha; (R group) and \u0026omega; (Z functional thiocarbonylthio group) groups of the CTA in the polymeric chains. The incorporation of the thiocarbonylthio group at the end of the polymeric chains enables this material to act as a RAFT macroagent in the polymerization of other suitable monomer leading to the formation of block copolymers by chain extension [38]. Besides linear block copolymers, other architectures can be formed, including star, branched or hyperbranched polymers. Moreover,\u0026nbsp;since the RAFT agent is retained in the polymer structure,\u0026nbsp;polymers with the desired end-functionality can be also obtained simply by choosing the appropriate RAFT agent. Subsequently, the end-functionalized polymers can be coupled with other polymers leading to a copolymerization. Generally, the \u003cem\u003ein situ\u003c/em\u003e synthesis of end-functional polymers is conducted by using the R-group of the CTA, as the Z groups are quite unstable to nucleophiles [39].\u003c/p\u003e\n\u003cp\u003e\u0026quot;Click Chemistry\u0026quot; was a concept introduced in 2001 by Sharpless that described several types of thermodynamically favorable reactions for coupling two molecules in a very simple, fast, regioselective way and with high yields, conducting under mild experimental conditions [40]. Among the numerous techniques that are included in the \u0026ldquo;Click Chemistry\u0026rdquo; concept, the 1,3-dipolar cycloaddition reaction between azides and alkynes catalyzed by Cu(I) species (Copper(I)-catalyzed Azide-Alkyne Cycloaddition - CuAAC) has been one of the most used due to its high yield and selectivity [41]. As shown in the literature, a wide variety of both PHBHV-based (e.g.:\u0026nbsp;PULL-\u003cem\u003eg\u003c/em\u003e-PHBHV\u0026nbsp;[42], dextran-\u003cem\u003eg\u003c/em\u003e-PHBHV [43], PHBHV-\u003cem\u003eb\u003c/em\u003e-poly(2-methyl-2-oxazoline [44])\u0026nbsp;and PNVCL-based (e.g.: PNVCL-\u003cem\u003eb\u003c/em\u003e-poly(\u0026epsilon;-caprolactone) [45], PNVCL-\u003cem\u003eb\u003c/em\u003e-poly(D,L-lactide) [16], PNVCL-\u003cem\u003eb\u003c/em\u003e-poly(oligo(ethylene oxide) methyl ether methacrylate [30]\u0026nbsp;copolymers have been successfully synthesized by the CuAAC reaction.\u003c/p\u003e\n\u003cp\u003eUp to now,\u0026nbsp;as far as we are aware, there have been no reports in the literature regarding the synthesis of block copolymers comprising both PNVCL and PHBHV segments. In this work, we performed the synthesis of the well-defined amphiphilic\u0026nbsp;block copolymer, PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV, using the combination of the RAFT/MADIX and click chemistry techniques. A\u0026nbsp;\u0026omega;-azido-functionalized xanthate agent, namely, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was first synthesized and subsequently used to mediate the RAFT/MADIX polymerization of NVCL to give rise to the well-defined PNVCL homopolymer comprising an azido-end group (PNVCL-N\u003csub\u003e3\u003c/sub\u003e). Meanwhile, alkyne-terminated PHBHV (alkyne-PHBHV) was prepared by the transesterification reaction between a PHBHV with high molar mass and propargyl alcohol using dibutyltin dilaurate as catalyst. PHBHV-\u003cem\u003eb\u003c/em\u003e-PNVCL block copolymers were then prepared by Copper(I)-catalyzed Azide Alkyne Cycloaddition (\u003cem\u003eCuAAC \u0026ndash;\u0026nbsp;\u003c/em\u003e\u0026ldquo;click chemistry\u0026rsquo;\u0026rsquo;). These (co)polymers were characterized by Size Exclusion Chromatography (SEC), Fourier-Transform Infrared spectroscopy (FTIR), Proton Nuclear Magnetic Resonance spectroscopy (\u003csup\u003e1\u003c/sup\u003eH NMR) and Differential Scanning Calorimetry (DSC) measurements, and the effect of PHBHV chain length on the amphiphilicity of the copolymers besides their ability of micelles formation were evaluated.\u003c/p\u003e"},{"header":"2. EXPEMIMENTAL SECTION","content":"\u003cp\u003e\u003cstrong\u003e2.1 Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eN\u003c/em\u003e-vinylcaprolactam (NVCL, kindly supplied by BASF) was distilled under reduced pressure. Poly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate) (PHBHV containing 8.6% of HV, \u003cem\u003eM\u003c/em\u003en \u003csub\u003eSEC\u003c/sub\u003e = 114873 g mol\u003csup\u003e-1\u003c/sup\u003e and \u003cem\u003eĐ\u003c/em\u003e = 2.86, kindly supplied by PHB Industrial S/A) was dispersed in a large amount of deionized water, recovered by filtration, washed with cold ethanol and, subsequently, with cold diethyl ether, and dried under vacuum. 2,2\u0026rsquo;-azobis(isobutyronitrile) (AIBN) was recrystallized from methanol. 2-bromoethanol (BE, Aldrich, 95%), sodium azide (NaN\u003csub\u003e3\u003c/sub\u003e, Synth, 99%), 2-chloro-2-phenylacetyl chloride (Aldrich, 90%), potassium ethyl xanthogenate (KEX, Aldrich, 96%), dibutyltin dilaurate (DBTD, 95%, Synth), propargyl alcohol (99%, Aldrich), copper bromide (CuBr, Aldrich, 98%), \u003cem\u003eN,N,N\u0026prime;,N\u0026prime;\u0026prime;,N\u0026prime;\u0026prime;\u003c/em\u003e-pentamethyldiethylenetriamine (PMDTA, Aldrich, 99%), sodium hydrogen carbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e, Synth, 99.7-100%), ammonium chloride (NH\u003csub\u003e4\u003c/sub\u003eCl, Synth, 100%), anhydrous magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e XH\u003csub\u003e2\u003c/sub\u003eO, Synth, 98%) and 1.3.5-trioxane (Aldrich,\u0026nbsp;≧99%) were used as received. Triethylamine (TEA, Aldrich, 99%), toluene (Synth, 99.5%), tetrahydrofuran (THF, Synth, 99%) and 1.4-dioxane (Synth, 99%) were dried and fractionally distilled from sodium. Dimethylformamide (DMF, Synth, 99.8%) was distilled under reduced pressure in the presence of calcium hydride (CaH\u003csub\u003e2\u003c/sub\u003e). Hexane (Synth, 98.5%), ethyl acetate (Synth, 99.5%), diethyl ether (Synth, 98%) and dichloromethane (DCM, Synth, 99.5%) were used without purification steps. The water used in the entire process was deionized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1 Synthesis of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSynthesis of RAFT agent,\u0026nbsp;\u003c/em\u003e2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate\u003c/p\u003e\n\u003cp\u003eThe RAFT agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was synthesized in three steps. The synthetic route is shown in Scheme 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ea) \u0026nbsp;Synthesis of 2-azidoethanol\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn a 100 mL round-bottom flask, 40 mmol of 2-bromoethanol was diluted in 20 mL of deionized water. The result solution was placed in an ice bath and kept under vigorous stirring. Meanwhile, 4 g of NaN\u003csub\u003e3\u003c/sub\u003ewas added in small portions in the flask. Then, the system was removed from the ice bath and left under stirring for 2 hours at room temperature. Then, 2.5667 g of NaN\u003csub\u003e3\u003c/sub\u003e was added to the mixture, and the flask was immersed in a glycerin bath previously heated at 80 \u0026deg;C and maintained at this temperature, under stirring, for 24 hours. The product was then extracted with ether (4x30 mL). The combined organic extracts were dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e, and concentrated under reduced pressure at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eb) \u0026nbsp;Synthesis of\u0026nbsp;\u003c/em\u003e\u003cem\u003e2-azidoethyl-2-chloro-2-phenylacetate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA solution of 2-chloro-2-phenylacetyl chloride (4.20 g, 20 mmol) in 35 mL of dry THF was prepared in a dried 100 mL round-bottom flask equipped with a magnetic stirring bar under nitrogen at room temperature. A mixture of 2-azidoethanol (1.39 g, 16 mmol) in 8 mL of dry THF and TEA (3.24 g, 32 mmol) in 7 mL of dry THF was then added dropwise to the above solution mixture that was cooled in an ice bath. Then, the reaction mixture was stirred for 24 hours at room temperature. The precipitated by-product (i.e., Et\u003csub\u003e3\u003c/sub\u003eN.HBr), was removed by filtration and THF was evaporated. After that, the concentrated mixture was dissolved in 20 mL of DCM and washed thoroughly with saturated sodium bicarbonate (4x30 mL). The organic layer was further washed with water (3x30 mL), dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e overnight, and filtered. Finally, the filtrate was concentrated under reduced pressure at room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ec) Synthesis of 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn a dried \u0026nbsp;500 mL round-bottom flask equipped with a magnetic stirring bar under nitrogen at room temperature, a solution of potassium ethyl xanthogenate (3.63 g, 22 mmol) in 330 mL of dry THF was prepared. \u0026nbsp;Then, a solution of 2-azidoethyl-2-chloro-2-phenylacetate (3.48 g, 14.5 mmol) in 20 mL of dry THF was added dropwise to the previous solution during stirring under nitrogen flow. The reaction mixture was then stirred for 24 hours at room temperature. The THF was evaporated under vacuum at room temperature. Then, the concentrated mixture was dissolved in 150 mL of ether and washed consecutively with saturated NH\u003csub\u003e4\u003c/sub\u003eCl solution (4x50 mL), saturated NaHCO\u003csub\u003e3\u003c/sub\u003e, solution (4x50 mL), and water (4x50 mL). The organic layer was dried over anhydrous MgSO\u003csub\u003e4\u003c/sub\u003e, and filtered. The filtrate was concentrated under reduced pressure at room temperature.\u003c/p\u003e\n\u003cp\u003eRAFT/MADIX polymerization of NVCL\u003c/p\u003e\n\u003cp\u003eWell-defined azido-end capped PNVCL (PNVCL-N\u003csub\u003e3\u003c/sub\u003e) was synthesized by RAFT/MADIX polymerization of NVCL mediated by the azido-functionalized xanthate chain transfer agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate at 70 \u0026deg;C, using AIBN as the initiator and 1.4-dioxane as solvent (Scheme 2). This reaction was performed with the feed molar ratio [NVCL]\u003csub\u003e0\u003c/sub\u003e:[CTA]\u003csub\u003e0\u003c/sub\u003e:[AIBN]\u003csub\u003e0\u003c/sub\u003e = 300:1:0.25.\u003c/p\u003e\n\u003cp\u003eIn a dried and nitrogen purged round-bottom flask, 39.0 mg of 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, 5 g of NVCL, 4.9 mg of AIBN and 101.1 mg (1.12 mmol) of 1,3,5-trioxane (internal standard) were dissolved in 2.5 mL of 1,4-dioxane. A homogeneous solution was obtained after stirring, and degassed under nitrogen for 30 min. The flask was immersed in an oil bath preheated at 70 \u0026deg;C.\u0026nbsp;In order to optimize the NVCL RAFT/MADIX polymerization, the NVCL polymerization rate, and the evolution of the number average molar mass (\u003cem\u003eM\u003c/em\u003en) and the dispersity (Đ) of the polymer were monitored by \u003csup\u003e1\u003c/sup\u003eH NMR and HPLC for 24 hours, respectively. The material was reproduced under the same conditions, but on a larger scale (all quantities were increased by a factor of 10) and for only 6 hours. This reaction was stopped by cooling the mixture in an ice bath. The resulting polymer was purified by precipitation in cold diethyl ether, recovered by filtration, dried under vacuum at room temperature for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2 Preparation of the alkyne-PHBHV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePHBHV comprising an alkyne-end group (alkyne-PHBHV) was obtained via transesterification reaction between a\u0026nbsp;high-molecular-weight PHBHV (\u003cem\u003eM\u003c/em\u003en \u003csub\u003eSEC\u0026nbsp;\u003c/sub\u003e= 114873 g.mol\u003csup\u003e-1\u003c/sup\u003e and \u003cem\u003eĐ\u003c/em\u003e = 2.86) and propargyl alcohol. The transesterification reaction was catalyzed by dibutyltin dilaurate (DBTD) at 110 \u0026deg;C, using toluene as solvent (Scheme 3).\u003c/p\u003e\n\u003cp\u003eTo obtain alkyne-PHBHV macrochains with two different lengths, a transesterification reaction (ReT (1)) was performed to evaluate the effect of time on the reduction of molar mass of the alkyne-PHBHV. During this first transesterification reaction samples were withdrawn at pre-established times (2, 4, 6, 8, 10 and 12 hours). In a 125 mL dried round-bottom flask equipped with a magnetic stirring bar under nitrogen flow, 5 g of high molar mass PHBHV was solubilized in 60 mL of toluene at 130 \u0026deg;C in a glycerin bath. After complete solubilization of the polymer, the reaction temperature was decreased to 110 \u0026deg;C and a solution of propargyl alcohol and dibutyltin dilaurate (DBTD) in 15 mL of toluene was immediately added into the flask. The polymers contained in the aliquots were precipitated in cold hexane and their \u003cem\u003eM\u003c/em\u003en was monitored by SEC (Table S1 in Supporting Information). Subsequently, two other reactions were conducted under the same conditions, but on a larger scale (all quantities were increased by a factor of 4), using different reaction times: 12 hours (alkyne-PHBHV (1)) and 7 hours (alkyne-PHBHV (2)). The polymers were precipitated in cold hexane, and a selective precipitation method of cold mixture of hexane and ethyl acetate (15/85 %, v/v) was used to improve the purification and to reduce the polymer dispersity (\u003cem\u003eĐ\u003c/em\u003e). In brief, the material recovered from the alkyne-PHBHV (1) reaction was dissolved in DCM and precipitated three times in the cold solvent mixture. On other side, the material obtained from the alkyne-PHBHV (2) reaction was precipitated four times in this cold solvent mixture. Finally, the polymers were dried under vacuum at room temperature for 24 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3 Synthesis of the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers were synthesized by click chemistry using CuBr as catalyst, \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e\u0026prime;,\u003cem\u003eN\u003c/em\u003e\u0026prime;\u0026prime;,\u003cem\u003eN\u003c/em\u003e\u0026prime;\u0026prime;-pentamethyldiethylenetriamine (PMDTA) as nitrogenous base and DMF as solvent (Scheme 4). The CuAAC reactions were performed in the presence of a slight excess of PNVCL-N\u003csub\u003e3\u003c/sub\u003e, using the [alkyne-PHBHV]\u003csub\u003e0\u003c/sub\u003e:[PNVCL-N\u003csub\u003e3\u003c/sub\u003e]\u003csub\u003e0\u003c/sub\u003e:[CuBr]\u003csub\u003e0\u003c/sub\u003e:[PMDTA]\u003csub\u003e0\u003c/sub\u003e molar ratio = 1:1.25:4:4. The ratio of DMF volume relative to the polymer weight used in these reactions was 10.0 mL g\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp skip=\"true\"\u003eIn a dried and nitrogen purged round-bottom flask, a solution containing 0.6 g of alkyne-PHBHV and 1.1 g of PNVCL-N\u003csub\u003e3\u003c/sub\u003e in dry DMF was prepared and kept under stirring at room temperature. Meanwhile, solutions of 73.9 mg of PMDETA and 61.8 mg of CuBr in DMF were prepared in two others dried and nitrogen purged round-bottom flask. After 30 minutes under stirring, the PMDTA and CuBr solutions were added dropwise into the polymer mixture that was cooled in an ice bath. After that, the flask was immersed in an oil bath preheated at 60 \u0026deg;C and kept for 120 hours. To eliminate the unreacted PNVCL-N\u003csub\u003e3\u003c/sub\u003e, the mixture was dialysis against water. Then, the reaction product was extracted with DCM. To eliminate residual copper salts, the organic layer was passed through a short aluminum oxide column. Finally, the polymer was precipitated in cold diethyl ether and dried under vacuum at room temperature for 24hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePolymeric particles preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe particles were prepared via the nanoprecipitation technique. 12.5 mg block copolymer was dissolved in 5 mL THF, thereafter, 1 mL of this polymeric solution was added dropwise into 45 mL of ultrapure water under stirred at 25 \u0026deg;C. The solvent THF was then removed using a rotary evaporator. Subsequently, the obtained dispersion was transferred into a 50 mL volumetric flask, followed by dilution to the calibration mark with water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePolymer Characterization\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe number average molar masses (\u003cem\u003eM\u003c/em\u003en) and dispersities (\u003cem\u003eĐ\u003c/em\u003e) were determined by SEC (Waters 1515) using chloroform or THF with triethylamine (0.3% v/v) as the solvent, at 35 \u0026deg;C, with a flow rate of 1.0 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e on three Phenogel columns (10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e6\u003c/sup\u003e \u0026Aring;) connected in series to a 2414 differential refractive index detector. A high-performance liquid chromatography (HPLC, Shimadzu) was also used to determine the \u003cem\u003eM\u003c/em\u003en and \u003cem\u003e\u0026ETH;\u003c/em\u003e using THF with triethylamine (0.3% v/v) as the solvent, at 35 \u0026deg;C with a flow rate of 0.8 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e on two Phenogel columns (10\u003csup\u003e4\u003c/sup\u003e and 10\u003csup\u003e6\u003c/sup\u003e \u0026Aring;) connected in series to a differential refractive index detector (Sil\u0026minus;20A Shimadzu). The columns were calibrated using polystyrene (PS) standards. The \u003csup\u003e1\u003c/sup\u003eH NMR spectra were obtained in a Varian Mercury-300 NMR spectrometer (300 MHz) at room temperature using deuterated dimethylsulfoxide (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) or chloroform (CDCl\u003csub\u003e3\u003c/sub\u003e) as solvents, and the chemical shifts are reported in parts per million (ppm) using tetramethylsilane (TMS) as the internal standard. FTIR spectra were collected using a Shimadzu IRPrestige-21 spectrometer using the KBr disk method. The (co)polymers thermal properties were evaluated using \u003cem\u003ea \u003cem\u003eTA Instruments Q20\u0026nbsp;\u003c/em\u003e\u003c/em\u003eDSC under nitrogen atmosphere. The instrument was calibrated with Indium before use. The samples were first heated from \u0026minus;80 to 180 \u0026deg;C at a 10 \u0026deg;C min\u003csup\u003e\u0026minus;1\u003c/sup\u003e heating rate, followed by quenching to \u0026minus;80 \u0026deg;C. Then, the samples were re-heated to 220 \u0026deg;C at a rate of 10 \u0026deg;C min\u003csup\u003e\u0026minus;1\u003c/sup\u003e. X-ray diffraction patterns were obtained by a PAN-analytical Empyrean ACMS 101 (Malvern, UK) diffractometer at room temperature. The diffractometer was used with a monochromatic radiation beam of the CuK\u0026alpha;. Experiments were conducted with a scan range from 10 to 90\u0026deg; (2\u0026theta;), a step size of 0.01\u0026deg; (2\u0026theta;), and a counting time of 10 s per step.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicelles characterization\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCritical micelle concentrations (cmc) of the block copolymers were determined by fluorescence measurements at 390 nm emission wavelength (Varian Cary Eclipse fluorescence spectrophotometer) using pyrene as a probe. A pyrene solution in acetone was added into a series of volumetric flasks in such an amount that the final concentration of pyrene in each solution was 6.10\u003csup\u003e-7\u0026nbsp;\u003c/sup\u003emol L\u003csup\u003e-1\u003c/sup\u003e. The acetone was then allowed to completely evaporate. The polymer solution, previously prepared with the aid of an ultrasound bath (30 min.), was added into the volumetric flasks and diluted to the calibration mark using deionized water to obtain different copolymer concentrations ranging from 10\u003csup\u003e-4\u003c/sup\u003e to 0.5 mg mL\u003csup\u003e-1\u003c/sup\u003e. The samples were stored at room temperature overnight to equilibrate micelles and pyrene. The size distribution of micelles (prepared by the nanoprecipitation technique) was determined by dynamic light scattering (DLS) using a Malvern Nano ZS instrument.\u003c/p\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e3.1 Synthesis of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.1.1 Synthesis of RAFT agent, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers were synthesized via the combination of RAFT/MADIX and click chemistry techniques as shown in the Scheme 4. In the first step, the xanthate agent comprising azide group, 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate, was synthesized and, subsequently, used to mediate the RAFT/MADIX polymerization of NVCL to the preparation of well-defined azido-terminated poly(\u003cem\u003eN\u003c/em\u003e-vinylcaprolactam) homopolymers (PNVCL-N\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectra of 2-azidoethanol, 2-azidoethyl-2-chloro-2-phenylacetate and 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate are shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003eThe resonance signals of the methylene (-CH\u003csub\u003e2\u003c/sub\u003e-, \u003cstrong\u003ea\u003c/strong\u003e e \u003cstrong\u003eb\u003c/strong\u003e), and hydroxyl (-OH, *) protons were observed at 3.44 and 3.76, and 2.6 ppm, respectively, confirming the achievement of 2-azidoethanol [46]. As for 2-azidoethyl-2-chloro-2-phenylacetate, the displacement of the methylene (-\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e, \u003cstrong\u003eb\u003c/strong\u003e) signal to 4,31ppm, and the appearance of the methane (CH) signals of the aliphatic chain (\u003cstrong\u003ec\u003c/strong\u003e) and the aromatic ring (\u003cstrong\u003ed\u003c/strong\u003e) at 5,40 and 7,46 ppm, respectively, confirmed the synthesis of this second intermediate product. Finally, the shift of the signal of the methylene (-CH\u003csub\u003e2\u003c/sub\u003e-, \u003cstrong\u003eb\u003c/strong\u003e) protons to 4.60 ppm, and the appearance of two new peaks of the methylene (-OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e, \u003cstrong\u003ee\u003c/strong\u003e) and methyl (-CH\u003csub\u003e3\u003c/sub\u003e, \u003cstrong\u003ef\u003c/strong\u003e) groups at 4.32 and 1.43 ppm, respectively, confirmed the synthesis of the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate. The production process yields for 2-azidoethanol, 2-azidoethyl-2-chloro-2-phenylacetate and 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate products were approximately 82, 95 and 93%, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.1.2\u0026nbsp;\u003c/em\u003eSynthesis of well-defined azido-terminated poly(\u003cem\u003eN\u003c/em\u003e-vinylcaprolactam) homopolymers (PNVCL-N\u003csub\u003e3\u003c/sub\u003e)\u003c/p\u003e\n\u003cp\u003eThe livingness of the RAFT/MADIX polymerization of NVCL in the presence of the RAFT agent\u0026nbsp;2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate\u0026nbsp;was evaluated by the polymerization kinetics using a [NVCL]:[CTA]:[AIBN] molar ratio of 300:1:0.25. The polymerization of NVCL proceeded slowly, reached a conversion limit of approximately 32.5% (Fig. S1(a) in Supporting Information). Although an induction period is often observed in RAFT/MADIX systems [47], such a period was not clearly observed in our system. The low chain transfer efficiency due to the steric hindrance caused by the caprolactam ring may be the reason for the low conversion rate. Similar findings were reported by Peng et al. (2015) [48]\u0026nbsp;during the RAFT copolymerization of NVCL with methacrylic acid N-hydroxysuccinimide ester (MNHS) mediated by the CTA methyl 2-(ethoxycarbonothioylthio)propanoate, where conversion values below 50% were attributed to high steric hindrance caused by the 7-membered group. Despite the linear increase in the number-average molar mass (\u003cem\u003eM\u003c/em\u003en\u003csub\u003eSEC\u003c/sub\u003e) of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e polymers with monomer conversion, the dispersity (\u003cem\u003e\u0026ETH;\u003c/em\u003e) of the polymer also slightly increased with the reaction time (Fig. S1(b) in the Supporting Information), suggesting that side reactions occurred under the experimental conditions, potentially due to the low thermal stability of the xanthate end group and/or impurities. Therefore, to keep the polymer dispersity (\u003cem\u003e\u0026ETH;\u003c/em\u003e) below 1.5, the NVCL conversion had to be limited to approximately 20% (reaction time = 6 hours).\u003c/p\u003e\n\u003cp\u003eSEC chromatogram of PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1) is depicted in Figure S2 in the \u003cem\u003eSupporting Information\u003c/em\u003e. The\u0026nbsp;number average molar masses (\u003cem\u003eM\u003c/em\u003en) and dispersity (\u003cem\u003eĐ\u003c/em\u003e) values obtained for this polymer are presented in Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Related data on PNVCL-N\u003csub\u003e3\u003c/sub\u003e synthesized by the RAFT/MADIX polymerization of NVCL mediated by the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"599\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.8963%;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.4281%;\"\u003e\n \u003cp\u003e[NVCL]\u003csub\u003e0\u003c/sub\u003e:[CTA]\u003csub\u003e0\u003c/sub\u003e:[AIBN]\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\n \u003cp\u003emolar ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.709%;\"\u003e\n \u003cp\u003eConv.\u003csup\u003ea\u003c/sup\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0368%;\"\u003e\n \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003en theo\u0026nbsp;\u003c/sub\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0368%;\"\u003e\n \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003en NMR\u0026nbsp;\u003c/sub\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8696%;\"\u003e\n \u003cp\u003e\u003cem\u003eM\u003c/em\u003e\u003csub\u003en SEC\u0026nbsp;\u003c/sub\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.02341%;\"\u003e\n \u003cp\u003e\u003cem\u003eĐ\u003c/em\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.8963%;\"\u003e\n \u003cp\u003ePNVCL-N\u003csub\u003e3\u003c/sub\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 28.4281%;\"\u003e\n \u003cp\u003e300:1:0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.709%;\"\u003e\n \u003cp\u003e19.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0368%;\"\u003e\n \u003cp\u003e8595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0368%;\"\u003e\n \u003cp\u003e8344\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 10.8696%;\"\u003e\n \u003cp\u003e3298\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.02341%;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eReaction time = 6h; \u003csup\u003ea\u003c/sup\u003e Determined by \u003csup\u003e1\u003c/sup\u003eH NMR; \u003csup\u003eb\u003c/sup\u003e\u003cem\u003eM\u003c/em\u003en \u003csub\u003etheo.\u003c/sub\u003e= ([NVCL]/[CTA] . MM\u003csub\u003eNVCL\u003c/sub\u003e . conv) + MM\u003csub\u003eCTA\u003c/sub\u003e; \u003csup\u003ec\u003c/sup\u003e determined by \u003csup\u003e1\u003c/sup\u003eH NMR by using the average peak area of methine protons from PNVCL backbone at 4.30 ppm and that one of the methine protons of the aromatic ring from the CTA present in the end of the PNVCL chain at 7.2 ppm; \u003csup\u003ed\u003c/sup\u003e determined by SEC in THF and TEA (0.3% v/v) at 35 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe experimental molar mass determined by NMR (\u003cem\u003eM\u003c/em\u003en \u003csub\u003eNMR\u003c/sub\u003e) agreed with its theoretical molar mass (\u003cem\u003eM\u003c/em\u003en \u003csub\u003etheo\u003c/sub\u003e) (Table 1). However, due to the use of PS as a calibration standard in the SEC analysis, there was a significant discrepancy between them and that one obtained by SEC (\u003cem\u003eM\u003c/em\u003en \u003csub\u003eSEC\u003c/sub\u003e). Nevertheless, the sample exhibits monomodal molar mass distribution and a relative low dispersity (\u003cem\u003eĐ\u0026nbsp;\u003c/em\u003e\u0026lt; 1.5), as desired.\u003c/p\u003e\n\u003cp\u003eFTIR spectrum of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1) presented in Fig. 2 showed absorption bands at 1631 cm\u003csup\u003e-1\u003c/sup\u003e due to the axial deformation of the carbonyl (C=O) of the caprolactam ring; at 2930 and 2858 cm\u003csup\u003e-1\u003c/sup\u003e relative to the C-H stretch; at 1442 cm\u003csup\u003e-1\u003c/sup\u003e referring to the -CH\u003csub\u003e2\u003c/sub\u003e- angular deformation and at 1480 cm\u003csup\u003e-1\u003c/sup\u003e relative to the C-N axial deformation. The shoulder at around 3250 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e is assigned to N-H stretching vibration. The broad absorption band in the region 3200\u0026ndash;3700 cm\u003csup\u003e\u0026minus;1\u0026nbsp;\u003c/sup\u003ehas arisen from the moisture adsorbed by the sample. The -N=N\u003csup\u003e+\u003c/sup\u003e=N\u003csup\u003e-\u003c/sup\u003e band present at around 2100 cm\u003csup\u003e-1\u003c/sup\u003e indicates the functionalization of the polymer with azide groups from the CTA.\u003c/p\u003e\n\u003cp\u003eAs seen in \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of PNVCL-N\u003csub\u003e3\u003c/sub\u003e (1) from Fig. 3, the sample gave the typical resonance signals of PNVCL at \u0026delta; (ppm) = 4.35 (1H, \u0026ndash;NCH\u0026ndash; \u0026alpha; position, \u003cstrong\u003ec\u003c/strong\u003e), 3.05 (\u0026ndash;NCH\u003csub\u003e2\u003c/sub\u003e\u0026ndash;, \u003cstrong\u003ed\u003c/strong\u003e), 2.25 (\u0026ndash;C(=O)CH\u003csub\u003e2\u003c/sub\u003e\u0026ndash;, \u003cstrong\u003eh\u003c/strong\u003e) and 1.0\u0026ndash;2.0 (6H, \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e\u0026ndash;, \u003cstrong\u003ee\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e of the caprolactam ring, and \u0026ndash;CH\u003csub\u003e2\u003c/sub\u003e\u0026ndash; of the backbone chain of the polymer, \u003cstrong\u003ei\u003c/strong\u003e). The resonance signals located at 7.23 and 5.0 ppm refer to the methine (5H, =CH-, \u003cstrong\u003ek\u003c/strong\u003e) groups of the aromatic ring, and the methylene (-C(=O)O\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003e, \u003cstrong\u003el\u003c/strong\u003e) group of the CTA, respectively. The peak located at 4.6 ppm is relative to the methylene (\u0026ndash;\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e, \u003cstrong\u003eb\u003c/strong\u003e) protons of the xanthate group. The other protons from CTA (\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;j\u003c/strong\u003e and \u003cstrong\u003em\u003c/strong\u003e) presented their signals overlapped by the characteristic peaks of the PNVCL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Synthesis of the alkyne end-capped PHBHV (alkyne-PHBHV)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlkyne end-capped PHBHV were prepared through a transesterification reaction between PHBHV (\u003cem\u003eM\u003c/em\u003en\u003csub\u003eSEC\u0026nbsp;\u003c/sub\u003e= 114873 g mol\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand \u003cem\u003eĐ\u003c/em\u003e = 2.86) and propargyl alcohol, in toluene, at 110 \u0026deg;C, using dibutyltin dilaurate (DBTD) as catalyst. SEC chromatograms obtained for the alkyne-PHBHV are shown in Fig. S4 in the \u003cem\u003eSupporting Information\u003c/em\u003e. The \u003cem\u003eM\u003c/em\u003en and \u003cem\u003e\u0026ETH;\u003c/em\u003e values for these materials are presented in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Related data on alkyne-PHBHV prepared by transesterification reaction between a high molar mass PHBHV and propargyl alcohol.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2617%;\"\u003e\n \u003cp\u003et (h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.0134%;\"\u003e\n \u003cp\u003e\u003cem\u003eM\u003c/em\u003en \u003csub\u003eNMR\u0026nbsp;\u003c/sub\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1477%;\"\u003e\n \u003cp\u003e\u003cem\u003eM\u003c/em\u003en \u003csub\u003eSEC\u0026nbsp;\u003c/sub\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026ETH;\u0026nbsp;\u003c/em\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eAlkyne-PHBHV (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2617%;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.0134%;\"\u003e\n \u003cp\u003e5687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1477%;\"\u003e\n \u003cp\u003e4536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eAlkyne-PHBHV (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.2617%;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 27.0134%;\"\u003e\n \u003cp\u003e9807\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.1477%;\"\u003e\n \u003cp\u003e9403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Determined by \u003csup\u003e1\u003c/sup\u003eH NMR by using the average peak areas of the methine and methyl protons from PHBHV backbone at 5.25 and 0.85 ppm, respectively, and methylene protons from the polymer chain termination at 4.68 ppm; \u003csup\u003eb\u003c/sup\u003e determined by SEC in THF and TEA (0.3 % v/v) at 35 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eAs expected, it was observed a decrease in the \u003cem\u003eM\u003c/em\u003en \u003csub\u003eSEC\u003c/sub\u003e of the PHBHV after its transesterification reaction with propargyl alcohol. Moreover, the alkyne-PHBHV (2) showed higher \u003cem\u003eM\u003c/em\u003en than alkyne-PHBHV (1), as expected, and both materials showed low dispersity values (\u003cem\u003e\u0026ETH;\u003c/em\u003e \u0026le; 1.5) and monomodal SEC chromatograms.\u003c/p\u003e\n\u003cp\u003eFig. 4 and 5 show the FTIR and \u003csup\u003e1\u003c/sup\u003eH NMR spectra of the alkyne-PHBHV (1), respectively. FTIR spectra of alkyne-PHBHV exhibited the characteristic strain bands of PHBHV, such as axial strain of the carbonyl (C=O) at 1722 cm\u003csup\u003e-1\u003c/sup\u003e, ester C-O at 1275 cm\u003csup\u003e-1\u003c/sup\u003e, O-H at 3431 cm\u003csup\u003e-1\u003c/sup\u003e and C-C at 976 cm\u003csup\u003e-1\u003c/sup\u003e. The spectra also showed the bands of symmetric angular deformation in the plane of the methyl (-CH\u003csub\u003e3\u003c/sub\u003e) groups at 1380 cm\u003csup\u003e-1\u003c/sup\u003e and the typical band of the chain spiral conformation at 1222 cm\u003csup\u003e-1\u003c/sup\u003e. The bands in the range between 2830 and 3070 cm\u003csup\u003e-1\u003c/sup\u003e correspond to the C-H axial strains of methyl and methylene groups, and the bands at 1130 and 1180 cm\u003csup\u003e-1\u003c/sup\u003e refer to the symmetric and asymmetric stretches of the -C-O-C- group, respectively. Moreover, the axial deformation vibration band of the \u0026equiv;C-H appears at 3255 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of the alkyne-PHBHV it was possible to attribute the chemical shifts that characterize the PHBHV structure, at: \u0026delta; (ppm) = 5.10-5.32 (-CH- of HB and HV, \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e respectively), 2.40-2.67 (-CH\u003csub\u003e2\u003c/sub\u003e- of HB and HV, \u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e, respectively), 1.5-1.7 (-CH\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e of HV, \u003cstrong\u003ee\u003c/strong\u003e), 1.20-1.35 (-CH\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e3\u003c/sub\u003e of HB, \u003cstrong\u003eb\u003c/strong\u003e) and 0.79-0.95 (-CHCH\u003csub\u003e2\u003c/sub\u003e\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e3\u0026nbsp;\u003c/sub\u003eof HV, \u003cstrong\u003ef\u003c/strong\u003e). Furthermore, it was possible to observe the resonance signals of the adjacent protons to the hydroxyl-terminal group characteristic of the PHBHV molecule at 4.16 ppm (-\u003cu\u003eCH\u003c/u\u003e(CH)\u003csub\u003e3\u003c/sub\u003eOH, \u003cstrong\u003ea\u0026apos;\u003c/strong\u003e), and the protons from the propargyl alcohol molecule coupled to the end of the PHBHV chain at 4.67 ppm (HC\u0026equiv;C-\u003cu\u003eCH\u003c/u\u003e\u003csub\u003e2\u003c/sub\u003e-, \u003cstrong\u003eh\u003c/strong\u003e) and 2.36 ppm (\u003cu\u003eHC\u003c/u\u003e\u0026equiv;C-CH\u003csub\u003e2\u003c/sub\u003e-, \u003cstrong\u003ei\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e3.3 \u003cstrong\u003eCoupling reaction between PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV homopolymers via CuAAC\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally,\u0026nbsp;PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV were reacted to give the corresponding PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers. Aiming to make all the coupling reactions completely, they were carried out by using an excess of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e homopolymer. After the click reactions, the unreacted PNVCL-N\u003csub\u003e3\u003c/sub\u003e was removed using dialysis method. PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers were characterized by SEC as shown in Fig. 6. Table 3 shows the \u003cem\u003eM\u003c/em\u003en and \u003cem\u003eĐ\u003c/em\u003e values obtained for these copolymers synthesized with different lengths of PHBHV hydrophobic segment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u003c/strong\u003e Related data on PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers synthesized by CuAAC\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45.9732%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eM\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003en\u003csub\u003eSEC\u0026nbsp;\u003c/sub\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003e(g.mol\u003csup\u003e-1\u003c/sup\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.2013%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eĐ\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45.9732%;\"\u003e\n \u003cp\u003ePNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003e6642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.2013%;\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 45.9732%;\"\u003e\n \u003cp\u003ePNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003e11190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 30.2013%;\"\u003e\n \u003cp\u003e1.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eUsing the [alkyne-PHBHV]\u003csub\u003e0\u003c/sub\u003e:[PNVCL-N\u003csub\u003e3\u003c/sub\u003e]\u003csub\u003e0\u003c/sub\u003e:[CuBr]\u003csub\u003e0\u003c/sub\u003e:[PMDTA]\u003csub\u003e0\u003c/sub\u003e = 1:1.25:4:4; \u003csup\u003ea\u003c/sup\u003e determined by SEC in THF and TEA (0.3 % v/v) at 35 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eAs seen in Fig. 4, the SEC chromatography of PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV shifted toward high molecular weight direction as compared to that of PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV, while neither PNVCL-N\u003csub\u003e3\u003c/sub\u003e nor alkyne-PHBHV traces were found. Furthermore, relatively low \u003cem\u003eĐ\u0026nbsp;\u003c/em\u003evalues were observed for both samples. Therefore, these results suggest the successful coupling between both macrochains, and the absence of residual PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV polymers.\u003c/p\u003e\n\u003cp\u003eFurther confirmation of the \u0026lsquo;\u0026lsquo;click\u0026rsquo;\u0026rsquo; coupling can be taken from FTIR spectroscopy. In Fig. 7, the IR spectra of PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers are compared to the spectra of starting polymers. As expected, the FTIR spectrum of PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV showed the characteristic vibration bands of both PNVCL and PHBHV segments including the axial deformation of the carbonyl (C=O) of the caprolactam ring at 1630 cm\u003csup\u003e-1\u003c/sup\u003e and the C=O stretching of esters at 1725 cm \u003csup\u003e-1\u003c/sup\u003e. The disappearance\u0026nbsp;of the band of the azide group (\u0026ndash;N=N\u003csup\u003e+\u003c/sup\u003e=N\u003csup\u003e-\u003c/sup\u003e) at 2100 cm\u003csup\u003e-1\u003c/sup\u003e in the spectra of the block copolymers suggest the formation of the desired block copolymers. However, it was not possible to confirm the disappearance of the \u0026equiv;CH band at 3255 cm\u003csup\u003e-1\u003c/sup\u003e because the materials exhibited a broad absorption in the region 3200\u0026ndash;3600 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e due to hydroxyl groups and/or moisture adsorbed by the sample and a band of NH stretching vibration at around 3275 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn \u003csup\u003e1\u003c/sup\u003eH NMR spectra of PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (Fig. 8), it was possible to observe the characteristic peaks of PHBHV (\u003cstrong\u003eu\u003c/strong\u003e, \u003cstrong\u003eq\u003c/strong\u003e, \u003cstrong\u003et\u003c/strong\u003e, \u003cstrong\u003ep\u003c/strong\u003e, \u003cstrong\u003ev\u003c/strong\u003e and \u003cstrong\u003es\u003c/strong\u003e) and PNVCL (\u003cstrong\u003ec\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ef\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e and \u003cstrong\u003ei\u003c/strong\u003e) segments, which were previously detailed. Comparing the spectrum of the alkyne-PHBHV with the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV,\u0026nbsp;the methylene protons resonance signal characteristics of the propargyl group of the alkyne-PHBHV at 4.68 ppm (represented by\u0026nbsp;\u0026ldquo;\u003cstrong\u003eh\u003c/strong\u003e\u0026rdquo; in Fig. 5) had clearly shifted to 5.22 ppm (represented by \u0026ldquo;\u003cstrong\u003eo\u003c/strong\u003e\u0026rdquo; in Fig. 8), which is overlapping by the \u003cstrong\u003eq\u003c/strong\u003e and \u003cstrong\u003eu\u003c/strong\u003e peaks. Moreover, the appearance of a new peak, relative to the methine (CH, \u003cstrong\u003en\u003c/strong\u003e) protons from the triazole ring at 7.82 ppm, confirms the coupling of PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV segments. Furthermore, these NMR results, in agreement with those obtained by SEC and FTIR analyses, also indicate a high degree of purity for the block copolymers, which is confirmed by the absence of PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV residue. It was not possible to determine the \u003cem\u003eM\u003c/em\u003en of these block copolymer by \u003csup\u003e1\u003c/sup\u003eH NMR because all characteristic peaks of the HV units (\u003cstrong\u003ep\u003c/strong\u003e, \u003cstrong\u003eq\u003c/strong\u003e, \u003cstrong\u003er\u003c/strong\u003e and \u003cstrong\u003es\u003c/strong\u003e) of the PHBHV segment are overlapping by other peaks of the block copolymer.\u003c/p\u003e\n\u003cp\u003ePNVCL\u0026ndash;N\u003csub\u003e3\u003c/sub\u003e, alkyne-PHBHV and the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers were characterized by DSC, as shown in Fig. 9, with the main transition temperature data summarized in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Thermal characterization of PNVCL-N\u003csub\u003e3\u003c/sub\u003e, alkyne-PHBHV and PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV by DSC\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003eTg \u003csub\u003ePNVCL\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003eTg \u003csub\u003ePHBHV\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003eTm\u003csub\u003e1 PHBHV\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003eTm\u003csub\u003e2 PHBHV\u0026nbsp;\u003c/sub\u003e(\u0026deg;C)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003e\u0026Delta;Hm\u003csub\u003e1\u003c/sub\u003e (J/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e\u0026Delta;Hm\u003csub\u003e2\u003c/sub\u003e (J/g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003eXc \u003csup\u003ea,b\u003c/sup\u003e (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003ePNVCL-N\u003csub\u003e3\u003c/sub\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e175.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eAlkyne-PHBHV (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e3.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e135.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e147.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003e21.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e49.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003e45.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003eAlkyne-PHBHV (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e146.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003e157.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003e39.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e51.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003e47.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003ePNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 23.8255%;\"\u003e\n \u003cp\u003ePNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003e14.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.7517%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.56376%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.0738%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 7.88591%;\"\u003e\n \u003cp\u003end\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNd = not determined, \u003csup\u003ea\u003c/sup\u003e Xc (%) = (\u0026Delta;Hm/ \u0026Delta;H\u0026deg;m).100; \u0026Delta;H\u0026deg;m = enthalpy fusion of 100% crystalline PHBHV = 109 J.g\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e[49]; \u003csup\u003eb\u003c/sup\u003e determined by using Tm\u003csub\u003e2PHBHV\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eThe glass transition temperature (Tg) of the PNVCL, an amorphous material, is commonly reported to be around 147 \u0026deg;C [50-52]. However, its Tg can be influenced by several factors, including molar mass, dispersity, purity [53], and the presence of water in the polymer [51, 52]. In our study, the determined Tg of the dried PNVCL\u0026ndash;N\u003csub\u003e3\u003c/sub\u003e was determined to be 175,4 \u0026deg;C, which is consistent with previous findings reported by our group [15] and in the literature, such as those by Usanmaz et al. (2009) [54] and Durkut et al. (2009) [55], who observed values of 177.2, 174.6 and 174.0 \u0026deg;C, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the PHBHV-alkyne samples, the Tg values were about 4.0 \u0026deg;C, which is close to the T\u003csub\u003eg\u003c/sub\u003e of PHBHV reported in the literature [56, 57]. Two peaks related to melting events (Tm\u003csub\u003e1\u003c/sub\u003e and Tm\u003csub\u003e2\u003c/sub\u003e) were observed in the DSC thermograms of PHBHV-alkyne. This thermal behavior referring to the PHBHV fusion agrees with the literature [58-60]. According to Liu et al. (2009) [61], these two melting peaks can be attributed to the polymer melting-recrystallization-remelt process. The second melting point (Tm\u003csub\u003e2\u003c/sub\u003e), the one with the highest temperature, is generally used as the Tm of the PHBHV. Furthermore, it is important to mention that the two endothermic peaks in the DSC thermogram have also been commonly observed for PHB [62-64]. Comparing the T\u003csub\u003em\u003c/sub\u003e and the crystallinity degree (Xc) of both PHBHV-alkyne, the PHBHV-alkyne (2) showed the highest T\u003csub\u003em\u003c/sub\u003e and crystallinity degree (Xc) values due to its higher molar mass, as expected.\u003c/p\u003e\n\u003cp\u003eThe thermograms of the block copolymers samples showed Tg values at 27.0 and 14.9 \u0026deg;C, corresponding to the PHBHV block segments in PNVCL-b-PHBHV (1) and PNVCL-b-PHBHV (2), respectively. However, the Tg associated with the PNVCL segment was not observed for both PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV copolymers, possibly due to the dominance of the PHBHV phase in these copolymers, or due to specific conditions of the DSC analysis. Additionally, the DSC thermograms of the block copolymers did not show endothermic peak related to melting, suggesting that these materials have a low degree of crystallinity or exhibit amorphous polymer characteristics. The PNVCL segment covalently bound to the PHBHV segment must have restricted the PHBHV crystallization, compromising the regularity of its crystalline structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Determination of the cmc of the block copolymers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe onset of micellization and the critical micelle concentration (cmc) of the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers were obtained using a fluorescence technique with pyrene as the fluorescence probe. The plot of the I337/I333 intensity ratio (from fluorescence measurement) \u003cem\u003eversus\u003c/em\u003e the logarithm of the corresponding concentration of the copolymer in water (in mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e) is shown in Fig. 10\u003cem\u003e.\u003c/em\u003e At a certain concentration, the intensity ratio started to increase dramatically due to the incorporation of pyrene into the hydrophobic core of the micelles. The intersection of the baseline and the rapidly rising I337/I333 line is considered as the cmc of the amphiphilic block copolymer [14]. From this plot, CMC values for both samples were close, being 3.89 and 3.09 10\u003csup\u003e-3\u003c/sup\u003e mg mL\u003csup\u003e-1\u003c/sup\u003e for PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1) and PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (2), respectively. In agreement with the literature for amphiphilic block copolymer [14-16, 65], the cmc decreased as the length of the hydrophobic block increased. This trend is strictly related to the hydrophilicity of the copolymer since a higher length of the hydrophobic segment results in stronger interactions between the hydrophobic chains; therefore, a lower concentration of polymer in water is necessary to induce micellization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5.\u003c/strong\u003e E\u003cstrong\u003effect of the hydrophobic block length on the hydrodynamic diameter of the polymeric micelles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of the PHBHV hydrophobic length on the average size of PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV-based polymeric micelles, micellar solutions, prepared with the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV copolymers by the precipitation technique, were analyzed by DLS. The hydrodynamic diameter (Zav) of the polymeric micelles formed from PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (1) and PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV (2) (0.05 mg mL\u003csup\u003e\u0026minus;1\u003c/sup\u003e) was about 173.3 nm (PDI 0.243) and 206.6 nm (PDI 0.372), respectively. In addition, both PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV micelles exhibited a monomodal size distribution (Fig. 11). As was expected, for the block copolymes synthesized in this study, with a fixed leght for the hydroplilic segment (PNVCL), an increase in the length of the hydrophobic segment of PHBHV led to micelles with larger diameters due to the greater hydrophobic interaction forces and packing density required to accommodate the longer PHBHV blocks within the micelle core. The increased hydrophobic block length results in a larger micelle core to minimize the unfavorable interactions between the hydrophobic PHBHV segments and the aqueous environment. These results are in agreement with the results reported in the literature for amphiphilic block copolymers [14-16, 65] and suggesting that the size of the micelle could be adjustable as a function of the length of the hydrophobic segment in the block copolymer.\u003c/p\u003e"},{"header":"4. CONCLUSION","content":"\u003cp\u003eA novel block copolymer, PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV, was successfully synthesized by combining the RAFT/MADIX polymerization and the click chemistry reaction. First, well-defined azido-group functionalized\u0026nbsp;PNVCL homopolymers (PNVCL-N\u003csub\u003e3\u003c/sub\u003e) was synthesized by RAFT/MADIX polymerization of NVCL mediated by the 2-azidoethyl 2-((ethoxycarbonothioyl)thio)-2-phenylacetate. The RAFT/MADIX polymerization of NVCL showed partial control by the CTA. Therefore, aiming to obtain PNVCL homopolymers with low dispersity, the NVCL conversion had to be limited to values below 20%. Alkyne-terminated PHBHV was prepared by transesterification reaction of the PHBHV with propargyl alcohol. SEC technique confirmed the transesterification reaction of PHBHV leading a material with low molecular mass. The chemical structures of the PNVCL-N\u003csub\u003e3\u003c/sub\u003e and alkyne-PHBHV were successfully confirmed by FTIR and \u003csup\u003e1\u003c/sup\u003eH NMR analyses. Then, PNVCL-N\u003csub\u003e3\u003c/sub\u003e and PHBHV-alkyne macrochains were coupled by the CuAAC technique, which was confirmed by the SEC analyzes. Furthermore, while FTIR analyzes showed the disappearance of the azide function of PNVCL-N\u003csub\u003e3\u003c/sub\u003e, after its reaction with PHBHV-alkyne, the formation of the triazole ring was verified by NMR analyses. The DSC analysis indicated that the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV block copolymers had characteristics of amorphous material or very low degrees of crystallinity. The amphiphilic character of the block copolymers and their ability to form micelles were successfully confirmed. The cmc values of these copolymers decreased with increasing the length of the PHBHV segment. Finally, the size of the polymeric micelles was investigated. The average diameter of the polymeric micelles determined by DLS increased with increasing the length of the PHBHV segment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCredit Authorship Contribution Statement\u0026nbsp;\u003c/strong\u003eRodolfo Minto de Moraes: Conceptualization, Methodology, Formal Analysis, Investigation, Validation, Data Curation, Writing-Original Draft Preparation, Writing—Review and Editing. Layde Teixeira de Carvalho: Writing-Original Draft Preparation. Gizelda Maria Alves: Formal Analysis and Data Curation. Simone de Fátima Medeiros: Writing-Original Draft Preparation, Writing—Review and Editing. Amilton Martins dos Santos: Writing—Review and Editing, Supervision, Project administration, Funding Acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAckonowledgments\u0026nbsp;\u003c/strong\u003eThis research was financially supported by the \u003cem\u003eSão Paulo State Research Support Foundation\u0026nbsp;\u003c/em\u003e(FAPESP, n° 2013/03355-4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe authors confirm that the data supporting the findings of this study are available within the article (and/or) its supplementary materials. If necessary, the extra data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDischer DE, Eisenberg A (2002) Polymer Vesicles. 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Iran Polym J 32:1627\u0026ndash;1641. https://doi.org/10.1007/s13726-023-01230-4.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Poly(N-vinylcaprolactam), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), Amphiphilic, Thermoresponsive, Block Copolymer, RAFT/MADIX, Click Chemistry","lastPublishedDoi":"10.21203/rs.3.rs-6328656/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6328656/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAmphiphilic copolymers have gained significant attention in the field of drug delivery systems (DDS). The key feature that makes them promising for such application is their ability to self-assemble into micelles in aqueous media. In this context, this work describes the synthesis of a novel amphiphilic well-defined block copolymer, poly(\u003cem\u003eN\u003c/em\u003e-vinylcaprolactam)-\u003cem\u003eb\u003c/em\u003e-poly(3-hydroxybutyrate-\u003cem\u003eco\u003c/em\u003e-3-hydroxyvalerate) (PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV), using a strategy that combines reversible addition-fragmentation chain-transfer macromolecular design via interchange of xanthates (RAFT/MADIX) polymerization and click chemistry reaction. Initially, azido-terminated PNVCL homopolymers (PNVCL-N\u003csub\u003e3\u003c/sub\u003e) were synthesized through RAFT/MADIX polymerization of the \u003cem\u003eN\u003c/em\u003e-vinylcaprolactam (NVCL) monomer, mediated by a chain transfer agent (CTA) bearing an azide group. Meanwhile, the alkyne-terminated PHBHV (alkyne-PHBHV) was prepared by the transesterification reaction between PHBHV and propargyl alcohol. Then, the 1,3-cyclo addition reaction between azide and alkyne (CuAAC) was used to obtain the block copolymer PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV. \u0026nbsp;Different size chains of PHBHV were evaluated as also their influence on the capacity of micelles formation. The chemical structures of all (co)polymers were assessed by Fourier-Transform Infrared spectroscopy (FTIR) and Proton Nuclear Magnetic Resonance spectroscopy (\u003csup\u003e1\u003c/sup\u003eH NMR) analysis, while their molar masses were determined by Size Exclusion Chromatography (SEC). Differential Scanning Calorimetry (DSC) measurement showed that the PNVCL-\u003cem\u003eb\u003c/em\u003e-PHBHV have lower degree of crystallinity than PHBHV. Additionally, it was observed that the critical micelle concentration (cmc) of the block copolymers in aqueous solution decreased as the length of the hydrophobic block increased, whereas the size of the polymeric micelles grew with a higher proportion of hydrophobic segments.\u003c/p\u003e","manuscriptTitle":"Synthesis and self-assembly of a novel block copolymer poly(N-vinylcaprolactam)-b-poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PNVCL-b-PHBHV) by the combination of RAFT/MADIX and click chemistry techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-24 15:01:37","doi":"10.21203/rs.3.rs-6328656/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-11T14:51:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-11T05:33:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153903982333057320817134546222037029043","date":"2025-06-02T07:03:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-24T03:50:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296905187083786670238018360159684323077","date":"2025-05-08T02:56:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-03T14:24:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-29T15:14:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-29T11:37:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Polymer Bulletin","date":"2025-03-28T13:35:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"polymer-bulletin","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pobu","sideBox":"Learn more about [Polymer Bulletin](http://link.springer.com/journal/289)","snPcode":"289","submissionUrl":"https://submission.nature.com/new-submission/289/3","title":"Polymer Bulletin","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b1d7c764-0e24-4b59-bc39-263385c5acff","owner":[],"postedDate":"April 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:04:14+00:00","versionOfRecord":{"articleIdentity":"rs-6328656","link":"https://doi.org/10.1007/s00289-025-05924-y","journal":{"identity":"polymer-bulletin","isVorOnly":false,"title":"Polymer Bulletin"},"publishedOn":"2025-07-10 15:58:00","publishedOnDateReadable":"July 10th, 2025"},"versionCreatedAt":"2025-04-24 15:01:37","video":"","vorDoi":"10.1007/s00289-025-05924-y","vorDoiUrl":"https://doi.org/10.1007/s00289-025-05924-y","workflowStages":[]},"version":"v1","identity":"rs-6328656","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6328656","identity":"rs-6328656","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}